Double-stranded oligonucleotide compositions and related methods

Abstract

The present disclosure provides double-stranded oligonucleotides, compositions and methods related thereto.The present disclosure encompasses the recognition that the structural elements of double-stranded oligonucleotides, such as base sequence, chemical modifications (e.g., sugar, base and / or internucleotide bond modifications) or patterns thereof, and / or stereochemistry (e.g., backbone chiral center (chiral internucleotide bond) stereochemistry) and / or patterns thereof, can have significant effects on the properties and activities of oligonucleotides, such as RNA interference (RNAi) activity, stability, delivery, etc.The present disclosure also provides methods of treating diseases using the provided double-stranded oligonucleotide compositions, for example, in RNA interference.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 246,756, filed September 21, 2021, the contents of which are incorporated by reference in their entirety. [Background technology]

[0002] background Gene-targeting oligonucleotides are useful in a variety of applications, including therapeutics, diagnostics, research, and nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) in such applications can be limited, for example, by their susceptibility to endo- and exonucleases. Therefore, various synthetic counterparts have been developed to avoid such drawbacks. These include synthetic oligonucleotides containing chemical modifications, such as base modifications, sugar modifications, and backbone modifications. However, there remains a need in the art for double-stranded (ds) oligonucleotides with improved properties for use in the above applications. Summary of the Invention [Means for solving the problem]

[0003] overview The present disclosure relates, in part, to the recognition that controlling the structural elements of double-stranded (ds) oligonucleotides can significantly affect the properties and / or activity of ds oligonucleotides. In certain embodiments, such structural elements include one or more of the following: (1) chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) and their patterns; and (2) alterations in stereochemistry (e.g., the stereochemistry of backbone chiral internucleotide linkages) and their patterns. One or more of such structural elements may, in certain embodiments, be independently present in one or both oligonucleotides of a ds oligonucleotide. In certain embodiments, properties and / or activities influenced by such structural elements include, but are not limited to, involvement in, or directionality of, a reduction in the expression, activity, or level of a gene or its gene product, mediated by, for example, RNA interference (RNAi interference), RNase H-mediated knockdown, steric hindrance of translation, etc.

[0004] 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.

[0005] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., the stereochemistry of backbone chiral centers, can unexpectedly maintain or improve the properties of ds oligonucleotides. For example, but not by way of limitation, the present disclosure provides, in part, (1) a guide strand containing backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand containing backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5′-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3′ direction and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (3) a guide strand containing one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide (the upstream backbone phosphorothioate chiral centers are in the Rp or Sp configuration); (4) a guide strand containing one or more backbone phosphorothioate chiral centers in the 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 between (a) the +3 and +4 nucleotides; and (b) the +5 and +6 nucleotides; (5) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in the Rp or Sp configuration; and (6) A ds oligonucleotide comprising one or more passenger strands in combination with one or more of the aforementioned guide strands, the passenger strands comprising backbone phosphorothioate chiral centers in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; The ds oligonucleotide further comprises: (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (i.e., the guide strand includes one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The present invention relates to ds oligonucleotides, wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bond incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is an Sp non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is an Sp non-negatively charged internucleotide bond. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0006] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., the stereochemistry of the chiral center in the 5'-terminal modification of the guide strand, can unexpectedly maintain or improve the properties of the ds oligonucleotides described herein. For example, but not by way of limitation, the present disclosure encompasses, in part, (1) phosphorothioate chiral centers in the Rp or Sp configuration; (2) Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in which the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage contains a 2' modification, e.g., a 2'F; and (3) (a) Including, but not limited to: [ka] 5'PO modifications such as; (b) Including, but not limited to: [ka] 5'VP modifications such as; (c) Including, but not limited to: [ka] 5'MeP modifications such as; (d) Including, but not limited to: [ka] 5'PN and 5'Trizol-P modifications such as; wherein the bases are selected from A, C, G, T, U, abasic and modified nucleobases; R 2’ is H, OH, O-alkyl, F, MOE, locked nucleic acid (LNA) bridges and bridged nucleic acid (BNA) bridges to 4'C, including, but not limited to: [ka] The present invention relates to a ds oligonucleotide comprising a guide strand comprising a 5'-end modification selected from the group consisting of: (selected from). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages.

[0007] In certain other embodiments, the present disclosure encompasses the recognition that the stereochemistry, e.g., the stereochemistry of the chiral center at the 5'-terminal nucleotide of the guide strand, can unexpectedly maintain or improve the properties of ds oligonucleotides in which the guide strand of the ds oligonucleotide also contains a phosphorothioate chiral center in the Rp or Sp configuration. For example, but not by way of limitation, the present disclosure encompasses, in part, (1) a phosphorothioate chiral center in the Rp or Sp configuration; (2) an Rp, Sp, or a sterically irregular non-negatively charged internucleotide linkage in which the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or a sterically irregular non-negatively charged internucleotide linkage contains a 2' modification, e.g., a 2'F; and (3) (a) Including, but not limited to: [ka] 5'PO nucleotides such as; (b) Including, but not limited to: [ka] 5'VP nucleotides such as; (c) Including, but not limited to: [ka] 5'MeP nucleotides such as; (d) Including, but not limited to: [ka] 5'PN and 5'Trizol-P nucleotides such as; (e) Including, but not limited to: [ka] 5' abasic VP and 5' abasic MeP nucleotides such as The present invention relates to a ds oligonucleotide comprising a guide strand comprising a 5'-end modification selected from the group consisting of: In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages.

[0008] In certain embodiments, the present disclosure encompasses the recognition that non-naturally occurring internucleotide linkages, such as neutral internucleotide linkages, can be used to attach one or more molecules to the double-stranded oligonucleotides described herein. In certain embodiments, such binding molecules can facilitate targeting and / or delivery of the double-stranded oligonucleotides. For example, but not limited to, such binding molecules include lipophilic molecules. In certain embodiments, the binding molecule is a molecule comprising one or more GalNAc moieties. In certain embodiments, the binding molecule is a receptor. In certain embodiments, the binding molecule is a receptor ligand.

[0009] In certain embodiments, the present disclosure provides techniques for incorporating various additional chemical moieties into ds oligonucleotides. In certain embodiments, the present disclosure provides, for example, reagents and methods for using nucleobases to introduce additional chemical moieties (e.g., by covalent attachment to sites on the nucleobases, optionally via linkers).

[0010] In certain embodiments, the present disclosure provides techniques, e.g., ds oligonucleotide compositions and methods, for achieving allele-specific suppression, in which transcripts from one allele of a particular target gene are selectively knocked down relative to at least one other allele of the same gene.

[0011] In particular, the present disclosure provides structural elements, techniques, and / or features that can be incorporated into a ds oligonucleotide to confer or adjust one or more properties thereof (e.g., compared to an otherwise identical ds oligonucleotide lacking the related technique or feature). In certain embodiments, the present disclosure describes that one or more of the provided techniques and / or features can be usefully incorporated into ds oligonucleotides of various sequences.

[0012] In certain embodiments, the present disclosure demonstrates that certain provided structural elements, techniques, and / or features are particularly useful for ds oligonucleotides that participate in and / or induce the RNAi mechanism (e.g., RNAi agents). Nevertheless, however, the teachings of the present disclosure are not limited to ds oligonucleotides that participate in or act by any particular mechanism. In certain embodiments, the present disclosure relates to any ds oligonucleotides that are useful for any purpose, that act via any mechanism, and that include any sequence, structure, or format (or a portion thereof) described herein. In certain embodiments, the present disclosure relates to ds oligonucleotides that are useful for any purpose, that act via any mechanism, and that include any sequence, structure, or format (or a portion thereof) described herein, (1) a guide strand containing backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand containing backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5′-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3′ direction and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (3) a guide strand containing one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide (the upstream backbone phosphorothioate chiral centers are in the Rp or Sp configuration); (4) a guide strand containing one or more backbone phosphorothioate chiral centers in the 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 between (a) the +3 and +4 nucleotides; and (b) the +5 and +6 nucleotides; (5) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in the Rp or Sp configuration; and 6) A ds oligonucleotide comprising one or more passenger strands in combination with one or more of the aforementioned guide strands, the passenger strands comprising a backbone phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; The ds oligonucleotide further comprises: (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand contains one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds downstream relative to the bond between the 5'-terminal dinucleotides, i.e., in the 3' direction, and / or upstream relative to the bond between the 3'-terminal dinucleotides, i.e., in the 5' direction); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and Provided are ds oligonucleotides, wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0013] In certain embodiments, provided ds oligonucleotides can participate (e.g., directly) in the RNAi machinery. In certain embodiments, provided ds oligonucleotides can participate in the RNase H (ribonuclease H) machinery. In certain embodiments, provided ds oligonucleotides can act as translation inhibitors (e.g., provide steric blocking of translation).

[0014] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, where n is from about 1 to 49. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0015] 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 (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0016] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers of Rp or Sp configuration upstream of a backbone phosphorothioate chiral center of 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; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0017] In certain embodiments, the guide strand has one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds occurring between the second (+2) and third (+3) nucleotides relative to the 5'-terminal nucleotide of the guide strand and an internucleotide bond to the penultimate 3' (N-1) nucleotide; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0018] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone phosphorothioate chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0019] 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 (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0020] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers of Rp or Sp configuration upstream of a backbone chiral center of 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; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0021] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in the 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 between (a) the (+3) nucleotide and the (+4) nucleotide; and (b) the (+5) nucleotide and the (+6) nucleotide, and (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0022] In certain embodiments, the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages present 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 (N is the 3'-terminal nucleotide), a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more of Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0023] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and 2' modifications, e.g., 2'F modifications, of the 3' nucleotide of the nucleotide pairs linked by Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0024] In certain embodiments, the guide strand comprises Rp, Sp, or alternating backbone phosphorothioate chiral centers between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage; and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0025] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage; and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0026] In certain embodiments, the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages present 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 (N is the 3'-terminal nucleotide), a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0027] In certain embodiments, provided ds oligonucleotides may be involved 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 proteins, small molecules, nucleic acids, or cells. In certain embodiments, provided ds oligonucleotides may be involved in the formation of triple helices with double-stranded nucleic acids within cells. In certain embodiments, provided ds oligonucleotides may bind to genomic (e.g., chromosomal) nucleic acids. In certain embodiments, provided ds oligonucleotides may bind to genomic (e.g., chromosomal) nucleic acids and thus prevent or reduce expression of the nucleic acid (e.g., by preventing or reducing transcription, transcription promotion, 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 include CpG sequences. In certain embodiments, provided ds oligonucleotides may be immunostimulatory, may contain CpG sequences, and may be useful as adjuvants. In certain embodiments, provided ds oligonucleotides may be immunostimulatory, may contain CpG sequences, and may be useful as adjuvants in the treatment of disease (e.g., 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 may be useful for therapeutic, diagnostic, research, and / or nanomaterial 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 probes, in microarrays, 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 the RNAi and RNase H mechanisms.

[0028] In certain embodiments, provided ds oligonucleotides are directed to a target (e.g., a target sequence, a target RNA, a target mRNA, a target pre-mRNA, a target gene, etc.). A target gene is a gene for which the expression and / or activity of one or more gene products (e.g., RNA and / or protein products) is intended to be altered. In certain embodiments, the target gene is intended to be inhibited. Thus, when the ds oligonucleotides described herein act on a particular target gene, the presence and / or activity of one or more gene products of that gene is altered in the presence of the ds oligonucleotide compared to when the ds oligonucleotide is absent.

[0029] In certain embodiments, a target is a specific allele for which the expression and / or activity of one or more products (e.g., RNA and / or protein products) is intended to be altered. In certain embodiments, a target allele is one whose presence and / or expression is associated with (e.g., correlates with) the presence, incidence, and / or severity of one or more diseases and / or conditions. Alternatively or additionally, in certain embodiments, a target allele is one whose alteration of the level and / or activity of one or more gene products is correlated with amelioration of one or more aspects of a disease and / or condition (e.g., delayed onset, reduced severity, responsiveness to other therapies, etc.).

[0030] In certain embodiments, for example, when the presence and / or activity of a particular allele (disease-associated allele) is associated (e.g., correlated) with the presence, occurrence, and / or severity of one or more disorders, diseases, and / or conditions, and different alleles of the same gene exist that are unassociated or associated to a lesser extent (e.g., exhibit a less significant or statistically insignificant correlation), the ds oligonucleotides and methods described herein may preferentially or specifically target the associated allele relative to one or more less associated / unassociated alleles, thus mediating allele-specific suppression.

[0031] In certain embodiments, the target sequence is a sequence to which an oligonucleotide described herein binds. In certain embodiments, the target gene is identical to or an exact complement of the sequence of a provided oligonucleotide or consecutive residues therein (e.g., a provided oligonucleotide comprises a target binding sequence that is identical to or an exact complement of the target sequence). In certain embodiments, the target binding sequence is an exact complement of a target sequence in a transcript (e.g., pre-mRNA, mRNA, etc.). The target binding sequence / target sequence can be of various lengths for the provided oligonucleotides for desired activity and / or properties. In certain embodiments, the target binding sequence / target sequence comprises 5-50 bases (e.g., 10-40, 15-30, 15-25, 16-25, 17-25, 18-25, 19-25, 20-25, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more bases). In certain embodiments, a small number of differences / mismatches are tolerated between (relevant portions of) the oligonucleotide and its target sequence, including, but not limited to, the 5' and / or 3'-terminal regions of the target and / or oligonucleotide sequence. In certain embodiments, the target sequence is present within a target gene. In certain embodiments, the target sequence is present within a transcript (e.g., mRNA and / or pre-mRNA) produced from the target gene.

[0032] In certain embodiments, a target gene includes one or more allelic sites (i.e., positions within the target gene where allelic variation occurs). In certain embodiments, the allelic sites are mutations. In certain embodiments, the allelic sites are SNPs. In some such embodiments, provided oligonucleotides bind preferentially or specifically to one allele over one or more other alleles. In certain embodiments, provided oligonucleotides bind preferentially to disease-associated alleles. For example, in certain embodiments, the oligonucleotides provided herein (or target binding sequence portions thereof) have a sequence that is completely or at least partially identical to or an exact complement of a particular allelic version of a target sequence.

[0033] In certain embodiments, the oligonucleotides provided herein (or target binding sequence portions thereof) have a sequence that is identical to or the exact complement of a target sequence comprising an allele or an allelic site of a disease-associated allele. In certain embodiments, the oligonucleotides provided herein have a target binding sequence that is the exact complement of a target sequence comprising an allelic site of a transcript of an allele (in certain embodiments, a disease-associated allele), where the allelic site is a variant. In certain embodiments, the oligonucleotides provided herein have a target binding sequence that is the exact complement of a target sequence comprising an allelic site of a transcript of an allele (in certain embodiments, a disease-associated allele), where the allelic site is a SNP. In certain embodiments, the sequence is any sequence disclosed herein.

[0034] Unless otherwise indicated, all sequences (including, but not limited to, base sequences and chemical, modification and / or stereochemical patterns) are presented in 5' to 3' order, with the 5' terminal nucleotide identified as the "+1" position, the 3' terminal nucleotide identified by the number of the nucleotide in the complete sequence or "N", the penultimate nucleotide identified as, for example, "N-1", etc.

[0035] In certain embodiments, the present disclosure provides compositions and methods relating to oligonucleotides that are specific for a target and have any of the formats, structural elements, or base sequences of any of the oligonucleotides disclosed herein.

[0036] In certain embodiments, the present disclosure provides compositions and methods relating to oligonucleotides that are specific for a target and have or include 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 optionally be replaced by a T or a DNA T.

[0037] In certain embodiments, the present disclosure provides compositions and methods for RNA interference induced by RNAi agents (also called RNAi oligonucleotides). In certain embodiments, the oligonucleotides of such compositions can have the format, structural elements, or base sequence of the oligonucleotides disclosed herein.

[0038] In certain embodiments, the present disclosure provides compositions and methods for RNase H-mediated knockdown of target gene RNA induced by oligonucleotides (eg, antisense oligonucleotides).

[0039] The provided oligonucleotides and oligonucleotide compositions can have any format, structural element, or base sequence of any of the oligonucleotides disclosed herein. In certain embodiments, the structural element is a 5'-terminal structure, a 5'-terminal region, a 5'-nucleotide, a seed region, a post-seed region, a 3'-terminal region, a 3'-terminal dinucleotide, a 3'-end cap, or any portion of these structures, GC content, a long GC stretch, and / or any modification, chemistry, stereochemistry, pattern of modification, chemistry or stereochemistry, or 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.

[0040] In certain embodiments, the present disclosure provides compositions and methods of use of oligonucleotides.

[0041] In certain embodiments, the present disclosure provides compositions and methods of use of oligonucleotides capable of inducing both RNA interference and RNase H-mediated knockdown of target gene RNA. In certain embodiments, the oligonucleotides of such compositions can have the format, structural elements, or base sequences of the oligonucleotides disclosed herein.

[0042] In certain embodiments, an oligonucleotide that induces a particular event or activity is involved in reducing the expression, level, or activity of a particular event or activity, such as a target gene or its gene product. In certain embodiments, an oligonucleotide is considered to "induce" a particular event or activity if the presence of the oligonucleotide in a system in which the event or activity may occur correlates with a detectable increase in the occurrence, frequency, intensity, and / or level of the event or activity.

[0043] In certain embodiments, the provided oligonucleotides comprise any one or more structural elements of the oligonucleotides described herein, such as a base sequence (or a portion thereof of at least 15 consecutive bases); an internucleotide linkage pattern (or a portion thereof of at least 5 consecutive internucleotide linkages); an internucleotide linkage stereochemistry pattern (or a portion thereof of at least 5 consecutive internucleotide linkages); a 5'-terminal structure; a 5'-terminal region; a first region; a second region; and a 3'-terminal region (which can be a 3'-terminal dinucleotide and / or a 3'-end cap); and any additional chemical moieties; in certain embodiments, at least one structural element comprises a chiral center that is chirally controlled. In certain embodiments, the 3'-terminal dinucleotide can comprise two whole nucleotides. In certain embodiments, the oligonucleotide further comprises a chemical moiety selected from, by way of non-limiting example, 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, the APGR-binding moiety is a GalNAc moiety, or a variant, derivative, or modified version thereof, as described herein and / or known in the art. In certain embodiments, the oligonucleotide is an RNAi agent. In certain embodiments, the first region is a seed region. In certain embodiments, the second region is a post-seed region.

