Oligonucleotide compositions and methods thereof

Abstract

To provide designed oligonucleotides, compositions, and methods thereof.SOLUTION: Provided is an oligonucleotide composition comprising a first plurality of oligonucleotides of a particular oligonucleotide type defined by: (1) a base sequence; (2) a pattern of backbone linkages; (3) a pattern of backbone chiral centers; and (4) a pattern of backbone phosphorus modifications, the composition being chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, for oligonucleotides of the particular oligonucleotide type, the first plurality of oligonucleotides comprising one or more natural phosphate bridges.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 62 / 195,779, filed July 22, 2015, 62 / 236,847, filed October 2, 2015, and 62 / 331,960, filed May 4, 2016, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Oligonucleotides are useful for therapeutic, diagnostic, research, and nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) for therapeutic agents can be limited, for example, by their instability to extracellular and intracellular nucleases, toxicity, and / or their poor cellular penetration and distribution. There is a need for new and improved oligonucleotides and oligonucleotide compositions, for example, new antisense and siRNA oligonucleotides and oligonucleotide compositions. Summary of the Invention [Problem to be solved by the invention]

[0003] In particular, the present disclosure encompasses the recognition that structural elements of an oligonucleotide, e.g., base sequence, chemical modifications (e.g., sugar, base, and / or internucleotide bridge modifications and their patterns), and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide bridges) and / or their patterns), can significantly affect oligonucleotide properties, e.g., activity, toxicity, as may be mediated, for example, by protein binding characteristics, stability, etc. In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements (e.g., controlled chemical modifications and / or controlled backbone stereochemical patterns) confer unexpected properties, including, but not limited to, specific activity, toxicity, etc. In some embodiments, the present disclosure demonstrates that oligonucleotide properties, e.g., activity, toxicity, etc., can be tailored by chemical modifications (e.g., sugar, base, internucleotide bridge modifications, etc.), chiral structure (e.g., chiral internucleotide bridge stereochemistry and its pattern, etc.), and / or combinations thereof. [Means for solving the problem]

[0004] The present disclosure recognizes the challenge of providing oligonucleotide compositions and methods with reduced toxicity. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced toxicity. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced immune response. In some embodiments, the present disclosure recognizes that various toxicities induced by oligonucleotides are related to complement activation. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced complement activation. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced complement activation via an alternative pathway. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced complement activation via a traditional pathway. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced drug-induced vascular damage. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced injection site inflammation. In some embodiments, reduced toxicity can be assessed by one or more assays commonly known and performed by those skilled in the art (e.g., assessing levels of complete activation product, protein binding, etc., as described herein).

[0005] In some embodiments, the present disclosure demonstrates that oligonucleotide properties, such as activity and toxicity, can be tailored by chemical modification. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides having a common base sequence and comprising one or more modified sugar moieties, one or more natural phosphate bridges, or a combination thereof. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides having a common base sequence and comprising one or more modified internucleotide bridges, one or more modified sugar moieties, one or more natural phosphate bridges, or a combination thereof. In some embodiments, the first plurality of oligonucleotides has a wing-core-wing structure. In some embodiments, each wing region independently comprises one or more natural phosphate bridges and, optionally, one or more modified internucleotide bridges, and the core comprises one or more modified internucleotide bridges and, optionally, one or more natural phosphate bridges. In some embodiments, each wing region independently comprises one or more native phosphate bridges and one or more modified internucleotide bridges, and the core comprises one or more modified internucleotide bridges and no native phosphate bridges. In some embodiments, the wing comprises a modified sugar moiety. In some embodiments, the modified internucleotide bridge is a phosphorothioate. In some embodiments, the modified internucleotide bridge is a substituted phosphorothioate. In some embodiments, the modified internucleotide bridge has the structure of Formula I described in the present disclosure. In some embodiments, the modified sugar moiety is 2'-modified. In some embodiments, the 2'-modification is a 2'-OR 1 In some embodiments, such provided compositions have low toxicity. In some embodiments, provided compositions have low complement activation.

[0006] In some embodiments, the present disclosure provides oligonucleotide compositions with improved protein binding profiles, e.g., reduced detrimental protein binding and / or increased beneficial protein binding. In some embodiments, the present disclosure provides methods for improved delivery of oligonucleotide compositions, including providing oligonucleotide compositions with improved protein binding profiles. In some embodiments, the present disclosure demonstrates that protein binding by oligonucleotide compositions can be tuned by chemical modification, stereochemistry, or a combination thereof. In some embodiments, protein binding by oligonucleotide compositions can be tuned by incorporating modified internucleotide crosslinks. In some embodiments, increasing the proportion of modified internucleotide crosslinks increases binding of the oligonucleotide to a specific protein. In some embodiments, replacing one or more modified internucleotide crosslinks with native phosphate crosslinks decreases binding to a specific protein. In some embodiments, replacing one or more native phosphate crosslinks with modified internucleotide crosslinks increases binding to a specific protein. In some embodiments, certain chemical modifications increase protein binding to a specific protein. In some embodiments, certain chemical modifications decrease protein binding to a specific protein. In some embodiments, different chemical modifications of the same type confer different protein binding. For example, in some embodiments, 2'-MOE reduces protein binding (eg, at least in certain contexts such as sequence, stereochemistry, etc.) compared to 2'-OMe.