[0044] In certain embodiments, the provided oligonucleotide comprises any one or more structural elements of the RNAi agent described herein, such as a 5'-terminal structure; a 5'-terminal region; a seed region; a post-seed region (the region between the seed region and the 3'-terminal region); and a 3'-terminal region (which can be a 3'-terminal dinucleotide and / or a 3'-end cap); and optional additional chemical moieties; in certain embodiments, at least one structural element comprises a chiral center that is chirally controlled. In certain embodiments, the 3'-terminal dinucleotide can comprise two full nucleotides. In certain embodiments, the oligonucleotide further comprises a chemical moiety selected from, by way of non-limiting example, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety. In certain embodiments, the APGR-binding moiety is any GalNAc, or variant, derivative, or modification thereof, as described herein or known in the art.

[0045] In certain embodiments, the provided oligonucleotides comprise any one or more structural elements of the oligonucleotides described herein, such as a 5'-terminal structure, a 5'-terminal region, a first region, a second region, a 3'-terminal region, and optional additional chemical moieties, wherein at least one structural element comprises a chiral center that is chiral-controlled. In certain embodiments, the oligonucleotide comprises a span of at least 5 full nucleotides without a 2'-modification. In certain embodiments, the oligonucleotide further comprises an additional chemical moiety selected from, by way of non-limiting example, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety. In certain embodiments, the provided oligonucleotides are capable of inducing RNA interference. In certain embodiments, the provided oligonucleotides are capable of inducing RNase H-mediated knockdown. In certain embodiments, the provided oligonucleotides are capable of inducing both RNA interference and RNase H-mediated knockdown. In certain embodiments, the first region is a seed region. In certain embodiments, the second region is a post-seed region.

[0046] In certain embodiments, the provided oligonucleotide comprises any one or more structural elements of an RNAi agent, such as a 5'-end structure, a 5'-end region, a seed region, a post-seed region, and a 3'-end region, and optionally additional chemical moieties, wherein at least one structural element comprises a chiral center that is chiral-controlled; in certain embodiments, the oligonucleotide is also capable of inducing RNase H-mediated knockdown of target gene RNA. In certain embodiments, the oligonucleotide comprises a span of at least five total 2'-deoxynucleotides. In certain embodiments, the oligonucleotide further comprises a chemical moiety selected from, by way of non-limiting example, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety, as well as any other additional chemical moiety described herein.

[0047] In certain embodiments, the present disclosure demonstrates that the properties of oligonucleotides can be tuned by chemical modification. In certain embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, sugar and / or base modifications. In certain embodiments, the present disclosure provides an oligonucleotide composition capable of inducing RNA interference, comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, and / or one or more sugar and / or one or more base modifications. In certain embodiments, the oligonucleotide or oligonucleotide composition can also induce RNase H-mediated knockdown of target gene RNA. In certain embodiments, the present disclosure demonstrates that the properties of oligonucleotides, such as activity, toxicity, etc., can be tuned through chemical modification of the sugar, nucleobase and / or internucleotide linkage. In certain embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more modified internucleotide linkages (or "non-natural internucleotide linkages"), such as the natural phosphate internucleotide linkage found in natural DNA and RNA (-OP(O)(OH)O-, which is in the salt form at physiological pH (-OP(O)(O -Oligonucleotide compositions are provided that include a plurality of oligonucleotides comprising a linkage that can be utilized in place of (O-), one or more modified sugar moieties, and / or one or more natural phosphate linkages. In certain embodiments, the provided oligonucleotides can include two or more types of modified internucleotide linkages. In certain embodiments, the provided oligonucleotides include a non-negatively charged internucleotide linkage. In certain embodiments, the non-negatively charged internucleotide linkage is a neutral internucleotide linkage. In certain embodiments, the neutral internucleotide linkage includes a cyclic guanidine moiety. Such a moiety is optionally substituted. In certain embodiments, the provided oligonucleotides include a neutral internucleotide linkage and another internucleotide linkage that is not a neutral backbone. In certain embodiments, the provided oligonucleotides include a neutral internucleotide linkage and a phosphorothioate internucleotide linkage. In certain embodiments, the provided oligonucleotide compositions that include a plurality of oligonucleotides are chiral-controlled, wherein the level of the plurality of oligonucleotides in the composition is controlled or predetermined, and the plurality of oligonucleotides share a common stereochemical configuration at one or more chiral internucleotide linkages. For example, in certain embodiments, a plurality of oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more chiral internucleotide linkages, each independently being Rp or Sp; in certain embodiments, a plurality of oligonucleotides share a common stereochemical configuration at each chiral internucleotide linkage. In certain embodiments, chiral internucleotide linkages in which a controlled level of oligonucleotides in a composition share a common stereochemical configuration (independently Rp or Sp configuration) are referred to as chiral-controlled internucleotide linkages.In certain embodiments, the modified internucleotide linkage exists predominantly (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.) in a neutral or cationic form, respectively (or an anionic form (e.g., -OP(O)(O). - )-O- (the anionic form of the natural phosphate bond), -OP(O)(S -In certain embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that it exists predominantly in its neutral form (as compared to, for example, -O- (the anionic form of a phosphorothioate linkage)). In certain embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that it exists predominantly in its neutral form at pH. In certain embodiments, the modified internucleotide linkage is a cationic internucleotide linkage in that it exists predominantly in its cationic form at pH. In certain embodiments, the pH is human physiological pH (about 7.4). In certain embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that at least 90% of the internucleotide linkage exists in its neutral form at pH 7.4 in aqueous solution. In certain embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the internucleotide linkage exists in its neutral form in an aqueous solution of the oligonucleotide. 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 internucleotide linkage, e.g., a neutral internucleotide linkage, when in its neutral form, has no moieties with a pKa of less than 8, 9, 10, 11, 12, 13, or 14. In certain embodiments, the pKa of an internucleotide linkage in the present disclosure can be represented by the pKa of a CH3-internucleotide linkage -CH3 (i.e., two -CH3 groups replace two nucleoside units linked by an internucleotide linkage). Without wishing to be bound by any particular theory, at least in some cases, neutral internucleotide linkages in oligonucleotides can result in improved properties and / or activity, such as improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape, and / or improved nuclear uptake, compared to an equivalent nucleic acid that does not contain a neutral internucleotide linkage.

[0048] In certain embodiments, the non-negatively charged internucleotide linkages are those described in, e.g., U.S. Patent Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, 9,982,257, 10,160,969, 10,479,995, U.S. Patent Application Publication No. 2020 / 0056173, U.S. Patent Application Publication No. 2018 / 0216107, U.S. Patent Application Publication No. 2019 / 0127733, U.S. Patent No. 10,450,568, U.S. Patent Application Publication No. 2019 / 0077817, U.S. Patent Application Publication No. 2019 / 0249173, U.S. Patent Application Publication No. 2019 / 0259173, U.S. Patent Application Publication No. 2019 / 0269173, U.S. Patent Application Publication No. 2019 / 0279173, U.S. Patent Application Publication No. 2019 / 026 ... and / or WO 2019 / 032612, having a structure of Formula In-1, In-2, In-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, as described in WO 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612, or the like. In certain embodiments, the non-negatively charged internucleotide linkage comprises a cyclic guanidine moiety. In certain embodiments, the modified internucleotide linkage comprising a cyclic guanidine moiety is [ka] In certain embodiments, the neutral internucleotide linkage comprising a cyclic guanidine moiety is chiral controlled. In certain embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage.

[0049] In certain embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate internucleotide linkage is a chiral-controlled internucleotide linkage of the Sp configuration.

[0050] In certain embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate is a chiral-controlled internucleotide linkage of the Rp configuration.

[0051] In certain embodiments, the present disclosure provides a Tmg group ( [ka] ) and at least one neutral internucleotide linkage comprising at least one phosphorothioate.

[0052] In certain embodiments, each internucleotide linkage in the oligonucleotide is independently selected from a natural phosphate linkage, a phosphorothioate linkage, and a non-negatively charged internucleotide linkage (e.g., n001, n003, n004, n006, n008, n009, n013, n020, n021, n025, n026, n029, n031, n033, n037, n043, n046, n047, n048, n054, n058, or n055). In some embodiments, each internucleotide linkage in the oligonucleotide is independently selected from a natural phosphate linkage, a phosphorothioate linkage, and a neutral internucleotide linkage (e.g., n001, n003, n004, n006, n008, n009, n013, n020, n021, n025, n026, n029, n031, n033, n037, n043, n046, n047, n048, n054, n058, or n055).

[0053] In certain embodiments, the present disclosure relates to compositions comprising an oligonucleotide comprising at least one neutral internucleotide linkage comprising a Tmg group and at least one phosphorothioate (phosphorothioates are chiral-controlled internucleotide linkages of the Sp configuration).

[0054] In certain embodiments, the present disclosure relates to compositions comprising an oligonucleotide comprising at least one neutral internucleotide linkage selected from neutral internucleotide linkages comprising a Tmg group and at least one phosphorothioate (phosphorothioates are chiral-controlled internucleotide linkages of the Rp configuration).

[0055] Various types of internucleotide linkages have different properties. Without wishing to be bound by any theory, the present disclosure notes that natural phosphate linkages (phosphodiester internucleotide linkages) are anionic and may be unstable when used alone in vivo without other chemical modifications. Phosphorothioate internucleotide linkages are anionic, generally more stable in vivo than natural phosphate linkages, and generally more hydrophobic; neutral internucleotide linkages, such as those exemplified in the present disclosure, that contain cyclic guanidine moieties are neutral at physiological pH and may be more stable and more hydrophobic in vivo than natural phosphate linkages.

[0056] In certain embodiments, the chiral-controlled neutral internucleotide linkage is neutral at physiological pH, chiral-controlled, stable in vivo, hydrophobic, and capable of increasing endosomal escape.

[0057] In certain embodiments, the provided oligonucleotides comprise one or more regions, such as block, wing, core, 5'-end, 3'-end, middle, seed, post-seed region, and the like. In certain embodiments, the regions (e.g., block, wing, core, 5'-end, 3'-end, intermediate region, etc.) may be selected from the group consisting of those described in, for example, U.S. Patent No. 9,394,333, U.S. Patent No. 9,744,183, U.S. Patent No. 9,605,019, U.S. Patent No. 9,598,458, U.S. Patent No. 9,982,257, U.S. Patent No. 10,160,969, U.S. Patent No. 10,479,995, U.S. Patent Application Publication No. 2020 / 0056173, U.S. Patent Application Publication No. 2018 / 0216107, U.S. Patent Application Publication No. 2019 / 0127733, U.S. Patent No. 10,450,568, U.S. Patent Application Publication No. 2019 / 0077817, U.S. Patent Application Publication No. 2019 / 0249173, U.S. Patent Application Publication No. 2020 / 0259174, U.S. Patent Application Publication No. 2020 / 0259175, U.S. Patent Application Publication No. 2020 / 0259176, U.S. Patent Application Publication No. 2020 / 0259177, U.S. Patent Application Publication No. 2020 / 0259178, U.S. Patent Application Publication No. 2020 / 0259179 ... 19 / 0375774, International Publication No. 2018 / 223056, International Publication No. 2018 / 223073, International Publication No. 2018 / 223081, International Publication No. 2018 / 237194, International Publication No. 2019 / 032607, International Publication No. 2019 / 055951, International Publication No. 2019 / 075357, International Publication No. 2019 / In certain embodiments, the region comprises a non-negatively charged internucleotide linkage of formula In-1, In-2, In-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, as set forth in WO 200185, WO 2019 / 217784, and / or WO 2019 / 032612. In certain embodiments, the region comprises a neutral internucleotide linkage. In certain embodiments, the region comprises an internucleotide linkage comprising a cyclic guanidine moiety. In certain embodiments, the region comprises an internucleotide linkage comprising a cyclic guanidine moiety. In certain embodiments, the region comprises [ka] In certain embodiments, such internucleotide linkages are chiral controlled.

[0058] In certain embodiments, the nucleotide is a natural nucleotide. In certain embodiments, the nucleotide is a modified nucleotide. In certain embodiments, the nucleotide is a nucleotide analog. In certain embodiments, the base is a modified base. In certain embodiments, the base is a protected nucleobase, such as a protected nucleobase used in oligonucleotide synthesis. In certain embodiments, the base is a base analog. In certain embodiments, the sugar is a modified sugar. In certain embodiments, the sugar is a sugar analog. In certain embodiments, the internucleotide linkage is a modified internucleotide linkage. In certain embodiments, a nucleotide comprises a base, a sugar, and an internucleotide linkage, wherein each of the base, sugar, and internucleotide linkage is independently and optionally naturally occurring or non-naturally occurring. In certain embodiments, a nucleotide comprises a base and a sugar, wherein each of the base and sugar is independently and optionally naturally occurring or non-naturally occurring. Non-limiting examples of nucleotides include DNA (2'-deoxy) and RNA (2'-OH) nucleotides; and those containing one or more modifications in the base, sugar, and / or internucleotide 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'-MOE modifications. In certain embodiments, the internucleotide linkage is a moiety that does not contain phosphorus but serves to join two natural or unnatural sugars.

[0059] In certain embodiments, the composition comprises any two or more multimers: a first plurality of oligonucleotides and / or a second plurality of oligonucleotides, wherein the first and second plurality of oligonucleotides are capable of independently knocking down the same or different targets via RNA interference and / or RNase H-mediated knockdown.

[0060] In certain embodiments, the present disclosure provides 1) common base sequence; 2) common patterns of skeletal bonding; 3) independently having a common stereochemistry at 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 internucleotide linkages ("chiral controlled internucleotide linkages"). The present invention provides an oligonucleotide composition comprising a first plurality of oligonucleotides sharing a common structure, wherein the level of the first plurality of oligonucleotides in the composition is chiral-controlled at a predetermined point.

[0061] In certain embodiments, an oligonucleotide composition comprising a plurality of oligonucleotides (e.g., a first plurality of oligonucleotides) is chiral-controlled in that the plurality of oligonucleotides independently share a common stereochemistry at one or more chiral internucleotide linkages. In certain embodiments, the plurality of oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more chiral internucleotide linkages, each of which is independently Rp or Sp. In certain embodiments, the plurality of oligonucleotides share a common stereochemical configuration at each chiral internucleotide linkage. In certain embodiments, chiral internucleotide linkages at which a given level of oligonucleotides in the composition share a common stereochemical configuration (independently Rp or Sp) are referred to as chiral-controlled internucleotide linkages.

[0062] In certain embodiments, the predetermined level of oligonucleotides of a provided composition, e.g., the first plurality of oligonucleotides of a particular exemplary composition, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral-controlled internucleotide linkages.

[0063] In certain embodiments, at least 5 internucleotide linkages are chiral controlled; in certain embodiments, at least 10 internucleotide linkages are chiral controlled; in certain embodiments, at least 15 internucleotide linkages are chiral controlled; in certain embodiments, each chiral internucleotide linkage is chiral controlled.

[0064] In certain embodiments, 1 to 100% of the chiral internucleotide linkages are chiral controlled, hi 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 the chiral internucleotide linkages are chiral controlled.

[0065] In certain embodiments, the present disclosure provides 1) common base sequence; 2) a common pattern of backbone bonding; and 3) Common patterns of skeletal chiral centers The present invention provides an oligonucleotide composition comprising a first plurality of oligonucleotides sharing a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers, wherein the composition is a substantially pure preparation of oligonucleotides. In certain embodiments, the common pattern of backbone chiral centers comprises at least one internucleotide linkage that includes a chiral center that is chirally controlled. In certain embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition. In certain embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in a provided composition that are of or contain 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, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in a provided composition that are of or contain a common base sequence, base modification, sugar modification, and / or modified internucleotide linkage.In certain embodiments, all of the oligonucleotides in a provided composition that are of or comprise a common base sequence, base modification, sugar modification, and / or modified internucleotide 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 of the oligonucleotides in the composition. In certain embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in a provided composition that are or contain a common base sequence, pattern of base modifications, pattern of sugar modifications, and / or pattern of modified internucleotide linkages. In certain embodiments, all of the oligonucleotides in a provided composition that are or contain a common base sequence, pattern of base modifications, pattern of sugar modifications and / or pattern of modified internucleotide linkages represent 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 of the oligonucleotides in the composition. In certain embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in a provided composition that share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, and / or a common pattern of modified internucleotide linkages.In certain embodiments, all oligonucleotides in a provided composition that share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, and / or a common pattern of modified internucleotide linkages represent 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, the predetermined level is between 1 and 100%. In certain embodiments, the predetermined level is at least 1%. In certain embodiments, the predetermined level is at least 5%. In certain embodiments, the predetermined level is at least 10%. In certain embodiments, the predetermined level is at least 20%. In certain embodiments, the predetermined level is at least 30%. In certain embodiments, the predetermined level is at least 40%. In certain embodiments, the predetermined level is at least 50%. In certain embodiments, the predetermined level is at least 60%. In certain embodiments, the predetermined level is at least 10%. In certain embodiments, the predetermined level is at least 70%. In certain embodiments, the predetermined level is at least 80%. In certain embodiments, the predetermined level is at least 90%. In certain embodiments, the predetermined level is at least 5*(½g), where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least 10*(½g), where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least 100*(½g), where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.80)g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.80) g, where g is the number of chiral controlled internucleotide linkages.In certain embodiments, the predetermined level is at least (0.80) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.85) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.90) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.95) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.96) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.97) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.98) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.99) g, where g is the number of chiral-controlled internucleotide linkages. In certain embodiments, to determine the level of an oligonucleotide having g chiral-controlled internucleotide linkages in a composition, the product of the diastereopurities of each of the g chiral-controlled internucleotide linkages is used as the level: (diastereopurity of chiral-controlled internucleotide linkage 1) * (diastereopurity of chiral-controlled internucleotide linkage 2) * * (diastereopurity of chiral-controlled internucleotide linkage g). In this formula, the diastereopurity of each chiral-controlled internucleotide linkage is independently represented by the diastereopurity of a dimer containing the same internucleotide linkage and nucleosides flanking the internucleotide linkage and prepared in a manner equivalent to the oligonucleotide (e.g., an equivalent or preferably identical oligonucleotide preparation cycle including equivalent or preferably identical reagents and reaction conditions). In certain embodiments, the level of oligonucleotide and / or diastereopurity can be determined by analytical methods, such as chromatographic methods, spectroscopy, spectroscopic methods, or any combination thereof.In particular, the present disclosure encompasses the recognition that stereoirregular oligonucleotide formulations contain multiple distinct chemical entities that differ from one another, for example, in terms of the stereochemical structure (or stereochemistry) of individual backbone chiral centers within the oligonucleotide chain. If the stereochemistry of the backbone chiral centers is not controlled, stereoirregular oligonucleotide formulations result in uncontrolled compositions containing indeterminate levels of oligonucleotide stereoisomers. Even if these stereoisomers have the same base sequence and / or chemical modifications, they are distinct chemical entities due at least to their different backbone stereochemistry, and they may have different properties, such as susceptibility to nucleases, activity, distribution, etc., as demonstrated herein. In certain embodiments, a particular stereoisomer can be defined, for example, by its base sequence, its length, its backbone bond pattern, and its backbone chiral center pattern. In certain embodiments, the present disclosure demonstrates that the improved properties and activity achieved by controlling the stereochemistry within an oligonucleotide can be comparable to or even better than those achieved by the use of chemical modifications.

[0066] In particular, the present disclosure encompasses the recognition that stereoirregular oligonucleotide formulations contain multiple distinct chemical entities that differ from one another, for example, in terms of the stereochemical structure (or stereochemistry) of individual backbone chiral centers within the oligonucleotide chain. If the stereochemistry of the backbone chiral centers is not controlled, stereoirregular oligonucleotide formulations result in uncontrolled compositions containing indeterminate levels of oligonucleotide stereoisomers. Even if these stereoisomers have the same base sequence and / or chemical modifications, they are distinct chemical entities due at least to their different backbone stereochemistry, and they may have different properties, such as susceptibility to nucleases, activity, distribution, etc., as demonstrated herein. In certain embodiments, a particular stereoisomer can be defined, for example, by its base sequence, its length, its backbone bond pattern, and its backbone chiral center pattern. In certain embodiments, the present disclosure demonstrates that the improved properties and activity achieved by controlling the stereochemistry within an oligonucleotide can be comparable to or even better than those achieved by the use of chemical modifications. DETAILED DESCRIPTION OF THE INVENTION

[0067] I. Detailed Description of Certain Embodiments The techniques of the present disclosure may be more readily understood by reference to the following detailed description of certain embodiments.

[0068] definition As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, elements are identified according to 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.

[0069] As used herein in this disclosure, unless otherwise clear from the 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 or not used in conjunction with "limited to"), and "include" (whether or not used in conjunction with "not limited to") may be understood to encompass the itemized elements or steps, whether presented by themselves or presented with one or more additional elements 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 allow for standard variations as understood by one of ordinary skill in the art; and (vi) when ranges are given, the endpoints are included.

[0070] Unless otherwise specified, oligonucleotides and their elements (e.g., base sequence, sugar modifications, internucleotide linkages, bond phosphorus stereochemistry, patterns thereof, etc.) are from 5' to 3', with the 5'-terminal nucleotide identified as the "+1" position, the 3'-terminal nucleotide identified either by the number of nucleotides in the complete sequence or by "N," the penultimate nucleotide identified as, for example, "N-1," etc. As one of skill in the art will understand, in certain embodiments, oligonucleotides may be provided and / or utilized as salt forms, particularly pharmaceutically acceptable salt forms, e.g., sodium salts. As one of skill in the art will also understand, in certain embodiments, individual oligonucleotides within a composition may be considered to be of the same constitution and / or structure, even within such a composition (e.g., a liquid composition), and certain such oligonucleotides may be in different salt forms at a particular moment (and may be dissolved, the oligonucleotide chain may exist, for example, in an anionic form when in a liquid composition). For example, one of skill in the art will understand that at a given pH, individual internucleotide bonds along an oligonucleotide chain may be in the acid (H) form or one of several possible salt forms (e.g., sodium salts, or salts of different cations depending on which ions may be present in the preparation or composition), and will consider those acid forms (e.g., all cations, if present, may be H). + It will be understood that so long as the nucleotides (replaced by ) are of the same composition and / or structure, such individual oligonucleotides may be considered to be of the same composition and / or structure, as appropriate.

[0071] Aliphatic: As used herein, "aliphatic" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated (but not aromatic) units, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more unsaturated (but not aromatic) units, or a combination thereof. In certain embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. In certain embodiments, an aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0072] Alkenyl: As used herein, the term "alkenyl" refers to an aliphatic group, as defined herein, having one or more double bonds.

[0073] Alkyl: As used herein, the term "alkyl" has its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight-chain or branched-chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C6 for a straight chain). 20, for branched chains C2 to C 20 ), or alternatively having about 1-10. In certain embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure, and where such rings are monocyclic, bicyclic, or polycyclic, and alternatively have about 5, 6 or 7 carbons in the ring structure. In certain embodiments, an alkyl group can be a lower alkyl group, where the lower alkyl group has from 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0074] Alkynyl: As used herein, the term "alkynyl" refers to an aliphatic group, as defined herein, having one or more triple bonds.

[0075] Analog: The term "analog" includes any chemical moiety that is structurally different from a reference chemical moiety or class of moieties, but that can perform at least one function of such reference chemical moiety or class of moieties. Non-limiting examples include a nucleotide analog that is structurally different from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog that is structurally different from a nucleobase but performs at least one function of a nucleobase, etc.

[0076] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In certain embodiments, "animal" refers to humans at any stage of development. In certain embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In certain embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In certain embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0077] Aryl: The term "aryl," used alone or as part of a larger moiety of "aralkyl," "aralkoxy," or "aryloxyalkyl," as used herein, refers to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, wherein at least one ring in such systems is aromatic. In certain embodiments, an aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. In certain embodiments, each monocyclic ring unit is aromatic. In certain embodiments, an aryl group is a biaryl group. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl, and the like, which may bear one or more substituents. As used herein, also included within the scope of the term "aryl" are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0078] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical assignment of the chiral linking phosphorus at a chiral internucleotide linkage within an oligonucleotide. As used herein, a chiral internucleotide linkage is an internucleotide linkage in which the linking phosphorus is chiral. In certain embodiments, the control is achieved by a chiral element not present in the sugar and base moieties of the oligonucleotide; for example, in certain embodiments, the control is achieved by the use of one or more chiral auxiliary agents during oligonucleotide preparation, which are often part of the chiral phosphoramidite used during oligonucleotide preparation. In contrast to chiral control, those skilled in the art will understand that conventional oligonucleotide synthesis without the use of a chiral auxiliary agent cannot control the stereochemistry at the chiral internucleotide linkage when such conventional oligonucleotide synthesis is used to form the chiral internucleotide linkage. In certain embodiments, the stereochemical assignment of each chiral linking phosphorus at each chiral internucleotide linkage within an oligonucleotide is controlled.

[0079] Chirality-controlled oligonucleotide composition: The terms "chirality-controlled oligonucleotide composition," "chirality-controlled nucleic acid composition," and the like, as used herein, refer to a composition comprising multiple oligonucleotides (or nucleic acids) that share a common base sequence, and the multiple oligonucleotides (or nucleic acids) share the same linking phosphorus stereochemistry at one or more chiral internucleotide linkages (chirality-controlled or sterically-restricted internucleotide linkages, where the chiral linking phosphorus in the composition is Rp or Sp ("sterically-restricted"), rather than a random mixture of Rp and Sp as in the case of non-chirality-controlled internucleotide linkages). In certain embodiments, a chiral controlled oligonucleotide composition comprises a plurality of oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, where the plurality of oligonucleotides (or nucleic acids) share the same linking phosphorus stereochemistry at one or more chiral internucleotide linkages (chiral controlled or sterically restricted internucleotide linkages, the chiral linking phosphorus is Rp or Sp ("sterically restricted") in the composition, rather than the random Rp and Sp mixtures present in non-chiral controlled internucleotide linkages). The level of the plurality of oligonucleotides (or nucleic acids) in a chiral controlled oligonucleotide composition is predetermined / controlled or enhanced (e.g., by preparing a chiral controlled oligonucleotide that stereoselectively forms one or more chiral internucleotide linkages) compared to the random level in a non-chiral controlled oligonucleotide composition.In certain embodiments, about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 50%, 50%, 60%, 70%, 80%, 90%, 95 ... %, 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%) is a plurality of oligonucleotides. In certain embodiments, about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95 ... Up to 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%) are a plurality of oligonucleotides.In certain embodiments, the level is between about 1% and 100% (e.g., between about 5% and 100%) of all oligonucleotides in a composition, or of all oligonucleotides in a composition (e.g., of multiple oligonucleotides or types of oligonucleotides) that share a common base sequence, 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 pattern of base modifications, a common pattern of sugar modifications, a common pattern of internucleotide linkage types, and / or a common pattern of internucleotide linkage modifications. 00%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 50% to 90% or approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%). In certain embodiments, the plurality of oligonucleotides share the same stereochemistry at at least about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotide linkages.In certain embodiments, the plurality of oligonucleotides have between about 1% and 100% (e.g., between about 5% and 100%, 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, 90% and 100%, 95% and 100%, 50% and 90%, about 5%, 10%, 15%, 20%, 30%, 30%, 40%, 40%, 50%, 50%, 60%, 70%, 80%, 90%, 90%, 95%, 50%, 50%, 60%, 70%, 80%, 90%, 95 ... %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% share the same stereochemistry. In certain embodiments, multiple oligonucleotides (or nucleic acids) all share the same pattern of sugar and / or nucleobase modifications. In certain embodiments, multiple oligonucleotides (or nucleic acids) are various forms of the same oligonucleotide (e.g., acid and / or various salts of the same oligonucleotide). In certain embodiments, multiple oligonucleotides (or nucleic acids) are of the same composition. In certain embodiments, the level of the plurality of oligonucleotides (or nucleic acids) is about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 10 ... 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, each chiral internucleotide linkage is a chiral controlled internucleotide linkage, and the composition is a completely chiral controlled oligonucleotide composition.In certain embodiments, the multiple oligonucleotides (or nucleic acids) are structurally identical. In certain embodiments, the chiral-controlled internucleotide linkage has a diastereomeric purity 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 certain embodiments, the chiral-controlled internucleotide linkage has a diastereomeric purity of at least 95%. In certain embodiments, the chiral-controlled internucleotide linkage has a diastereomeric purity of at least 96%. In certain embodiments, the chiral-controlled internucleotide linkage has a diastereomeric purity of at least 97%. In certain embodiments, the chiral-controlled internucleotide linkage has a diastereomeric purity of at least 98%. In certain embodiments, the chiral controlled internucleotide linkage has a diastereomeric purity of at least 99%. In certain embodiments, the percentage level is (DS). nc or at least (DS) nc where DS is the diastereomeric purity as described herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater), and nc is the number of chiral internucleotide linkages as described herein (e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 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 greater). In certain embodiments, the percentage level is (DS) nc or at least (DS) nc and DS is 95% to 100%. For example, DS is 99%, nc is 10, and the percentage is 90% or at least 90% (99%). 10≈0.90=90%). In certain embodiments, the level of multiple oligonucleotides in a composition is expressed as the product of the diastereopurities of each chiral-controlled internucleotide linkage in the oligonucleotide. In certain embodiments, the diastereopurity of an internucleotide linkage linking two nucleosides in an oligonucleotide (or nucleic acid) is expressed by the diastereopurity of the internucleotide linkage of a dimer linking the same two nucleosides, the dimer being prepared using equivalent conditions, and in some instances, identical synthesis cycle conditions (e.g., for a linkage between Nx and Ny in an oligonucleotide...NxNy...., the dimer is NxNy). In certain embodiments, not all chiral internucleotide linkages are chiral-controlled internucleotide linkages, and the composition is a partially chiral-controlled oligonucleotide composition. In certain embodiments, the non-chirally controlled internucleotide linkages have a diastereomeric purity of less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50%, as typically observed in sterically irregular oligonucleotide compositions (e.g., from conventional oligonucleotide synthesis, e.g., phosphoramidite methods, as will be understood by those of skill in the art). In certain embodiments, the multiple oligonucleotides (or nucleic acids) are of the same type. In certain embodiments, the chirality controlled oligonucleotide composition comprises a non-random or controlled level of distinct oligonucleotide types or nucleic acid types. For example, in certain embodiments, the chirality controlled oligonucleotide composition comprises only one oligonucleotide type. In certain embodiments, the chirality controlled oligonucleotide composition comprises two or more oligonucleotide types. In certain embodiments, the chirality controlled oligonucleotide composition comprises multiple oligonucleotide types. In certain embodiments, the chirality controlled oligonucleotide composition is a composition consisting of oligonucleotides of a certain oligonucleotide type, which composition does not comprise a non-random or controlled level of distinct oligonucleotide types. The method comprises the steps of: (a) detecting a plurality of oligonucleotides at different levels;

[0080] Equivalent: The term "equivalent" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit a comparison of the results obtained or the phenomena observed. In certain embodiments, equivalent sets of conditions or circumstances are characterized by a plurality of substantially identical characteristics and one or a small number of variable characteristics. One skilled in the art will understand that sets of conditions are equivalent to one another when they are characterized by a sufficient number and variety of substantially identical characteristics to provide a basis for a reasonable conclusion that differences in the results obtained or phenomena observed under the various sets of conditions or circumstances are attributable to or indicative of differences in the variable characteristics.

[0081] Alicyclic: The terms "alicyclic," "carbocycle," "carbocyclyl," "carbocyclic group," and "carbocyclic ring" are used interchangeably and, as used herein, unless otherwise specified, refer to a saturated or partially unsaturated but non-aromatic cycloaliphatic monocyclic, bicyclic, or polycyclic ring system as described herein, having 3 to 30 ring members. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In certain embodiments, an alicyclic group has 3 to 6 carbons. In certain embodiments, an alicyclic group is saturated and is cycloalkyl. The term "alicyclic" can also include alicyclic rings fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In certain embodiments, alicyclic groups are bicyclic. In certain embodiments, alicyclic groups are tricyclic. In certain embodiments, alicyclic groups are polycyclic. In certain embodiments, "alicyclic" refers to a C3-C6 monocyclic hydrocarbon having a single point of attachment to the rest of the molecule, or a C8-C6 monocyclic hydrocarbon having a single point of attachment to the rest of the molecule, which is fully saturated or contains one or more units of unsaturation, but is not aromatic. 10Bicyclic or polycyclic hydrocarbons, or C9-C, which are fully saturated or contain one or more unsaturated units, but are not aromatic, and have a single point of attachment to the rest of the molecule 16 Refers to polycyclic hydrocarbons.

[0082] Heteroaliphatic: The term "heteroaliphatic," as used herein, has its ordinary meaning in the art and refers to an aliphatic group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In certain embodiments, one or more units selected from C, CH, CH, and CH are independently replaced with one or more heteroatoms (including oxidized and / or substituted forms thereof). In certain embodiments, a heteroaliphatic group is a heteroalkyl. In certain embodiments, a heteroaliphatic group is a heteroalkenyl.

[0083] Heteroalkyl: The term "heteroalkyl," as used herein, has its ordinary meaning in the art and refers to an alkyl group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0084] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy," as used herein, refer to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, in which at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In certain embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), and in certain embodiments, 5, 6, 9, or 10 ring atoms. In certain embodiments, each monocyclic ring unit is aromatic. In certain embodiments, a heteroaryl group has 6, 10, or 14 pi electrons shared in the cyclic arrangement; and 1 to 5 heteroatoms in addition to the carbon atoms. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In certain embodiments, a heteroaryl is a heterobiaryl group such as bipyridyl. The terms "heteroaryl" and "heteroar-," as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic.The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl group, where the alkyl and heteroaryl portions independently are optionally substituted.

[0085] Heteroatom: As used herein, the term "heteroatom" refers to an atom that is not carbon or hydrogen. In certain embodiments, the heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized forms of nitrogen, sulfur, phosphorus, or silicon; nitrogen (e.g., quaternized forms, forms such as iminium groups, etc.), phosphorus, sulfur, charged forms of oxygen, etc.). In certain embodiments, the heteroatom is silicon, phosphorus, oxygen, sulfur, or nitrogen. In certain embodiments, the heteroatom is silicon, oxygen, sulfur, or nitrogen. In certain embodiments, the heteroatom is oxygen, sulfur, or nitrogen.

[0086] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic group," and "heterocyclic ring" are used interchangeably and refer to a monocyclic, bicyclic, or polycyclic ring moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In certain embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +It can be NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic group" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. Heterocyclyl groups can be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions independently are optionally substituted.

[0087] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and / or between polypeptide molecules. In certain embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Next, the nucleotides at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons performed with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0088] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to the bond connecting the nucleoside units of an oligonucleotide or nucleic acid. In certain embodiments, the internucleotide linkage is a phosphodiester bond (a natural phosphate bond (-OP(=O)(OH)O-), which, as will be understood by those of skill in the art, may exist as a salt form) that is widely found in naturally occurring DNA and RNA molecules. In certain embodiments, the internucleotide linkage is a modified internucleotide linkage (not a natural phosphate bond). In certain embodiments, the internucleotide linkage is a "modified internucleotide linkage," in which at least one oxygen atom or -OH of the phosphodiester bond is replaced with a different organic or inorganic moiety. In certain embodiments, such organic or inorganic moiety is selected from =S, =Se, =NR', -SR', -SeR', -N(R')2, B(R')3, -S-, -Se-, and -N(R')-, where each R' is independently as defined and described in this disclosure. In certain embodiments, the internucleotide linkage is a phosphodiester linkage, a phosphorothioate linkage (or a phosphorothioate diester linkage, -OP(=O)(SH)O-, which may exist as a salt form as will be understood by those skilled in the art), or a phosphorothioate triester linkage. In certain embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In certain embodiments, the internucleotide linkage is, for example, one of a PNA (peptide nucleic acid) or PMO (phosphorodiamidate morpholino oligomer) linkage. In certain embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In certain embodiments, the modified internucleotide linkage is a neutral internucleotide linkage (e.g., n001 in certain provided oligonucleotides). Those skilled in the art will understand that internucleotide linkages can exist as anions or cations at a given pH due to the presence of acidic or basic moieties in the linkage.In certain embodiments, the modified internucleotide linkages are those designated s, s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 and s18 as described in WO 2017 / 210647.

[0089] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment (e.g., in a test tube or reaction vessel, in cell culture, etc.) rather than within an organism (e.g., an animal, plant, and / or microorganism).

[0090] In vivo: As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, a plant, and / or a microorganism).

[0091] Bound phosphorus: As defined herein, the phrase "bound phosphorus" is used to indicate that the particular phosphorus atom being referenced is a phosphorus atom present in an internucleotide linkage, where the phosphorus atom corresponds to the phosphorus atom of a phosphodiester internucleotide linkage as it occurs in naturally occurring DNA and RNA. In certain embodiments, the bound phosphorus atom is present in a modified internucleotide linkage, where each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In certain embodiments, the bound phosphorus atom is chiral (e.g., as in a phosphorothioate internucleotide linkage). In certain embodiments, the bound phosphorus atom is achiral (e.g., as in a natural phosphate linkage).

[0092] Modified nucleobase: The terms "modified nucleobase," "modified base," and the like refer to a chemical moiety that is chemically different from a nucleobase but can perform at least one function of a nucleobase. In certain embodiments, a modified nucleobase is a nucleobase that includes a modification. In certain embodiments, a modified nucleobase retains the ability of at least one function of a nucleobase, for example, the ability to form a moiety in a polymer that has base pairing ability with a nucleic acid that contains at least a complementary base sequence. In certain embodiments, a modified nucleobase is a substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In certain embodiments, in the context of an oligonucleotide, a modified nucleobase refers to a nucleobase that is not A, T, C, G, or U.

[0093] Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but containing a chemical modification that distinguishes it from a natural nucleoside. Non-limiting examples of modified nucleosides include those containing modifications at the base and / or sugar. Non-limiting examples of modified nucleosides include those having 2' modifications at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (lacking a nucleobase). In certain embodiments, modified nucleosides retain the capability of at least one function of a nucleoside, e.g., the capability of forming a moiety in a polymer that has base pairing capability with a nucleic acid containing at least a complementary base sequence.

[0094] Modified Nucleotide: The term "modified nucleotide" includes any chemical moiety that is structurally different from a natural nucleotide but that can perform at least one function of a natural nucleotide. In certain embodiments, a modified nucleotide comprises a modification to the sugar, base, and / or internucleotide linkage. In certain embodiments, a modified nucleotide comprises a modified sugar, a modified nucleobase, and / or a modified internucleotide linkage. In certain embodiments, a modified nucleotide retains the capability of at least one function of a nucleotide, e.g., the ability to form a subunit in a polymer that has base-pairing capability with a nucleic acid comprising at least a complementary base sequence.

[0095] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electrical properties, or some other physicochemical property of the sugar. In certain embodiments, as described in this disclosure, the modified sugar is a substituted ribose or deoxyribose. In certain embodiments, the modified sugar comprises a 2'-modification. Examples of useful 2'-modifications are widely available in the art and described herein. In certain embodiments, the 2'-modification is 2'-F. In certain embodiments, the 2'-modification is 2'-OR (where R is an optionally substituted C 1~10 In certain embodiments, the 2'-modification is 2'-OMe. In certain embodiments, the 2'-modification is 2'-MOE. In certain embodiments, the modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.). In certain embodiments, in the context of oligonucleotides, the modified sugar is a sugar that is not a ribose or deoxyribose as typically found in natural RNA or DNA.

[0096] Nucleic Acid: As used herein, the term "nucleic acid" includes any nucleotide 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 molecule 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, including modified nucleotides and / or modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. These terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from nucleobases and / or modified nucleobase N-glycosides or C-glycosides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotide linkages. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified internucleotide linkages. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to nucleic acids containing from 2 to about 10,000 nucleotide monomer units, and the prefix oligo- refers to nucleic acids containing from 2 to about 200 nucleotide monomer units.

[0097] Nucleobase: The term "nucleobase" refers to the portion of a nucleic acid that participates in hydrogen bonding to link 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 certain embodiments, naturally occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In certain embodiments, naturally occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In certain embodiments, a nucleobase comprises a heteroaryl ring, the ring atom of which is nitrogen, and in the case of a nucleoside, the nitrogen is linked to the sugar moiety. In certain embodiments, a nucleobase comprises a heterocyclic ring, the ring atom of which is nitrogen, and in the case of a nucleoside, the nitrogen is linked to the sugar moiety. In certain embodiments, the nucleobase is a "modified nucleobase," a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In certain embodiments, the modified nucleobase is a substituted A, T, C, G, or U. In certain embodiments, the modified nucleobase is a substituted tautomer of A, T, C, G, or U. In certain embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In certain embodiments, the modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the hydrogen bonding properties that bind one nucleic acid strand to another in a sequence-specific manner. In certain embodiments, the modified nucleobase is capable of pairing with all 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 that are used in place of natural or naturally occurring nucleotides, such as modified nucleobases and nucleobase analogs. In certain embodiments, the nucleobase is an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U.In certain embodiments, "nucleobase" refers to a nucleobase unit in an oligonucleotide or nucleic acid (e.g., A, T, C, G, or U as in an oligonucleotide or nucleic acid).

[0098] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar. In certain embodiments, the nucleoside is a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In certain embodiments, the nucleoside is a modified nucleoside, such as a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In certain embodiments, the 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 certain embodiments, "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid.

[0099] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a nucleobase, a sugar, and one or more internucleotide linkages (e.g., phosphate linkages in natural DNA and RNA). Naturally occurring bases [guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)] are derivatives of purines or pyrimidines, although it should be understood that naturally occurring and non-naturally occurring base analogs are also included. Naturally occurring sugars are the pentose (five-carbon sugar) deoxyribose (forming DNA) or ribose (forming RNA), although it should be understood that naturally occurring and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotide linkages to form nucleic acids, or polynucleotides. Many internucleotide linkages are known in the art (such as, but not limited to, via phosphate, phosphorothioate, boranophosphate, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriester, phosphorothioate, H-phosphonate, phosphoramidate, boranophosphate, methylphosphonate, phosphonoacetate, thiophosphonoacetate, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In certain embodiments, natural nucleotides contain naturally occurring bases, sugars, and internucleotide linkages. As used herein, the term "nucleotide" also encompasses structural analogs that are used in place of natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In certain embodiments, "nucleotide" refers to a nucleotide unit in an oligonucleotide or nucleic acid.

[0100] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, which may contain any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0101] Oligonucleotides can be single-stranded or double-stranded. Single-stranded oligonucleotides can have a double-stranded region (formed by two portions of the single-stranded oligonucleotide), and double-stranded oligonucleotides containing two oligonucleotide strands can have a single-stranded region, for example, in the region where the two oligonucleotide strands are not complementary to each other. Examples of oligonucleotides include, but are not limited to, structural genes, genes including regulatory and termination regions, self-replicating systems such as viruses 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, Ul adapters, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0102] Oligonucleotides of the present disclosure can be of various lengths. In certain embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, single-stranded, double-stranded, or triple-stranded oligonucleotides can range in length from about 4 to about 10 nucleosides, about 10 to about 50 nucleosides, about 20 to about 50 nucleosides, about 15 to about 30 nucleosides, or about 20 to about 30 nucleosides in length. In certain embodiments, oligonucleotides are about 9 to about 39 nucleosides in length. In certain embodiments, oligonucleotides are 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 certain embodiments, oligonucleotides are at least 4 nucleosides in length. In certain embodiments, the oligonucleotide is at least 5 nucleosides in length. In certain embodiments, the oligonucleotide is at least 6 nucleosides in length. In certain embodiments, the oligonucleotide is at least 7 nucleosides in length. In certain embodiments, the oligonucleotide is at least 8 nucleosides in length. In certain embodiments, the oligonucleotide is at least 9 nucleosides in length. In certain embodiments, the oligonucleotide is at least 10 nucleosides in length. In certain embodiments, the oligonucleotide is at least 11 nucleosides in length. In certain embodiments, the oligonucleotide is at least 12 nucleosides in length. In certain embodiments, the oligonucleotide is at least 15 nucleosides in length. In certain embodiments, the oligonucleotide is at least 15 nucleosides in length. In certain embodiments, the oligonucleotide is at least 16 nucleosides in length. In certain embodiments, the oligonucleotide is at least 17 nucleosides in length. In certain embodiments, the oligonucleotide is at least 18 nucleosides in length. In certain embodiments, the oligonucleotide is at least 19 nucleosides in length. In certain embodiments, the oligonucleotide is at least 20 nucleosides in length.In certain embodiments, the oligonucleotide is at least 25 nucleosides in length. In certain embodiments, the oligonucleotide is at least 30 nucleosides in length. In certain embodiments, each nucleoside counted in the length of the oligonucleotide independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In certain embodiments, each nucleoside counted in the length of the oligonucleotide independently comprises A, T, C, G, or U, or an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U.

[0103] Oligonucleotide Type: As used herein, the phrase "oligonucleotide type" is used to define oligonucleotides having a particular base sequence, backbone linkage pattern (i.e., pattern of internucleotide linkage types, e.g., phosphate, phosphorothioate, phosphorothioate triester, etc.), pattern of backbone chiral centers (i.e., pattern of phosphorus stereochemistry (Rp / Sp)), and pattern of backbone phosphorus modifications. In certain embodiments, oligonucleotides of a commonly designated "type" are structurally identical to each other.

[0104] Those skilled in the art will appreciate that the synthetic methods of the present disclosure provide a degree of control during the synthesis of an oligonucleotide chain, as each nucleotide unit of the oligonucleotide chain can be designed and / or selected in advance to have a specific stereochemistry at the linking phosphorus and / or a specific modification at the linking phosphorus, and / or a specific base and / or a specific sugar. In certain embodiments, the oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the linking phosphorus. In certain embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the linking phosphorus. In certain embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In certain embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the structural features described above. In certain embodiments, the present disclosure provides compositions (e.g., chiral controlled oligonucleotide compositions) comprising or consisting of a plurality of oligonucleotide molecules. In certain embodiments, such molecules are all of the same type (i.e., structurally identical to one another). However, in certain embodiments, the provided compositions comprise a plurality of oligonucleotides of different types, typically in predetermined relative amounts.

[0105] Optionally substituted: As described herein, compounds (e.g., oligonucleotides) of the present disclosure may contain optionally substituted and / or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety have been 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 substituents may be the same or different at all positions. In certain embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by the present disclosure preferably result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that remain substantially unchanged when subjected to conditions that permit their production, detection, and, in certain embodiments, their collection, purification, and use for one or more of the purposes disclosed herein. Certain substituents are described below.

[0106] Suitable monovalent substituents on substitutable atoms, for example suitable carbon atoms, are independently halogen; -(CH) 0~4 R°;-(CH2) 0~4 OR°;-O(CH2) 0~4 R°, -O-(CH2) 0~4 C(O)OR°;-(CH2) 0~4 CH(OR°)2; R° can be substituted with -(CH2) 0~4 Ph; R° may be substituted with -(CH2) 0~4 O(CH2) 0~1 Ph; may be substituted with R° -CH=CHPh; may be substituted with R° -(CH 0~4 O(CH2) 0~1 -pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0~4 N(R°)2;-(CH2) 0~4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0~4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2) 0~4 N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2) 0~4 C(O)R°; -C(S)R°; -(CH2) 0~4 C(O)OR°; -(CH2) 0~4 C(O)SR°; -(CH2) 0~4 C(O)OSiR°3; -(CH2) 0~4 OC(O)R°; -OC(O)(CH2) 0~4 SR°、 -SC(S)SR°; -(CH2) 0~4 SC(O)R°; -(CH2) 0~4 C(O)NR°2; -C(S)NR°2; -C(S)SR°; -(CH2) 0~4 OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2) 0~4 SSR°; -(CH2) 0~4 S(O)2R°; -(CH2) 0~4 S(O)2OR°; -(CH2) 0~4 OS(O)2R°; -S(O)2NR°2; -(CH2) 0~4 S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -Si(R°)3; -OSi(R°)3; -B(R°)2; -OB(R°)2; -OB(OR°)2; -P(R°)2; -P(OR°)2; -P(R°)(OR°); -OP(R°)2; -OP(OR°)2; -OP(R°)(OR°); -P(O)(R°)2; -P(O)(OR°)2; -OP(O)(R°)2; -OP(O)(OR°)2; -OP(O)(OR°)(SR°); -SP(O)(R°)2; -SP(O)(OR°)2; -N(R°)P(O)(R°)2; -N(R°)P(O)(OR°)2; -P(R°)2[B(R°)3]; -P(OR°)2[B(R°)3]; -OP(R°)2[B(R°)3]; -OP(OR°)2[B(R°)3]; -(C 1~4 linear or branched alkylene)O-N(R°)2; or -(C1~4 linear or branched alkylene)C(O)ON(R°), where each R° may be substituted as defined herein and independently represents hydrogen, C 1~20 C having 1 to 5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon, and phosphorus 1~20 Heteroaliphatic, -CH2-(C 6~14 aryl), -O(CH2) 0~1 (C 6~14 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 above definitions, two independent occurrences of R° taken together with their intervening atoms 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.

[0107] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° taken together with their intervening atoms) are independently halogen, —(CH) 0~2 R · ,-(Halo R · ), -(CH2) 0~2 OH, -(CH2) 0~2 OR · , -(CH2) 0~2 CH(OR · )2;-O(HaloR · ), -CN, -N3, -(CH2) 0~2 C(O)R · , -(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR · , -(CH2) 0~2 SR · , -(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR · , -(CH2) 0~2 NR· 2, -NO2, -SiR · 3. -OSiR · 3. -C(O)SR · , -(C 1~4 Linear or branched alkylene)C(O)OR · , or -SSR · (In the formula, each R · is unsubstituted or, if preceded by "halo", substituted only with one or more halogens), and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph 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.

[0108] For example, suitable divalent substituents on suitable carbon atoms are, independently, the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2~3 O- or -S(C(R * 2)) 2~3 S- and R * Each independent occurrence of is hydrogen, C which may be substituted as defined below 1~6 Aliphatic and unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Preferred divalent substituents attached to adjacent substitutable carbon atoms of an "optionally substituted" group include -O(CR * 2) 2~3 O- and R * Each independent occurrence of is hydrogen, C which may be substituted as defined below 1~6 It is selected from aliphatic and unsubstituted 5-6 membered saturated, partially unsaturated and aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0109] R * Suitable substituents on the aliphatic group are independently halogen, —R · ,-(Halo R · ), -OH, -OR · , -O(HaloR · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2 or -NO2 (each R · is unsubstituted or, if preceded by "halo", substituted only with one or more halogens), and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0110] In certain embodiments, suitable substituents on a substitutable nitrogen are independently —R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † and each R † are independently hydrogen, C which may be substituted as defined below 1~6 an aliphatic, 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 above definitions, R †two independent occurrences of are taken together with their intervening atoms to form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0111] R † Suitable substituents on the aliphatic group are independently halogen, —R · ,-(Halo R · ), -OH, -OR · , -O(HaloR · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2 or -NO2 (each R · is unsubstituted or, if preceded by "halo", substituted only with one or more halogens), and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0112] P-modification: As used herein, the term "P-modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In certain embodiments, a P-modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus.

[0113] 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 with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0114] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In certain embodiments, the active agent is present in a unit dose amount suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In certain embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including: oral administration, e.g., 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, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained-release formulation; topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingual; intraocular; transdermal; or adapted for the nasal, pulmonary, and other mucosal surfaces.

[0115] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that 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.

[0116] 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 that encapsulates a material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. 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 that 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 carboxymethylcellulose, ethylcellulose, 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, corn 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; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0117] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in a pharmaceutical context, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. provide a detailed description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 66: 1-19 (1977). In certain embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts of amino groups 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 formed by other methods used in the art, such as ion exchange. In certain embodiments, pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, These include, but are not limited to, salts of 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, and the like.In certain embodiments, provided compounds contain one or more acidic groups, e.g., oligonucleotides, and pharmaceutically acceptable salts are alkali salts, alkaline earth metal salts, or ammonium salts (e.g., ammonium salts of N(R)3, where each R is independently as defined and described in this disclosure). Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt. In certain embodiments, the pharmaceutically acceptable salt is a potassium salt. In certain embodiments, the pharmaceutically acceptable salt is a calcium salt. In certain embodiments, pharmaceutically acceptable salts include amine cations formed, where appropriate, using non-toxic ammonium, quaternary ammonium, and counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates. In certain embodiments, provided compounds contain two or more acid groups; for example, oligonucleotides may contain two or more acid groups (e.g., in natural phosphate linkages and / or modified internucleotide linkages). In certain embodiments, pharmaceutically acceptable salts, or salts in general, of such compounds contain two or more cations, which may be the same or different. In certain embodiments, in a pharmaceutically acceptable salt (or salt in general), all ionizable hydrogens in the acid groups are replaced with cations (e.g., in an aqueous solution having a pKa of about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less; in certain embodiments, about 7 or less; in certain embodiments, about 6 or less; in certain embodiments, about 5 or less; in certain embodiments, about 4 or less; in certain embodiments, about 3 or less). In certain embodiments, each phosphorothioate and phosphate group independently exists in its salt form (e.g., in the case of sodium salts, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively).In certain embodiments, each phosphorothioate and phosphate internucleotide linkage is independently present in its salt form (e.g., in the case of sodium salts, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). In certain embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, and each acidic phosphate group and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if present, is present as a salt form (all sodium salts).

[0118] Predetermined: Predetermined (or pre-determined) means deliberately selected or non-random or controlled, as opposed to, for example, randomly occurring, irregular, or achieved without control. Those skilled in the art who read this specification will understand that the present disclosure provides techniques that allow for the selection of specific chemical and / or stereochemical features to be incorporated into oligonucleotide compositions, and further allow for the controlled preparation of oligonucleotide compositions having such chemical and / or stereochemical features. A composition so provided is "predetermined" as described herein. A composition that may contain a particular oligonucleotide is not a "predetermined" composition because it is accidentally generated through a process that is not controlled to intentionally produce specific chemical and / or stereochemical features. In certain embodiments, a predetermined composition is one that can be intentionally reproduced (e.g., by repeating a controlled process). In certain embodiments, a predetermining level of a plurality of oligonucleotides in a composition means that the absolute and / or relative amounts (ratios, percentages, etc.) of the plurality of oligonucleotides in the composition are controlled. In certain embodiments, a predetermined level of multiple oligonucleotides in a composition is achieved by the preparation of chiral controlled oligonucleotides.

[0119] Protecting Group: The term "protecting group" as used herein refers to a group that is well known in the art and is described in Organic Synthesis, TW Greene and PGM Wuts, 3 rdedition, John Wiley & Sons, 1999. Also included are those protecting groups specifically adapted for nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012, Chapter 2 of which is incorporated herein by reference in its entirety. Suitable amino-protecting groups include methyl carbamate, ethyl carbamate (carbamante), 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), and 2-trimethylsilylethyl carbamate (Teoc). , 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamido)ethyl, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, carbamate Alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitribenzyl carbamate (nitobenzyl), p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylsulfinylbenzyl carbamate -methylthioethyl, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-Dimethoxy-6-nitrobenzyl, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate , 2,2-Dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenyl) carbamate p-(phenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- Butylphenyl, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide,(N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3 -acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamine (Fcm), N -2-Picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)carbonyl]amine, N-copper chelate compounds, N-zinc chelate compounds, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridine sulfenamide (Npys), p-toluenesulfonamide (Ts), benzene Sulfonamides, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4 -methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0120] Suitable protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

[0121] Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, and 4-methoxytetrahydrothiopyranyl. S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-furanyl Oroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyl hydroxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxamate Acetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamanoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-Trichloroethyl (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthothyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl methylthiomethoxymethyl)benzoate, 4-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). To protect 1,2- or 1,3-diols, protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,Examples of the hydroxybenzoates include 4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl borate, and phenyl borate.

[0122] In certain embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4′-dimethoxytrityl, ( DMTr) and 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (Pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In certain embodiments, the hydroxyl protecting groups are selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.In certain embodiments, the phosphorus-linked protecting group is a group that is added to a phosphorus linkage (e.g., an internucleotide linkage) throughout oligonucleotide synthesis. In certain embodiments, the protecting group is added to the sulfur atom of a phosphorothioate linkage. In certain embodiments, the protecting group is added to the oxygen atom of a phosphorothioate internucleotide linkage. In certain embodiments, the protecting group is added to the oxygen atom of a phosphate internucleotide linkage. In certain embodiments, the protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0123] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a compound (e.g., an 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; parasites, etc.) and plants. In certain embodiments, the subject is a human. In certain embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0124] Substantially: As used herein, the term "substantially" refers to the qualitative condition of indicating the entire or nearly entire extent or degree of a desired characteristic or property. A base sequence that is substantially identical to or complementary to a second sequence is not completely identical to or complementary to the second sequence, but is largely or nearly identical to or complementary to the second sequence. In certain embodiments, an oligonucleotide having a sequence substantially complementary to another oligonucleotide or nucleic acid will form a duplex with that oligonucleotide or nucleic acid in a manner similar to an oligonucleotide having a completely complementary sequence. Additionally, those skilled in the art of biology and / or chemistry will understand that biological and chemical events rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, as used herein, the term "substantially" is used to capture the potential lack of completeness inherent in many biological and / or chemical events.

[0125] Sugar: The term "sugar" refers to closed and / or open monosaccharides or polysaccharides. In certain embodiments, a sugar is a monosaccharide. In certain embodiments, a sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term "sugar" also encompasses structural analogs that are used in place of traditional sugar molecules, such as glycols, polymers that form the backbone of nucleic acid analogs, glycol nucleic acids ("GNAs"). As used herein, the term "sugar" also encompasses structural analogs that are used in place of natural or naturally occurring nucleotides, such as modified sugars and nucleotide sugars. In certain embodiments, a sugar is an RNA or DNA sugar (ribose or deoxyribose). In certain embodiments, a sugar is a modified ribose or deoxyribose sugar, e.g., 2'-modified, 5'-modified, etc. As described herein, in certain embodiments, modified sugars, when used in oligonucleotides and / or nucleic acids, can provide one or more desirable properties, activities, etc. In certain embodiments, the sugar is an optionally substituted ribose or deoxyribose. In certain embodiments, "sugar" refers to the sugar unit in an oligonucleotide or nucleic acid.

[0126] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who is at a higher risk than the general population of individuals of developing the disease, disorder, and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition has a predisposition to having the disease, disorder, and / or condition. In certain 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 certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In certain 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 certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0127] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any agent that induces a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In certain embodiments, an agent, e.g., a dsRNAi agent, is considered to be a therapeutic agent if it exhibits a statistically significant effect across a relevant population. In certain embodiments, the relevant population is a population of subjects suffering from and / or susceptible to a disease, disorder, or condition. In certain embodiments, the relevant population is a population of model organisms. In certain embodiments, the relevant population may be defined by one or more criteria, such as age group, sex, genetic background, pre-existing clinical conditions, etc., prior to receiving therapy. In certain embodiments, a therapeutic agent is a substance that, when administered to a subject in an effective amount, relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition in the subject. In certain embodiments, a "therapeutic agent" is a drug that has been approved, or is required to be approved, by a government agency before it can be marketed for administration to humans. In certain embodiments, a "therapeutic agent" is a drug for which a prescription is required for administration to humans. In certain embodiments, a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.

[0128] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to 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 treatment regimen. In certain embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be understood by one of skill in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In certain embodiments, a therapeutically effective amount is administered in a single dose; in certain embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0129] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition. In certain embodiments, treatment may be administered to subjects who show very early signs of the disease, disorder, and / or condition, e.g., to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0130] Unsaturated: The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0131] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning, which refers to an entity that has structure and / or activity as found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or situation. Those of skill in the art will understand that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0132] As will be understood by those of skill in the art, the methods and compositions described herein relating to provided compounds (e.g., oligonucleotides) generally also apply to pharmaceutically acceptable salts of such compounds.

[0133] 1. Description of Certain Embodiments Oligonucleotides are useful tools for a wide variety of applications. For example, RNAi oligonucleotides are useful in therapeutic, diagnostic, and research applications, including the treatment of various conditions, disorders, and diseases. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endo- and exonucleases. Therefore, various synthetic counterparts have been developed to circumvent these drawbacks and / or further improve various properties and activities. These include, in particular, synthetic oligonucleotides containing chemical modifications (e.g., base modifications, sugar modifications, backbone modifications, etc.) that reduce the susceptibility of these molecules to degradation and improve other properties and / or activities. From a structural perspective, modifications to internucleotide linkages can introduce chirality and / or alter charge, and certain properties can be influenced by the positioning of the oligonucleotide's binding phosphorus atom. For example, binding affinity, sequence-specific binding to complementary RNA, stability against nucleases, cleavage of target nucleic acids, delivery, pharmacokinetics, etc., can be influenced, inter alia, by the chirality and / or charge of the backbone binding atoms.

[0134] 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.

[0135] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, for example, the stereochemistry of backbone chiral center, can unexpectedly maintain or improve the properties of ds oligonucleotide.Contrary to many previous observations that some structural elements that enhance stability also reduce activity, for example, RNA interference, the present disclosure demonstrates that, surprisingly, by controlling stereochemistry, it can maintain increased stability without significantly reducing activity.For example, but not limited to, the present disclosure is, in part, for example, but not limited to, the present disclosure is, in part, (1) a guide strand containing backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand containing backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5′-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3′ direction and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (3) a guide strand containing one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide (the upstream backbone phosphorothioate chiral centers are in the Rp or Sp configuration); (4) a guide strand containing one or more backbone phosphorothioate chiral centers in the 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 between (a) the +3 and +4 nucleotides; and (b) the +5 and +6 nucleotides; (5) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in the Rp or Sp configuration; and 6) A ds oligonucleotide comprising one or more passenger strands in combination with one or more of the aforementioned guide strands, the passenger strands comprising a backbone phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; The ds oligonucleotide further comprises: (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The present invention relates to ds oligonucleotides, wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bond incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is an Sp non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is an Sp non-negatively charged internucleotide bond. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0136] In certain embodiments, the present disclosure encompasses the recognition that the stereochemistry, e.g., the stereochemistry of the chiral center in the 5'-terminal modification of the guide strand, can unexpectedly maintain or improve the properties of ds oligonucleotides in which the guide strand of the ds oligonucleotide also contains a phosphorothioate chiral center in the Rp or Sp configuration. For example, but not by way of limitation, the present disclosure encompasses, in part, (1) phosphorothioate chiral centers in the Rp or Sp configuration; (2) Rp, Sp, or a sterically irregular non-negatively charged internucleotide linkage in which the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or a sterically irregular non-negatively charged internucleotide linkage contains a 2' modification, e.g., a 2'F; and (3) (a) Including, but not limited to: [ka] 5'PO modifications such as; (b) Including, but not limited to: [ka] 5'VP modifications such as; (c) Including, but not limited to: [ka] 5'MeP modifications such as; (d) Including, but not limited to: [ka] 5'PN and 5'Trizol-P modifications such as; wherein the bases are selected from A, C, G, T, U, abasic and modified nucleobases; R 2’ is H, OH, O-alkyl, F, MOE, locked nucleic acid (LNA) bridges, and bridged nucleic acid (BNA) bridges to the 4'C, including, but not limited to: [ka] The present invention relates to a ds oligonucleotide comprising a guide strand comprising a 5'-end modification selected from the group consisting of: (selected from). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages.

[0137] In certain other embodiments, the present disclosure encompasses the recognition that the stereochemistry, e.g., the stereochemistry of the chiral center at the 5'-terminal nucleotide of the guide strand, can unexpectedly maintain or improve the properties of ds oligonucleotides in which the guide strand of the ds oligonucleotide also contains a phosphorothioate chiral center in the Rp or Sp configuration. For example, but not by way of limitation, the present disclosure encompasses, in part, (1) a phosphorothioate chiral center in the Rp or Sp configuration; (2) an Rp, Sp, or a sterically irregular non-negatively charged internucleotide linkage in which the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or a sterically irregular non-negatively charged internucleotide linkage contains a 2' modification, e.g., a 2'F; and (3) (a) Including, but not limited to: [ka] 5'PO nucleotides such as; (b) Including, but not limited to: [ka] 5'VP nucleotides such as; (c) Including, but not limited to: [ka] 5'MeP nucleotides such as; (d) Including, but not limited to: [ka] 5'PN and 5'Trizol-P nucleotides such as; (e) Including, but not limited to: [ka] 5' abasic VP and 5' abasic MeP nucleotides such as The present invention relates to a ds oligonucleotide comprising a guide strand comprising a 5'-end modification selected from the group consisting of: In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages.

[0138] In certain embodiments, the present disclosure encompasses the recognition that Rp, Sp, or sterically irregular non-naturally occurring internucleotide linkages, such as neutral internucleotide linkages, can unexpectedly maintain or improve the properties of ds oligonucleotides. For example, the present disclosure demonstrates that modified internucleotide linkages can be introduced into ds oligonucleotides without significantly reducing the activity of the ds oligonucleotides. For example, but not limited to, the present disclosure provides, in part: (1) a guide strand containing backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand containing backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5′-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3′ direction and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (3) a guide strand containing one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide (the upstream backbone phosphorothioate chiral centers are in the Rp or Sp configuration); (4) a guide strand containing one or more backbone phosphorothioate chiral centers in the 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 between (a) the +3 and +4 nucleotides; and (b) the +5 and +6 nucleotides; (5) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in the Rp or Sp configuration; and 6) comprising one or more passenger strands in combination with one or more of the aforementioned guide strands, which contain backbone phosphorothioate chiral centers in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; The ds oligonucleotide further comprises: (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0139] In certain embodiments, the present disclosure encompasses the recognition that non-naturally occurring internucleotide linkages, such as neutral internucleotide linkages, can be used to attach one or more molecules to the double-stranded oligonucleotides described herein. In certain embodiments, such binding molecules can facilitate targeting and / or delivery of the double-stranded oligonucleotides. For example, but not limited to, such binding molecules include lipophilic molecules. In certain embodiments, the binding molecule is a molecule comprising one or more GalNAc moieties. In certain embodiments, the binding molecule is a receptor. In certain embodiments, the binding molecule is a receptor ligand.

[0140] In certain embodiments, the present disclosure provides techniques (e.g., compounds, methods, etc.) for improving oligonucleotide stability while maintaining or increasing activity, including compositions of oligonucleotides with improved stability.

[0141] In certain embodiments, the present disclosure provides techniques for incorporating various additional chemical moieties into ds oligonucleotides. In certain embodiments, the present disclosure provides, for example, reagents and methods for using nucleobases to introduce additional chemical moieties (e.g., by covalent attachment to sites on the nucleobases, optionally via linkers).

[0142] In certain embodiments, the present disclosure provides techniques, e.g., ds oligonucleotide compositions and methods, for achieving allele-specific suppression, in which transcripts from one allele of a particular target gene are selectively knocked down relative to at least one other allele of the same gene.

[0143] In particular, the present disclosure provides structural elements, techniques, and / or features that can be incorporated into a ds oligonucleotide to confer or adjust one or more properties thereof (e.g., compared to an otherwise identical ds oligonucleotide lacking the related technique or feature). In certain embodiments, the present disclosure describes that one or more of the provided techniques and / or features can be usefully incorporated into ds oligonucleotides of various sequences.

[0144] In certain embodiments, the present disclosure demonstrates that certain provided structural elements, techniques, and / or features are particularly useful for ds oligonucleotides that participate in and / or induce the RNAi machinery (e.g., RNAi agents). Nevertheless, the teachings of the present disclosure are not limited to ds oligonucleotides that participate in or act by any particular mechanism.

[0145] In certain embodiments, the present disclosure relates to any ds oligonucleotide useful for any purpose, acting via any mechanism, and comprising any sequence, structure, or format (or portion thereof) described herein.

[0146] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, acting via any mechanism, and comprising any sequence, structure, or format (or a portion thereof) described herein. In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0147] 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 (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0148] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers of Rp or Sp configuration upstream of a backbone phosphorothioate chiral center of 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; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0149] In certain embodiments, the guide strand has one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds occurring between the second (+2) and third (+3) nucleotides relative to the 5'-terminal nucleotide of the guide strand and an internucleotide bond to the penultimate 3' (N-1) nucleotide; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49). In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0150] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone phosphorothioate chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0151] 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 (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0152] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers of Rp or Sp configuration upstream of a backbone chiral center of 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; (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0153] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in the 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 between (a) the (+3) nucleotide and the (+4) nucleotide; and (b) the (+5) nucleotide and the (+6) nucleotide, and (1) a guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond (i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides); (2) a guide strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds exist 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 (N is the 3'-terminal nucleotide); (3) a guide strand in which an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond is present between the third (+3) and fourth (+4) nucleotide relative to the 5'-terminal nucleotide of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotide relative to the 5'-end; (4) a passenger strand in which one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are present upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (5) one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages in the passenger strand are present downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in an Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are Sp, non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular, non-negatively charged internucleotide linkages are sterically irregular, non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0154] In certain embodiments, the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages present 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 (N is the 3'-terminal nucleotide), a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0155] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and 2'-modifications, e.g., 2'F-modifications, of the 3'-nucleotide of nucleotide pairs linked by Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0156] In certain embodiments, the guide strand comprises Rp, Sp, or alternating backbone phosphorothioate chiral centers between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage; and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0157] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage; and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0158] In certain embodiments, the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages present 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 (N is the 3'-terminal nucleotide), a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or sterically irregular non-negatively charged internucleotide linkage, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages (n is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages are sterically irregular non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises a backbone phosphorothioate chiral center of Sp between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and a backbone phosphorothioate chiral center of Sp between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0159] In certain embodiments, the RNAi oligonucleotide comprises a sequence that is completely or substantially identical to or completely or substantially complementary to 10 or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous bases of a target genomic sequence or a transcript therefrom (e.g., mRNA (e.g., pre-mRNA, spliced ​​mRNA, etc.)). In certain embodiments, the RNAi oligonucleotide comprises a sequence that is completely complementary to 10 or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous bases of the target transcript. In certain embodiments, the number of contiguous bases is about 15-20. In certain embodiments, the number of contiguous bases is about 20. In certain embodiments, the RNAi oligonucleotide can hybridize to a target transcript (e.g., pre-mRNA, RNA, etc.) and reduce the level of the target transcript and / or the protein encoded by the target transcript.

[0160] In certain embodiments, the present disclosure provides dsRNAi oligonucleotides as disclosed herein, for example, in Table 1. In certain embodiments, the present disclosure provides dsRNAi oligonucleotides having a base sequence as disclosed herein, for example, in Table 1, or a portion thereof comprising at least 10 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive bases, wherein the RNAi oligonucleotide is sterically disordered or not chiral, and each T can be independently replaced by U, or vice versa.

[0161] In certain embodiments, the internucleotide linkages of the oligonucleotide comprise or consist of 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, 1 to 50, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirality-controlled internucleotide linkages. In certain embodiments, the present disclosure provides dsRNAi oligonucleotide compositions in which the dsRNAi oligonucleotide comprises at least one chirality-controlled internucleotide linkage. In certain embodiments, the present disclosure provides dsRNAi oligonucleotide compositions in which the dsRNAi oligonucleotide is sterically irregular or not chiral. In certain embodiments, at least one internucleotide linkage in the dsRNAi oligonucleotide is sterically irregular and at least one internucleotide linkage is chirality-controlled.

[0162] In certain embodiments, the internucleotide linkages of the oligonucleotide comprise or consist of one or more neutrally charged internucleotide linkages.

[0163] 1.1 Double-stranded oligonucleotides In certain embodiments, the present disclosure provides oligonucleotides of various designs, which may include various nucleobases and their patterns, sugars and their patterns, internucleotide linkages and their patterns, and / or additional chemical moieties and their patterns, as described herein. In certain embodiments, the provided dsRNAi oligonucleotides can induce a decrease in the expression, level, and / or activity of a gene and / or one or more of its products (e.g., transcripts, mRNA, proteins, etc.). In certain embodiments, the provided dsRNAi oligonucleotides can induce a decrease in the expression, level, and / or activity of a gene and / or one or more of its products in cells of a subject or patient. In certain embodiments, the cells normally express or produce proteins. In certain embodiments, provided dsRNAi oligonucleotides are capable of inducing a reduction in the expression, level, and / or activity of a target gene or gene product, and consist of, comprise, or comprise a portion (e.g., 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or a span of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous bases) of the base sequence of a dsRNAi oligonucleotide disclosed herein, wherein each T can be independently replaced by U, or vice versa, and wherein the dsRNAi oligonucleotide comprises at least one non-naturally occurring modification of the base, sugar, and / or internucleotide linkage.

[0164] In certain embodiments, dsRNAi oligonucleotides can induce a reduction in the expression, level, and / or activity of a target gene, e.g., a target gene, or its product. In certain embodiments, provided ds oligonucleotides can induce a reduction in the expression and / or level of a target gene or its gene product. In certain embodiments, provided ds oligonucleotides can induce a reduction in the level of a target product. In certain embodiments, provided ds oligonucleotides can reduce the level of a target gene transcript. In certain embodiments, provided ds oligonucleotides can reduce the level of a target gene mRNA. In certain embodiments, provided ds oligonucleotides can reduce the level of a protein encoded by a target gene. In certain embodiments, provided ds oligonucleotides can induce a reduction in the expression and / or level of a target gene or its gene product via RNA interference. In certain embodiments, provided ds oligonucleotides can induce a reduction in the expression and / or level of a target gene or its gene product via RNA interference or a RISC-free biochemical mechanism (including, but not limited to, RNase H-mediated knockdown or steric hindrance of gene expression). In certain embodiments, provided ds oligonucleotides can induce a reduction in the expression and / or levels of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In certain embodiments, provided ds oligonucleotides can induce a reduction in the expression and / or levels of a target gene or its gene product by sterically blocking translation after binding to the target gene mRNA and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion.In certain embodiments, the provided ds oligonucleotides comprise one or more structural elements described herein or known in the art according to the present disclosure, such as base sequence; modifications; stereochemistry; internucleotide linkage pattern; GC content; long GC stretches; backbone linkage pattern; backbone chiral center pattern; backbone phosphorus modification pattern; additional chemical moieties, including, but not limited to, one or more targeting moieties, lipid moieties, and / or carbohydrate moieties; seed region; post-seed region; 5'-terminal structure; 5'-terminal region; 5'-nucleotide moiety; 3'-terminal region; 3'-terminal dinucleotide; 3'-end cap; etc. In certain embodiments, the seed region of the oligonucleotide is or comprises the 2nd to 8th, 2nd to 7th, 2nd to 6th, 3rd to 8th, 3rd to 7th, or 4th to 8th, or 4th to 7th nucleotides counting from the 5' end; and the post-seed region of the oligonucleotide is the region immediately 3' from the seed region, interposed between the seed region and the 3'-terminal region. In certain embodiments, the provided compositions comprise ds oligonucleotides. In certain embodiments, the provided compositions comprise one or more lipid moieties, one or more carbohydrate moieties (other than the sugar moieties of the nucleoside units forming the oligonucleotide chain with internucleotide linkages, unless otherwise specified), and / or one or more targeting components. In certain embodiments, the dsRNAi oligonucleotides can induce a reduction in the expression, level, and / or activity of a target gene or its product by sterically blocking translation after binding to the target gene mRNA and / or by altering or interfering with mRNA splicing. Nevertheless, the present disclosure is not limited to any particular mechanism. In certain embodiments, the present disclosure provides ds oligonucleotides, compositions, methods, etc. that can act via double-stranded RNA interference, single-stranded RNA interference, RNase H-mediated knockdown, steric hindrance of translation, or a combination of two or more such mechanisms.

[0165] In certain embodiments, dsRNAi oligonucleotides comprise structural elements or portions thereof described herein, e.g., in Table 1. In certain embodiments, dsRNAi oligonucleotides comprise a base sequence (or portion thereof), chemical modification or pattern of chemical modifications (or portion thereof), and / or format or portion thereof described herein, where each T may be independently replaced by U, and vice versa. In certain embodiments, dsRNAi oligonucleotides have a base sequence comprising a base sequence (or portion thereof) of an oligonucleotide disclosed herein, e.g., in Table 1, or otherwise disclosed herein, where each T may be independently replaced by U, chemical modification pattern (or portion thereof), and / or format. In certain embodiments, such ds oligonucleotides, e.g., dsRNAi oligonucleotides, reduce the expression, level, and / or activity of a gene, e.g., a gene or its gene product.

[0166] In particular, dsRNAi oligonucleotides can hybridize to their target nucleic acids (e.g., pre-mRNA, mature mRNA, etc.). For example, in certain embodiments, dsRNAi oligonucleotides can hybridize to nucleic acids derived from a DNA strand (either strand of a gene). In certain embodiments, dsRNAi oligonucleotides can hybridize to transcripts. In certain embodiments, dsRNAi oligonucleotides can hybridize to target nucleic acids at any stage of RNA processing, including, but not limited to, pre-mRNA or mature mRNA. In certain embodiments, dsRNAi oligonucleotides can hybridize to any element or complement of an oligonucleotide nucleic acid, including, but not limited to, a promoter region, an enhancer region, a transcription termination region, a translation initiation signal, a translation termination signal, a coding region, a non-coding region, an exon, an intron, an intron / exon or exon / intron junction, a 5'UTR, or a 3'UTR. In certain embodiments, dsRNAi oligonucleotides can hybridize to their targets with no more than two mismatches. In certain embodiments, the dsRNAi oligonucleotide can hybridize to its target with no more than one mismatch. In certain embodiments, the dsRNAi oligonucleotide can hybridize to its target without any mismatches (e.g., all CG and / or AT / U base pairings).

[0167] In certain embodiments, ds oligonucleotide can hybridize with two or more transcript variants.In certain embodiments, dsRNAi oligonucleotide can hybridize with two or more or all transcript variants.In certain embodiments, dsRNAi oligonucleotide can hybridize with two or more or all transcript variants derived from sense strand.

[0168] In certain embodiments, the target of the dsRNAi oligonucleotide is an RNA that is not an mRNA.

[0169] In certain embodiments, ds oligonucleotides, for example, dsRNAi oligonucleotides, contain increased levels of one or more isotopes. In certain embodiments, ds oligonucleotides, for example, dsRNAi oligonucleotides, are labeled with one or more isotopes of, for example, one or more elements, for example, hydrogen, carbon, nitrogen, etc. In certain embodiments, the ds oligonucleotides, for example, dsRNAi oligonucleotides, in the provided compositions, for example, a plurality of ds oligonucleotides in the composition, comprise base modifications, sugar modifications, and / or internucleotide bond modifications, and the ds oligonucleotides contain enriched levels of deuterium. In certain embodiments, the oligonucleotides, for example, RNAi oligonucleotides, are labeled with deuterium at one or more positions (- 1 H- 2 In certain embodiments, one or more of the ds oligonucleotide strand or any moiety conjugated to the ds oligonucleotide strand (e.g., targeting moiety, etc.) 1 H is 2 Substituted with H. Such ds oligonucleotides can be used in the compositions and methods described herein.

[0170] In certain embodiments, the present disclosure provides 1) have a common base sequence that is complementary to a sequence in the transcript (e.g., a target sequence); and 2) A ds oligonucleotide composition is provided that includes a plurality of ds oligonucleotides that include one or more modified sugar moieties and / or modified internucleotide linkages.

[0171] In certain embodiments, dsRNAi oligonucleotides with a common base sequence may have the same nucleoside modification pattern, such as sugar modification, base modification, etc. In certain embodiments, the nucleoside modification pattern may be represented by the position and combination of modifications. In certain embodiments, the backbone bond pattern includes the position and type (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.) of each internucleotide bond.

[0172] In certain embodiments, multiple ds oligonucleotides, for example in the provided compositions, are of the same ds oligonucleotide type. In certain embodiments, the ds oligonucleotides of a ds oligonucleotide type have a common pattern of sugar modifications. In certain embodiments, the ds oligonucleotides of a ds oligonucleotide type have a common pattern of base modifications. In certain embodiments, the ds oligonucleotides of a ds oligonucleotide type have a common pattern of nucleoside modifications. In certain embodiments, the ds oligonucleotides of a ds oligonucleotide type have the same structure. In certain embodiments, the ds oligonucleotides of a ds oligonucleotide type are identical. In certain embodiments, multiple ds oligonucleotides are identical. In certain embodiments, multiple ds oligonucleotides share the same structure.

[0173] In certain embodiments, as exemplified herein, dsRNAi oligonucleotide is chiral controlled and comprises one or more chiral controlled internucleotide bonds.In certain embodiments, dsRNAi oligonucleotide is stereochemically pure.In certain embodiments, dsRNAi oligonucleotide is substantially separated from other stereoisomers.

[0174] In certain embodiments, the RNAi oligonucleotide comprises one or more modified nucleobases, one or more modified sugars, and / or one or more modified internucleotide linkages.

[0175] In certain embodiments, dsRNAi oligonucleotide comprises one or more modified sugars.In certain embodiments, ds oligonucleotide comprises one or more modified nucleobases.Various modifications can be introduced into sugar and / or nucleobase according to the present disclosure.For example, in certain embodiments, modification is the modification described in US Patent No. 9,006,198. In certain embodiments, modifications are made to the nucleotides described in U.S. Patent Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, 9,982,257, 10,160,969, 10,479,995, U.S. Patent Application Publication No. 2020 / 0056173, U.S. Patent Application Publication No. 2018 / 0216107, U.S. Patent Application Publication No. 2019 / 0127733, U.S. Patent No. 10,450,568 ...20 / 0056173, U.S. Patent Application Publication No. 2020 / 0056173, U.S. Patent Application Publication No. 2020 / 0056173, U.S. Patent Application Publication No. 2020 / 0056173, U.S. Patent Application Publication No. 2020 / 0056173, U.S. Patent Application Publication No and / or WO 2019 / 0077817, U.S. Patent Application Publication No. 2019 / 0249173, U.S. Patent Application Publication No. 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612.

[0176] As used in this disclosure, in certain embodiments, "one or more" is 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 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 certain embodiments, "one or more" is 1. In certain embodiments, "one or more" is 2. In certain embodiments, "one or more" is 3. In certain embodiments, "one or more" is 4. In certain embodiments, "one or more" is 5. In certain embodiments, "one or more" is 6. In certain embodiments, "one or more" is 7. In certain embodiments, "one or more" is 8. In certain embodiments, "one or more" is 9. In certain embodiments, "one or more" is 10. In certain embodiments, "one or more" is at least 1. In certain embodiments, "one or more" is at least 2. In certain embodiments, "one or more" is at least 3. In certain embodiments, "one or more" is at least 4. In certain embodiments, "one or more" is at least 5. In certain embodiments, "one or more" is at least 6. In certain embodiments, "one or more" is at least 7. In certain embodiments, "one or more" is at least 8. In certain embodiments, "one or more" is at least 9. In certain embodiments, "one or more" is at least 10.

[0177] As used in this disclosure, in certain embodiments, "at least one" is 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 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 certain embodiments, "at least one" is 1. In certain embodiments, "at least one" is 2. In certain embodiments, "at least one" is 3. In certain embodiments, "at least one" is 4. In certain embodiments, "at least one" is 5. In certain embodiments, "at least one" is 6. In certain embodiments, "at least one" is 7. In certain embodiments, "at least one" is 8. In certain embodiments, "at least one" is 9. In certain embodiments, "at least one" is ten.

[0178] In certain embodiments, the dsRNAi oligonucleotide is or comprises a dsRNAi oligonucleotide described in Table 1.

[0179] As demonstrated in the present disclosure, in certain embodiments, a provided ds oligonucleotide (e.g., a dsRNAi oligonucleotide) is characterized in that it knocks down its target (e.g., a transcript for the target oligonucleotide) when contacted with the transcript in a knockdown system.

[0180] In certain embodiments, ds oligonucleotides are provided as salt forms. In certain embodiments, ds oligonucleotides are provided as salts containing negatively charged internucleotide linkages (e.g., phosphorothioate internucleotide linkages, natural phosphate linkages, etc.) present in their salt forms. In certain embodiments, ds oligonucleotides are provided as pharmaceutically acceptable salts. In certain embodiments, ds oligonucleotides are provided as metal salts. In certain embodiments, ds oligonucleotides are provided as sodium salts. In certain embodiments, ds oligonucleotides are provided as metal salts, e.g., sodium salts, where each negatively charged internucleotide linkage is independently present in salt form (e.g., -OP(O)(SNa)-O- for a phosphorothioate internucleotide linkage, -OP(O)(ONa)-O- for a natural phosphate linkage, etc., for the sodium salt).

[0181] 1.2 Double-stranded oligonucleotide regions 1.2.1 Nucleotide sequence In certain embodiments, the dsRNAi oligonucleotides comprise a base sequence described herein or a portion thereof (e.g., 5-50, 5-40, 5-30, 5-20, or a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 20, or at least 10, at least 15 contiguous nucleobases) with 0-5 (e.g., 0, 1, 2, 3, 4, or 5) mismatches, wherein each T can be independently replaced with U, or vice versa. In certain embodiments, the dsRNAi oligonucleotides comprise a base sequence described herein or a portion thereof, wherein the portion is a span of at least 10 contiguous nucleobases, or a span of at least 15 contiguous nucleobases with 1-5 mismatches. In certain embodiments, the dsRNAi oligonucleotide comprises a base sequence described herein or a portion thereof, wherein the portion is a span of at least 10 contiguous nucleobases, or a span of at least 10 contiguous nucleobases with 1 to 5 mismatches, and each T can be independently replaced with a U, or vice versa. In certain embodiments, the base sequence of the ds oligonucleotide comprises or consists of 10 to 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 certain embodiments, at least 15; in certain embodiments, at least 16; in certain embodiments, at least 17; in certain embodiments, at least 18; in certain embodiments, at least 19; in certain embodiments, at least 20; in certain embodiments, at least 21; in certain embodiments, at least 22; in certain embodiments, at least 23; in certain embodiments, at least 24; in certain embodiments, at least 25) consecutive bases of a base sequence identical to or complementary to a base sequence of a gene or its transcript (e.g., mRNA).

[0182] As will be understood by those skilled in the art, the base sequence of the guide strand of a dsRNAi oligonucleotide typically has sufficient length and complementarity to its target, such as an RNA transcript (e.g., pre-mRNA, mature mRNA, etc.), to mediate target-specific knockdown. In certain embodiments, the base sequence of the dsRNAi oligonucleotide guide strand has sufficient length and identity to the transcript target to mediate target-specific knockdown. In certain embodiments, the dsRNAi oligonucleotide guide strand is complementary to a portion of the transcript (transcript target sequence). In certain embodiments, the base sequence of the dsRNAi oligonucleotide has 90% or more identity with the base sequence of the ds oligonucleotide disclosed in Table 1 (wherein each T can be independently replaced with U, and vice versa). In certain embodiments, the base sequence of the dsRNAi oligonucleotide has 95% or more identity with the base sequence of the oligonucleotide disclosed in Table 1 (wherein each T can be independently replaced with U, and vice versa). In certain embodiments, the base sequence of the dsRNAi oligonucleotide comprises a contiguous span of 15 or more bases of the oligonucleotides disclosed in Table 1 (wherein each T may be independently replaced with U, or vice versa), except where one or more bases within the span are abasic (e.g., a nucleobase is absent from the nucleotide). In certain embodiments, the base sequence of the dsRNAi oligonucleotide comprises a contiguous span of 19 or more bases of the dsRNAi oligonucleotides disclosed herein, except where one or more bases within the span are abasic (e.g., a nucleobase is absent from the nucleotide). In certain embodiments, the base sequence of the dsRNAi oligonucleotide comprises a contiguous span of 19 or more bases of the dsRNAi oligonucleotides disclosed herein, except where one or two bases at the 5' and / or 3' ends of the base sequence are different, and each T may be independently replaced with U, or vice versa.

[0183] In certain embodiments, the present disclosure relates to ds oligonucleotides having a base sequence comprising the base sequence of any ds oligonucleotide disclosed herein, wherein each T can be independently replaced with a U, or vice versa.

[0184] In certain embodiments, the present disclosure relates to ds oligonucleotides having a base sequence comprising at least 15 consecutive bases of the base sequence of any ds oligonucleotide disclosed herein, wherein each T can be independently replaced with a U, or vice versa.

[0185] In certain embodiments, the present disclosure relates to ds oligonucleotides having a base sequence that is at least 90% identical to the base sequence of any ds oligonucleotide disclosed herein, wherein each T can be independently replaced with a U, or vice versa.

[0186] In certain embodiments, the present disclosure relates to ds oligonucleotides having a base sequence at least 95% identical to the base sequence of any ds oligonucleotide disclosed herein, wherein each T can be independently replaced with a U, or vice versa.

[0187] In certain embodiments, the base sequence of the ds oligonucleotide is, comprises, or includes 10 to 20, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, consecutive bases of any ds oligonucleotide base sequence described herein, and each T can be independently replaced with U, or vice versa.

[0188] In certain embodiments, the dsRNAi oligonucleotide is selected from Table 1.

[0189] In certain embodiments, the dsRNAi oligonucleotide targets two or more or all alleles (if multiple alleles are present in the relevant system). In certain embodiments, the dsRNAi oligonucleotide reduces the expression, level, and / or activity of both the wild-type and mutant alleles and / or their transcripts and / or products.

[0190] In certain embodiments, the base sequences of the provided ds oligonucleotides are perfectly complementary to both human and non-human primate (NHP) target sequences. In certain embodiments, such sequences can be particularly useful because they can be easily evaluated in both humans and non-human primates.

[0191] In certain embodiments, the dsRNAi oligonucleotide comprises a base sequence or a portion thereof set forth in Table 1 (wherein each T may independently be replaced by U, and vice versa), and / or a sugar, nucleobase and / or internucleotide linkage modification and / or pattern thereof set forth in Table 1, and / or an additional chemical moiety (e.g., targeting moiety, lipid moiety, carbohydrate moiety, etc., in addition to the oligonucleotide chain) set forth in Table 1.

[0192] In certain embodiments, the terms "complementary," "fully complementary," and "substantially complementary" can be used in reference to matching bases between a ds oligonucleotide (e.g., a dsRNAi oligonucleotide) base sequence and a target sequence, as understood by those skilled in the art in connection with their use. Substitution of U with T, or vice versa, is generally recognized as not changing the amount of complementarity. As described herein, a ds polynucleotide that is "substantially complementary" to a target sequence is primarily or mostly complementary, but not 100% complementary. In certain embodiments, a substantially complementary sequence (e.g., a dsRNAi oligonucleotide) has one, two, three, four, or five mismatches when aligned with its target sequence. In certain embodiments, a dsRNAi oligonucleotide has a base sequence that is substantially complementary to a target sequence. In certain embodiments, a dsRNAi oligonucleotide has a base sequence that is substantially complementary to the complement of the sequence of a dsRNAi oligonucleotide disclosed herein. As will be understood by those skilled in the art, in certain embodiments, the sequences of ds oligonucleotides need not be 100% complementary to their targets in order for the ds oligonucleotides to perform their function (e.g., knockdown of a target nucleic acid). Typically, when determining complementarity, A and T (or U) are complementary nucleobases, and C and G are complementary nucleobases.

[0193] In certain embodiments, a "portion" (e.g., of a base sequence or modification pattern) is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units in length (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 in length). In certain embodiments, a "portion" of a base sequence is at least 5 bases in length. In certain embodiments, a "portion" of a base sequence is at least 10 bases in length. In certain embodiments, a "portion" of a base sequence is at least 15 bases in length. In certain embodiments, a "portion" of a base sequence is at least 16, 17, 18, 19, or 20 bases in length. In certain embodiments, a "portion" of a base sequence is at least 20 bases in length. In certain embodiments, the portion of the base sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more consecutive bases. In certain embodiments, the portion of the base sequence is 15 or more consecutive bases. In certain embodiments, the portion of the base sequence is 16, 17, 18, 19 or 20 or more consecutive bases. In certain embodiments, the portion of the base sequence is 20 or more consecutive bases.

[0194] In certain embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides. In certain embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides comprising 0 to 3 mismatches. In certain embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides with 0 to 3 mismatches, where a span with 0 mismatches is complementary and a span with one or more mismatches is a non-limiting example of substantial complementarity. In certain embodiments, the base comprises a portion that is characteristic of nucleic acid (for example, gene) in that a portion thereof is identical to or complementary to a portion of nucleic acid or its transcription product, and is not identical to or complementary to any other nucleic acid (for example, gene) or a portion of its transcription product in the same genome.In certain embodiments, the portion is characteristic of human dsRNAi.

[0195] In certain embodiments, provided oligonucleotides, e.g., dsRNAi oligonucleotides, as described herein, have a total length of no more than about 49, 45, 40, 30, 35, 25, or 23 nucleotides. In certain embodiments where the 5' end of the sequences described herein begins with a U or T, the U can be deleted and / or substituted with another base.

[0196] In certain embodiments, ds oligonucleotides, such as dsRNAi oligonucleotides, are stereoirregular. In certain embodiments, RNAi oligonucleotides are chiral-controlled. In certain embodiments, dsRNAi oligonucleotides are chirally pure (or "stereically pure" or "stereochemically pure"), and ds oligonucleotides exist as a single stereoisomeric form (often a single diastereomeric (or "diastereomeric") form, since multiple chiral centers may exist in ds oligonucleotides, for example, at the phosphorus, sugar, carbon, etc.). As will be understood by those skilled in the art, chirally pure ds oligonucleotides are separated from other stereoisomeric forms (to the extent that some impurities may exist, since chemical and biological processes, selectivity and / or purification, etc., are rarely, if ever, absolutely perfect). In chirally pure ds oligonucleotides, each chiral center is independently defined with respect to its configuration (for chirally pure ds oligonucleotides, each internucleotide bond is independently stereorestricted or chiral-controlled). In contrast to chirally controlled and chirally pure ds oligonucleotides containing sterically restricted linking phosphorus, racemic (or "sterically irregular," "chirally uncontrolled") ds oligonucleotides containing chiral linking phosphorus, derived, for example, from conventional phosphoramidite oligonucleotide synthesis without stereochemical control during the coupling step in combination with conventional sulfurization (which produces a stereoirregular phosphorothioate internucleotide linkage), refer to an irregular mixture of various stereoisomers (typically diastereoisomers (or "diastereomers"), since multiple chiral centers are present in the ds oligonucleotide; for example, derived from conventional ds oligonucleotide preparations using reagents that contain no chiral elements other than those at the nucleotides and linking phosphorus). For example, for A*A*A (where * is the phosphorothioate internucleotide linkage (containing the chiral linking phosphorus)), the preparation of racemic oligonucleotides can yield four diastereomers [2 2= 4, considering two chiral linking phosphorus, each of which can exist in either of two configurations (Sp or Rp): A*SA*SA, A*SA*RA, A*RA*SA, and A*RA*RA (*S represents the phosphorothioate internucleotide linkage of Sp, and *R represents the phosphorothioate internucleotide linkage of Rp). With respect to chirally pure oligonucleotides (e.g., A*SA*SA), the oligonucleotide exists in a single stereoisomeric form, and the oligonucleotide is separated from other stereoisomers (e.g., the diastereomers A*SA*RA, A*RA*SA, and A*RA*RA).

[0197] In certain embodiments, the dsRNAi oligonucleotides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sterically irregular internucleotide linkages (a mixture of Rp- and Sp-linked phosphorus at the internucleotide linkage, e.g., derived from conventional, non-chiral oligonucleotide synthesis). In certain embodiments, the dsRNAi oligonucleotides contain one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) chiral-controlled internucleotide linkages (Rp- or Sp-linked phosphorus at the internucleotide linkage, e.g., derived from chiral-controlled oligonucleotide synthesis).

[0198] In certain embodiments, the internucleotide linkage is a phosphorothioate internucleotide linkage. In certain embodiments, the internucleotide linkage is a sterically disordered phosphorothioate internucleotide linkage. In certain embodiments, the internucleotide linkage is a chiral controlled phosphorothioate internucleotide linkage.

[0199] In particular, the present disclosure provides techniques for preparing chiral controlled (and in certain embodiments stereochemically pure) ds oligonucleotides. In certain embodiments, the ds oligonucleotides are stereochemically pure. In certain embodiments, the ds oligonucleotides of the present disclosure have a chirality of about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 90%, 95%, 50%, ...50%, 60%, 70%, 80%, 90%, 90%, 95%, 50%, 50%, 60%, 70%, 80%, 90%, 90%, 95%, 50%, 60%, 70%, 80%, 90%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 95%, 90%, 9 %, 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 certain embodiments, the internucleotide linkages of the ds oligonucleotide are one or more (e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 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 nucleotides. In certain embodiments, the ds oligonucleotides of the present disclosure, e.g., dsRNAi oligonucleotides, comprise or consist of (DS) diastereomeric internucleotide linkages, each of which independently has a diastereomeric purity 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%. CILwherein DS is a diastereomeric purity as described herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more), and CIL is the number of chiral controlled internucleotide linkages (e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 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 certain embodiments, DS is 95% to 100%. In certain embodiments, each internucleotide linkage is independently chiral controlled, and CIL is the number of chiral controlled internucleotide linkages.

[0200] By way of example, certain dsRNAi oligonucleotides, including certain exemplary base sequences, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, internucleotide linkages and patterns thereof, linked phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties, are shown below in Table 1. In particular, ds oligonucleotides, such as those in Table 1A, can be utilized to target transcripts, e.g., to reduce the levels of the transcripts and / or their products.

[0201] [Table 1]

[0202] [Table 2]

[0203] [Table 3]

[0204] [Table 4]

[0205]

Table 5

[0206]

Table 6

[0207]

Table 7

[0208]

Table 8

[0209]

Table 9

[0210]

Table 10

[0211]

Table 11

[0212]

Table 12

[0213]

Table 13

[0214]

Table 14

[0215]

Table 15

[0216] Table 16

[0217] Table 17

[0218] Table 18

[0219]

Table 19

[0220] Table 20

[0221] Table 21

[0222] Table 22

[0223] Table 23

[0224] Table 24

[0225] Table 25

[0226] Table 26

[0227] Table 27

[0228] Table 28

[0229] Table 29

[0230]

Table 30

[0231] Table 31

[0232] Table 32

[0233] Table 33

[0234] Table 34

[0235] Table 35

[0236] Table 36

[0237] Table 37

[0238] Table 38

[0239] Table 39

[0240] Table 40

[0241] Table 41

[0242] Table 42

[0243] Table 43

[0244] Table 44

[0245] Table 45

[0246] Table 46

[0247] Notes: Descriptions, base sequences, and stereochemistry / linkages can be categorized into multiple series in Table 1 depending on their length. Unless otherwise specified, all oligonucleotides in Table 1 are single-stranded. As will be understood by those skilled in the art, nucleoside units are unmodified unless otherwise specified (e.g., r, m, etc.) and contain unmodified nucleobases and 2'-deoxy sugars; linkages are natural phosphate linkages unless otherwise specified; and acidic / basic groups may independently be present in their salt form. If the sugar is not specified, the sugar is the natural DNA sugar; if the internucleotide linkage is not specified, the internucleotide linkage is the natural phosphate linkage. Moieties and Modifications: m:2'-OMe; f or [fl2r]:2'-F; O, PO, p: phosphodiester (phosphate). This can be a bond or an end group (or a component thereof), e.g., the bond between the linker and the oligonucleotide chain, the internucleotide bond (natural phosphate bond), etc. A phosphodiester is usually indicated with an "O" in the stereochemistry / bonding column and is usually not labeled in the description column (if it is an end group, e.g., the 5' end group, it is indicated in the description and usually not in the stereochemistry / bonding); if a bond is not indicated in the description column, it is usually a phosphodiester unless otherwise indicated. Note that the phosphate bond between the linker (e.g., L001) and the oligonucleotide chain may not be labeled in the description column, but may be indicated with an "O" in the stereochemistry / bonding column; *, PS, sp: phosphorothioate. This can be an end group (if it is an end group, e.g., the 5' end group, this is indicated in the description and usually not in the stereochemistry / bonding), or a bond, e.g., a bond between a linker (e.g., L001) and an oligonucleotide chain, an internucleotide bond (phosphorothioate internucleotide bond), etc.; R, Rp, or [Rsp]: phosphorothioate in the Rp configuration. Note that *R in the description indicates a single phosphorothioate bond in the Rp configuration; S, Sp, or [Ssp]: phosphorothioate in the Sp configuration. Note that *S in the description indicates a single phosphorothioate bond in the Sp configuration; X: sterically irregular phosphorothioate; CHEM1:ligand; CHEM2: 5′-linker; n001: [ka] ; nX:sterically irregular n001; nR or n001R or [n001R]: n001 in Rp configuration; nS or n001S or [n001S]: n001 in Sp configuration; n009: [ka] ; nX:sterically irregular n009; nR or n009R: n009 in Rp configuration; nS or n009S: n009 in Sp configuration; n031: [ka] ; nX:sterically irregular n031; nR or n031R: n031 in Rp configuration; nS or n031S: n031 in Sp configuration; n033: [ka] ; nX:sterically irregular n033; nR or n033R: n033 in Rp configuration; nS or n033S: n033 in Sp configuration; n037: [ka] ; nX:sterically irregular n037; nR or n037R: n037 in Rp configuration; nS or n037S: n037 in Sp configuration; n046: [ka] ; nX:sterically irregular n046; nR or n046R: n046 in Rp configuration; nS or n046S: n046 in Sp configuration; n047: [ka] ; nX:sterically irregular n047; nR or n047R: n047 in Rp configuration; nS or n047S: n047 in Sp configuration; n025: [ka] ; nX:sterically irregular n025; nR or n025R:n025 in Rp configuration: nS or n025S: n025 in Sp configuration; n054: [ka] ; nX:sterically irregular n054; nR or n054R: n054 in Rp configuration; nS or n054S: n054 in Sp configuration; n055: [ka] ; nX:sterically irregular n055; nR or n055R: n055 in Rp configuration; nS or n055S: n055 in Sp configuration; n026: [ka] ; nX:sterically irregular n001; nR or n026R: n026 in Rp configuration; nS or n026S: n026 in Sp configuration; n004: [ka] ; nX:sterically irregular n004; nR or n004R: n004 in Rp configuration; nS or n004S: n004 in Sp configuration; n003: [ka] ; nX:sterically irregular n003; nR or n003R: n003 in Rp configuration; nS or n003S: n003 in Sp configuration; n008: [ka] ; nX:sterically irregular n008; nR or nR:nRp configuration: nS or n008S: n008 in Sp configuration; n029: [ka] ; nX:sterically irregular n029; nR or n029R: n029 in Rp configuration; nS or n029S: n029 in Sp configuration; n021: [ka] ; nX:sterically irregular n021; nR or n021R: n021 in Rp configuration; nS or n021S: n021 in Sp configuration; n006: [ka] ; nX:sterically irregular n006; nR or n006R: n006 in Rp configuration; nS or n006S: n006 in Sp configuration; n020: [ka] ; nX:sterically irregular n020; nR or n020R: n020 in Rp configuration; nS or n020S: n020 in Sp configuration; n043: [ka] ; nX:sterically irregular n043; nR or n043R: n043 in Rp configuration; nS or n043S: n043 in Sp configuration; n058: [ka] ; nX:sterically irregular n058; nR or n058R: n058 in Rp configuration; nS or n058S: n058 in Sp configuration; X: sterically irregular phosphorothioate; [ka] [ka] [ka] [ka] (wherein -C(O)- is attached to nitrogen); sm01n013: [ka] i.e., morpholine carbamate internucleotide linkage (sm01n013) [ka] ; [ka] L001 or nC6o: -NH-(CH2)6- linker (C6 linker, C6 amine linker, or C6 amino linker) linked to Mod (e.g., Mod001) via -NH- and, in the case of WV-38061, to the 5' end of the oligonucleotide chain via a phosphate bond (O or PO). For example, in WV-38061, L001 is linked to Mod001 via -NH- (forming an amide group -C(O)-NH-) and linked to the oligonucleotide chain via a phosphate bond (O). L010: [ka] In some embodiments, when L010 is present in the middle of an oligonucleotide, it is attached to an internucleotide linkage as another sugar (e.g., a DNA sugar), e.g., its 5'-carbon is linked to another unit (e.g., the 3' of the sugar) and its 3'-carbon is linked to another unit (e.g., the 5'-carbon of the carbon), independently, e.g., via linkages (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be chiral non-controlled or chiral controlled (Sp or Rp))). L012: -CH2CH2OCH2CH2OCH2CH2-. When L012 is present in the middle of an oligonucleotide, each of its two ends is independently linked to an internucleotide bond (e.g., a phosphate bond (O or PO) or a phosphorothioate bond (which may be non-chiral or chiral (Sp or Rp))); L022: [ka] where L022 is linked to the rest of the molecule via a phosphate unless otherwise indicated; L023: HO-(CH2)6- (where CH2 is linked to the rest of the molecule via a phosphate unless otherwise indicated), e.g., WV-42644 (where the O in OnRnRnRnRSSSSSSSSSSSSSSSSnRSSSSSnRSSnR represents the phosphate bond connecting L023 to the rest of the molecule); L025: [ka] where the -CH2- linkage serves as the C5 linkage of the sugar (e.g., DNA sugar) and is linked to another unit (e.g., the 3' of the sugar), and the linkage on the ring serves as the C3 linkage and is linked to another unit (the 5'-carbon of the carbon), each independently, for example, via a bond (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be chiral non-controlled or chiral controlled (Sp or Rp)). When L025 is at the 5' end without any modifications, this -CH2- linkage is attached to -OH. For example, L025L025L025- in various oligonucleotides is [ka] (which may exist as various salt forms) and is linked to the 5'-carbon of the oligonucleotide chain via a designated bond (e.g., a phosphate bond (O or PO) or a phosphorothioate bond (which may be chiral non-controlled or chiral controlled (Sp or Rp))); L016: [ka] where L016 is linked to the remainder of the molecule via the phosphate unless otherwise indicated; L016 is utilized with n001 to form L016n001, which has the following structure: [ka] .

[0248] 1.2.2 Double-stranded oligonucleotide length As will be understood by those skilled in the art, ds oligonucleotides can be of various lengths to provide desired properties and / or activities for various applications. Many techniques for evaluating, selecting, and / or optimizing the length of ds oligonucleotides are available in the art and can be utilized in accordance with the present disclosure. As demonstrated herein, in certain embodiments, dsRNAi oligonucleotides are of a length suitable for hybridizing with their targets and reducing the levels of the targets and / or their encoded products. In certain embodiments, ds oligonucleotides are of sufficient length to recognize target nucleic acids (e.g., target mRNAs). In certain embodiments, ds oligonucleotides are of sufficient length to distinguish the target nucleic acid from other nucleic acids (e.g., nucleic acids having base sequences other than the target sequence) and reduce off-target effects. In certain embodiments, dsRNAi oligonucleotides are sufficiently short to reduce the complexity of manufacturing or production and reduce the cost of the product.

[0249] In certain embodiments, the base sequence of the ds oligonucleotide is about 10 to 500 nucleobases in length. In certain embodiments, the base sequence is about 10 to 500 nucleobases in length. In certain embodiments, the base sequence is about 10 to 50 nucleobases in length. In certain embodiments, the base sequence is about 15 to 50 nucleobases in length. In certain embodiments, the base sequence is about 15 to about 30 nucleobases in length. In certain embodiments, the base sequence is about 10 to about 25 nucleobases in length. In certain embodiments, the base sequence is about 15 to about 22 nucleobases in length. In certain embodiments, the 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 certain embodiments, the base sequence is about 18 nucleobases in length. In certain embodiments, the base sequence is about 19 nucleobases in length. In certain embodiments, the base sequence is about 20 nucleobases in length. In certain embodiments, the base sequence is about 21 nucleobases in length. In certain embodiments, the base sequence is about 22 nucleobases in length. In certain embodiments, the base sequence is about 23 nucleobases in length. In certain embodiments, the base sequence is about 24 nucleobases in length. In certain embodiments, the base sequence is about 25 nucleobases in length. In certain embodiments, each nucleobase is optionally substituted A, T, C, G, U, or an optionally substituted tautomer of A, T, C, G, or U.

[0250] 2.2.3. Internucleotide bond In certain embodiments, ds oligonucleotides contain base modifications, sugar modifications, and / or internucleotide linkage modifications. A variety of internucleotide linkages can be utilized in accordance with the present disclosure to link nucleobase-containing units (e.g., nucleosides). In certain embodiments, provided ds oligonucleotides contain both one or more modified internucleotide linkages and one or more natural phosphate linkages. As is well known by those skilled in the art, natural phosphate linkages are widely found in natural DNA and RNA molecules; they have the structure -OP(O)(OH)O-, link sugars in nucleosides in DNA and RNA, and can exist in various salt forms, for example, at physiological pH (about 7.4), where natural phosphate linkages exist primarily in the salt form, with the anion being -OP(O)(O - )O-. A modified internucleotide linkage or non-natural phosphate linkage is an internucleotide linkage that is not a natural phosphate linkage or its salt form. Modified internucleotide linkages can exist in their salt forms depending on their structure. For example, as will be understood by those skilled in the art, a phosphorothioate internucleotide linkage having the structure -OP(O)(SH)O- can exist in various salt forms, for example, at physiological pH (about 7.4), and the anion is -OP(O)(S - )O-.

[0251] In certain embodiments, the ds oligonucleotide comprises an internucleotide linkage that is a modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, thiophosphate, 3'-thiophosphate, or 5'-thiophosphate.

[0252] In certain embodiments, the modified internucleotide linkage is a chiral internucleotide linkage comprising a chiral linking phosphorus. In certain embodiments, the chiral internucleotide linkage is a phosphorothioate linkage. In certain embodiments, the chiral internucleotide linkage is a non-negatively charged internucleotide linkage. In certain embodiments, the chiral internucleotide linkage is a neutral internucleotide linkage. In certain embodiments, the chiral internucleotide linkage is chiral controlled with respect to its chiral linking phosphorus. In certain embodiments, the chiral internucleotide linkage is stereochemically pure with respect to its chiral linking phosphorus. In certain embodiments, the chiral internucleotide linkage is not chiral controlled. In certain embodiments, the pattern of chiral centers in the backbone comprises or consists of the position of a chiral controlled internucleotide linkage and the configuration of the linking phosphorus (Rp or Sp) and the position of an achiral internucleotide linkage (e.g., a natural phosphate linkage).

[0253] In certain embodiments, the internucleotide linkage comprises a P-modification, which is a modification at the linked phosphorus. In certain embodiments, the modified internucleotide linkage does not contain phosphorus, as in, for example, peptide nucleic acids (PNAs), but is a moiety that serves to link two sugars or two moieties, each independently comprising a nucleobase.

[0254] In certain embodiments, the ds oligonucleotide has a modified internucleotide linkage, e.g., a structure of Formula I, Ia, Ib, or Ic, as described herein and / or in WO 2018 / 022473, WO 2018 / 098264, WO 2018 / 22305, each of which is independently incorporated by reference. 6, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612. In certain embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In certain embodiments, the modified internucleotide linkage is a phosphorothioate internucleotide linkage.

[0255] In certain embodiments, the modified internucleotide bond is a non-negatively charged internucleotide bond. In certain embodiments, the provided ds oligonucleotide comprises one or more non-negatively charged internucleotide bonds. In certain embodiments, the non-negatively charged internucleotide bond is a positively charged internucleotide bond. In certain embodiments, the non-negatively charged internucleotide bond is a neutral internucleotide bond. In certain embodiments, the present disclosure provides an oligonucleotide comprising one or more neutral internucleotide bonds.In certain embodiments, the non-negatively charged internucleotide linkage is selected from the group consisting of nucleotides of formula (I) and nucleotides of formula (II ... Patent No. 9394333, U.S. Patent No. 9744183, U.S. Patent No. 9605019, U.S. Patent No. 9982257, U.S. Patent Application Publication No. 20170037399, U.S. Patent Application Publication No. 20180216108, U.S. Patent Application Publication No. 20180216107, U.S. Patent No. 9598458, International Publication No. 2017 / 062862, International Publication No. 2018 / 067973, International Publication No. 2017 / 160741, International Publication No. 2017 / 192679, International Publication No. 2017 / 210647, International Publication No. 2018 / 098264, International Publication No. 2018 / 022473, International Publication No. 2018 / 223056, International Publication No. 2018 / 223073, International Publication No. 2018 / 223081, International Publication No. 2018 / 237194, International Publication No. 2019 / 032607, International Publication No. 2019 / 032612, International Publication No. 2019 / and / or WO 2019 / 032612, or a salt form thereof.

[0256] In certain embodiments, non-negatively charged internucleotide linkages can improve delivery and / or activity (e.g., adenosine editing activity).

[0257] In certain embodiments, the modified internucleotide linkage (e.g., a non-negatively charged internucleotide linkage) comprises an optionally substituted triazolyl. In certain embodiments, the modified internucleotide linkage (e.g., a non-negatively charged internucleotide linkage) comprises an optionally substituted alkynyl. In certain embodiments, the modified internucleotide linkage comprises a triazole or alkyne moiety. In certain embodiments, the triazole moiety, e.g., a triazolyl group, is optionally substituted. In certain embodiments, the triazole moiety, e.g., a triazolyl group, is substituted. In certain embodiments, the triazole moiety is unsubstituted. In certain embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety. In certain embodiments, the modified internucleotide linkage is [ka] and optionally chiral controlled, R 1 is -L-R', where L is L as described herein. B and R' is as described herein. In certain embodiments, each R 1 is independently R'. In certain embodiments, each R' is independently R. In certain embodiments, two R 1 and R together form a ring as described herein. In certain embodiments, two R on two different nitrogen atoms 1 is R and together form a ring as described herein. In certain embodiments, R 1 is independently an optionally substituted C as described herein. 1~6 In certain embodiments, R 1 is methyl. In certain embodiments, two R' on the same nitrogen atom are R and together form a ring as described herein. In certain embodiments, the modified internucleotide linkage is [ka] and optionally chiral controlled. In certain embodiments, [ka] teeth, [ka] In certain embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety, [ka] wherein W is O or S. In certain embodiments, W is O. In certain embodiments, W is S. In certain embodiments, the non-negatively charged internucleotide linkage is stereochemically controlled.

[0258] In certain embodiments, the non-negatively charged or neutral internucleotide linkage is an internucleotide linkage comprising a triazole moiety. In some embodiments, the internucleotide linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group) is [ka] In some embodiments, the internucleotide linkage comprising a triazole moiety has the structure: [ka] In some embodiments, the internucleotide linkage comprising a triazole moiety has the structure: [ka] where W is O or S. In some embodiments, an internucleotide linkage comprising an alkyne moiety (e.g., an optionally substituted alkynyl group) has the formula: [ka] wherein W is O or S. In some embodiments, the internucleotide linkage, e.g., a non-negatively charged internucleotide linkage, a neutral internucleotide linkage, comprises a cyclic guanidine moiety. In some embodiments, the internucleotide linkage comprising a cyclic guanidine moiety is [ka] In some embodiments, the non-negatively charged or neutral internucleotide linkage has the structure: [ka] wherein W is O or S. In certain embodiments, the internucleotide linkage, e.g., a non-negatively charged internucleotide linkage, a neutral internucleotide linkage, comprises a cyclic guanidine moiety. In certain embodiments, the internucleotide linkage comprising a cyclic guanidine moiety is [ka] In certain embodiments, the non-negatively charged or neutral internucleotide bond has the structure: [ka] wherein W is O or S. In certain embodiments, the internucleotide linkage is or comprises a Tmg group ( [ka] In certain embodiments, the internucleotide linkage comprises a Tmg group, [ka] ("Tmg internucleotide linkage"). In certain embodiments, neutral internucleotide linkages include PNA and PMO internucleotide linkages and Tmg internucleotide linkages.

[0259] In certain embodiments, the non-negatively charged internucleotide linkage has a structure of Formula I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 3-20-membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In certain embodiments, the non-negatively charged internucleotide 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 heterocyclyl or heteroaryl groups are 5-membered rings. In certain embodiments, such heterocyclyl or heteroaryl groups are 6-membered rings.

[0260] In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5- to 20-membered heteroaryl group having 1 to 10 heteroatoms. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5- to 20-membered heteroaryl group having 1 to 10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5- to 6-membered heteroaryl group having 1 to 4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-membered heteroaryl group having 1 to 4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the heteroaryl group is directly bonded to the linking phosphorus.

[0261] In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5- to 20-membered heterocyclyl group having 1 to 10 heteroatoms. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5- to 20-membered heterocyclyl group having 1 to 10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5- to 6-membered heterocyclyl group having 1 to 4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-membered heterocyclyl group having 1 to 4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodime...

Claims

1. A double-stranded RNAi (dsRNAi) agent comprising a guide strand and a passenger strand, a) the guide strand is complementary or substantially complementary to a target RNA sequence; and i. backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; ii. backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide; iii. one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration upstream of a backbone phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; and / or iv. containing one or more backbone phosphorothioate chiral centers of Rp or Sp configuration at one or both of the following: between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide, and between (a) the (+3) nucleotide and the (+4) nucleotide; and (b) the (+5) nucleotide and the (+6) nucleotide; b) the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds 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; c) the guide strand comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond; d) the passenger strand is i. 0 to n Rp, Sp, or non-negatively charged internucleotide linkages, where n is about 1 to 49; and ii. one or more backbone chiral centers of Rp or Sp configuration One or both of the following are included: e) each strand of the dsRNAi agent independently has a length of from about 15 to about 49 nucleotides; f) A double-stranded RNAi (dsRNAi) agent, wherein said dsRNAi is capable of inducing target-specific RNA interference.

2. 1. A chiral controlled oligonucleotide composition comprising a double-stranded oligonucleotide, the guide strand and passenger strand of the double-stranded oligonucleotide being independently: a) the common base sequence and length; b) a common pattern of backbone bonds; and c) Common patterns of backbone chiral centers Characterized by The composition is chiral controlled in that it is enriched for oligonucleotides having a common pattern of chiral centers relative to a substantially racemic preparation of guide strands having the same common base sequence and length; and a) the guide strand is complementary or substantially complementary to a target RNA sequence; and i. backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; ii. backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide; iii. one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration upstream of a backbone phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; and / or iv. comprises one or more backbone phosphorothioate chiral centers of Rp or Sp configuration in one or both of the following: between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide; and between (a) the (+3) nucleotide and the (+4) nucleotide; and (b) the (+5) nucleotide and the (+6) nucleotide; or b) the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds 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; c) the guide strand comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or sterically irregular non-negatively charged internucleotide bond; d) the passenger strand is i. 0 to n Rp, Sp, or non-negatively charged internucleotide linkages, where n is about 1 to 49; and ii. one or more backbone chiral centers of Rp or Sp configuration One or both of the following are included: e) the guide and passenger strands have a length of about 15 to about 49 nucleotides; and f) A chiral controlled oligonucleotide composition, wherein said guide and passenger strands are capable of inducing target-specific RNA interference.

3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center of 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 the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, where n is from about 1 to 49.

4. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises an Rp, Sp, or alternating backbone phosphorothioate chiral center 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 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, where n is about 1 to 49.

5. 3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers of Rp or Sp configuration upstream of a backbone phosphorothioate chiral center of 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 the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, where n is from about 1 to 49.

6. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds 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 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide bonds, where n is about 1 to 49.

7. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration.

8. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in the Rp, Sp, or alternating 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 the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration.

9. 3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers of the Rp or Sp configuration upstream of a backbone phosphorothioate chiral center of the 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 the passenger strand comprises one or more backbone chiral centers of the Rp or Sp configuration.

10. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers of Rp or Sp configuration in one or both of the following positions: between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the (+2) nucleotide and the immediately downstream (+3) nucleotide, and (a) between the (+3) nucleotide and the (+4) nucleotide; and (b) between the (+5) nucleotide and the (+6) nucleotide.

11. The guide strand is [Formula 1] Bases: A, C, G, T, U, abasic and modified nucleobases; R: H, OH, O-alkyl, F, MOE, LNA bridge at 4' position, BNA bridge at 4' position The double-stranded oligonucleotide of claim 1 or the composition of claim 2, comprising a 5'-end modification selected from:

12. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages 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 of Rp or Sp configuration.

13. 3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, where n is from about 1 to 49, and one or more backbone chiral centers in the Rp or Sp configuration.

14. 3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in an Rp, Sp, or alternating 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 the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, where n is about 1 to 49, and one or more backbone chiral centers in the Rp or Sp configuration.

15. 3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration upstream of a backbone phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0 to n Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages, where n is from about 1 to 49, and one or more backbone chiral centers in the Rp or Sp configuration.

16. 3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more Rp, Sp, or sterically irregular non-negatively charged internucleotide linkages 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 to n non-negatively charged internucleotide linkages, where n is about 1 to 49, and one or more backbone chiral centers of Rp or Sp configuration.

17. The double-stranded oligonucleotide or composition according to any one of claims 1 to 16, wherein the Rp, Sp or non-sterically disordered negatively charged backbone internucleotide linkages have a neutral charge.

18. The neutral backbone internucleotide linkages are [Case 2] 18. The double-stranded oligonucleotide or composition of claim 17, wherein:

19. The guide strand has the following structure between 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: [C3] 20. The double-stranded oligonucleotide or composition of claim 18, comprising a linkage having the formula:

20. The passenger strand has the structure [C4] 20. The double-stranded oligonucleotide or composition of claim 19, comprising a linkage having the formula:

21. 3. The composition of claim 2, wherein the guide and passenger strands in the composition that independently share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications and / or a common pattern of internucleotide linkages are at least 90% of all the guide and passenger strands in the composition.

22. The double-stranded oligonucleotide or composition according to any of claims 1 to 21, wherein the double-stranded oligonucleotide comprises a carbohydrate moiety linked at a nucleoside or internucleotide bond, optionally via a linker.

23. The double-stranded oligonucleotide or composition according to any one of claims 1 to 22, wherein the double-stranded oligonucleotide comprises a lipid moiety linked to the double-stranded oligonucleotide at a nucleoside or internucleotide bond, optionally via a linker.

24. The double-stranded oligonucleotide or composition according to any of claims 1 to 23, wherein one or both strands of the double-stranded oligonucleotide comprise a targeting moiety linked at an internucleoside or internucleotide bond, optionally via a linker.

25. 25. The double-stranded oligonucleotide or composition of any one of claims 1-24, wherein at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the internucleotide linkages of the double-stranded oligonucleotide are independently chiral internucleotide linkages.

26. 26. The double-stranded oligonucleotide or composition of any one of claims 1-25, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% of the nucleotide units of the double-stranded oligonucleotide independently comprise a 2'-substitution.

27. The double stranded oligonucleotide or composition according to any one of claims 1 to 26, wherein the 2'-substitution of said oligonucleotide is 2'-F.

28. The double-stranded oligonucleotide or composition according to any one of claims 1 to 27, wherein the 2'-substitution of said oligonucleotide is 2'-OR1.

29. The double-stranded oligonucleotide or composition of any one of claims 1 to 28, wherein the 2'-substitution of the oligonucleotide is -L-, where L links C2 and C4 of the sugar unit.

30. 30. The double-stranded oligonucleotide or composition of any one of claims 1 to 29, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% of the nucleotide units of the double-stranded oligonucleotide do not contain a 2'-substitution.

31. 31. The double-stranded oligonucleotide or composition of any one of claims 1 to 30, wherein the guide strand comprises a target binding sequence that is perfectly complementary to a target sequence, the target binding sequence having a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases, each base being an optionally substituted adenine, cytosine, guanosine, thymine or uracil, and the target sequence comprises one or more allelic sites, wherein the allelic site is a SNP or mutation.

32. The double-stranded oligonucleotide or composition according to any one of claims 1 to 31, wherein the target sequence comprises two SNPs.

33. 33. The double-stranded oligonucleotide or composition of any one of claims 1 to 32, wherein the target sequence comprises an allelic site and the target binding sequence is perfectly complementary to the target sequence of a disease associated allele, but is not complementary to the sequence of an allele less associated with the disease.

34. the double-stranded oligonucleotide comprises a guide strand that binds to a transcription product of a target nucleic acid sequence, the alleles of which exist within a population, each allele containing a specific nucleotide signature sequence element that defines the allele relative to other alleles of the same target nucleic acid sequence; the base sequence of the guide strand is or comprises a sequence complementary to the characteristic sequence element that defines a particular allele; and The double-stranded oligonucleotide or composition of any one of claims 1 to 33, characterized in that the guide strand, when contacted with a cell containing a transcript of the target nucleic acid sequence, exhibits suppression of the transcript of the specific allele or the protein encoded thereby at a level that exceeds the level of suppression observed for another allele of the same nucleic acid sequence.

35. The passenger strand is an Sp backbone phosphorothioate chiral center between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; and Sp backbone phosphorothioate chiral center between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide The double-stranded oligonucleotide or composition according to any one of claims 1 to 34, comprising:

36. 36. A method for reducing the level and / or activity of a transcript or the protein encoded thereby, comprising administering to a cell expressing said transcript a double-stranded oligonucleotide or composition according to any one of claims 1 to 35, wherein the guide strand of the double-stranded oligonucleotide or composition comprises a target binding sequence that is perfectly complementary to a target sequence of said transcript.

37. 37. The method of claim 36, wherein the cell is an immune cell, a blood cell, a heart cell, a lung cell, a photoreceptor cell, a muscle cell, a liver cell, a kidney cell, a brain cell, a cell of the central nervous system or a cell of the peripheral nervous system.

38. 1. A method for allele-specific suppression of a transcript from a nucleic acid sequence, wherein a plurality of alleles are present in a population, each allele containing a specific nucleotide signature sequence element that defines said allele relative to other alleles of the same target nucleic acid sequence, comprising: Contacting a sample containing a transcript of the target nucleic acid sequence with the double-stranded oligonucleotide or composition according to any one of claims 1 to 35. Including, the guide strand of the double-stranded oligonucleotide or composition comprises a target binding sequence that is identical to or completely complementary to a target sequence in the nucleic acid sequence, the target sequence comprising a characteristic sequence element that defines a particular allele; A method wherein when the guide strand of the double-stranded oligonucleotide or composition is contacted with a cell containing transcripts of both the target allele and another allele of the same nucleic acid sequence, the transcript of the particular allele is suppressed at a level greater than the level of suppression observed for the other allele of the same nucleic acid sequence.

39. 1. A method for allele-specific suppression of a transcript from a nucleic acid sequence, wherein a plurality of alleles are present in a population, each allele containing a specific nucleotide signature sequence element that defines said allele relative to other alleles of the same target nucleic acid sequence, comprising: Administering to a subject comprising a transcript of said target nucleic acid sequence a double-stranded oligonucleotide or a composition according to any one of claims 1 to 35. Including, the guide strand of the double-stranded oligonucleotide or composition comprises a target binding sequence that is identical to or completely complementary to a target sequence in the nucleic acid sequence, the target sequence comprising a characteristic sequence element that defines a particular allele; A method wherein when the guide strand of the double-stranded oligonucleotide or composition is contacted with a cell containing transcripts of both the target allele and another allele of the same nucleic acid sequence, the transcript of the particular allele is suppressed at a level greater than the level of suppression observed for the other allele of the same nucleic acid sequence.

40. When the oligonucleotide or the oligonucleotide of the composition is contacted with a cell that contains transcripts of both the target allele and another allele of the same nucleic acid sequence, a) a level greater than that in the absence of said composition; b) a level of suppression that exceeds the level of suppression observed for another allele of the same nucleic acid sequence; or c) a level that is greater than the absence of the composition and that is greater than the level of suppression observed for another allele of the same nucleic acid sequence.

40. The method of any one of claims 36 to 39, wherein the method exhibits suppression of the transcript of said specific allele.

41. 41. The method of claim 40, wherein the cell is an immune cell, a blood cell, a heart cell, a lung cell, a photoreceptor cell, a muscle cell, a liver cell, a kidney cell, a brain cell, a cell of the central nervous system or a cell of the peripheral nervous system.

42. 40. The method of any one of claims 36-39, wherein repression of transcription of the specific allele is greater than in the absence of the composition and at a level that exceeds the level of repression observed for another allele of the same nucleic acid sequence.