[0007] In particular, the present disclosure embraces the recognition that stereo-randomized oligonucleotide preparations contain multiple distinct chemical moieties that differ from one another, for example, in the stereochemical configuration of individual backbone chiral centers in the oligonucleotide chain. Without controlling the stereochemistry of the backbone chiral centers, stereo-randomized oligonucleotide preparations result in uncontrolled compositions containing undetermined amounts of oligonucleotide stereoisomers. While these stereoisomers may have the same base sequence and / or chemical modifications, they are at least different chemical moieties due to their different backbone stereochemistry and may have different properties (e.g., activity, toxicity, etc.), as demonstrated herein. In particular, the present disclosure provides chirality-controlled compositions that are or contain specific stereoisomers of a given oligonucleotide. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, its length, its backbone crosslinking pattern, and its backbone chiral center pattern. As understood in the art, in some embodiments, base sequence may refer to the identity and / or modification state of nucleoside residues in an oligonucleotide (e.g., the identity and / or modification state of the sugar and / or base elements relative to standard naturally occurring nucleotides, e.g., adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize with a particular complementary residue). In some embodiments, the present disclosure demonstrates that the improved properties (e.g., improved activity, reduced toxicity, etc.) achieved by including and / or arranging particular chiral structures in an oligonucleotide can be comparable to or better than those achieved by using chemical modifications, such as particular backbone crosslinks, residue modifications, etc. (e.g., by using particular types of modified phosphates (e.g., phosphorothioates, substituted phosphorothioates, etc.), sugar modifications (e.g., 2'-modifications, etc.), and / or base modifications (e.g., methylation, etc.).

[0008] In particular, the present disclosure demonstrates that stereochemistry can be used to tailor the toxicity of oligonucleotide compositions. In some embodiments, the present disclosure provides chirality-controlled oligonucleotide compositions that have lower toxicity compared to corresponding sterically random (or non-chirality-controlled) oligonucleotide compositions of oligonucleotides with the same base sequence and chemical modification. In some embodiments, chirality-controlled oligonucleotide compositions of oligonucleotides containing more Rp chiral internucleotide bridges have lower toxicity. In some embodiments, chirality-controlled oligonucleotide compositions of oligonucleotides containing one Rp chiral internucleotide bridge have higher toxicity compared to other chirality-controlled oligonucleotide compositions and / or corresponding sterically random oligonucleotide compositions of oligonucleotides with the same base sequence and chemical modification. In some embodiments, one Rp chiral internucleotide bridge is located in the center of the sequence. In some embodiments, chirality-controlled oligonucleotide compositions of oligonucleotides containing one or more Rp chiral internucleotide bridges at the 5'-end and / or 3'-end have lower toxicity. In some embodiments, chirality-controlled oligonucleotide compositions of oligonucleotides containing one or more natural phosphate bridges at the 5'-end and / or 3'-end have low toxicity. In some embodiments, the chiral internucleotide bridge has the structure of Formula I. In some embodiments, the chiral internucleotide bridge is a phosphorothioate bridge. In some embodiments, the chiral internucleotide bridge is a substituted phosphorothioate bridge.

[0009] In particular, the present disclosure recognizes that, in some embodiments, the properties (e.g., activity, toxicity, etc.) of an oligonucleotide can be adjusted by optimizing the pattern of chiral centers in the backbone, optionally in combination with adjusting / optimizing one or more other characteristics of the oligonucleotide (e.g., chemical modification, modification pattern, e.g., bridge pattern, nucleoside modification pattern, etc.). In some embodiments, the present disclosure recognizes and demonstrates that chemical modifications, e.g., modifications of nucleosides and internucleotide bridges, can confer enhanced properties. In some embodiments, the present disclosure demonstrates that a combination of chemical modifications and stereochemistry can confer unexpected and significantly improved properties (e.g., activity, toxicity, etc.). In some embodiments, a chemical combination (e.g., modifications of sugars, bases, and / or internucleotide bridges) combined with a stereochemical pattern provides oligonucleotides and compositions thereof with surprisingly enhanced properties, including reduced toxicity and better protein binding profiles. In some embodiments, provided oligonucleotide compositions comprising a first plurality of oligonucleotides have controlled chirality, wherein the first plurality of oligonucleotides comprises a combination of one or more 2'-modifications of sugar moieties, one or more natural phosphate bridges, and one or more chiral internucleotide bridges. In some embodiments, provided oligonucleotide compositions comprising a first plurality of oligonucleotides have controlled chirality, wherein the first plurality of oligonucleotides comprises a combination of one or more 2'-modifications of sugar moieties, one or more natural phosphate bridges, and one or more chiral internucleotide bridges, and the 5'- and / or 3'-terminal internucleotide bridges are chiral. In some embodiments, both the 5'- and 3'-terminal internucleotide bridges are chiral. In some embodiments, both the 5'- and 3'-terminal internucleotide bridges are chiral and Sp.In some embodiments, provided oligonucleotide compositions comprising a first plurality of oligonucleotides have controlled chirality, wherein the first plurality of oligonucleotides comprises a combination of one or more 2'-modifications of sugar moieties, one or more natural phosphate bridges, and one or more chiral internucleotide bridges and a stereochemical pattern of (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, where m>2. In some embodiments, the chiral internucleotide bridge has the structure of Formula I. In some embodiments, the chiral internucleotide bridge is a phosphorothioate bridge. In some embodiments, the chiral internucleotide bridge is a substituted phosphorothioate bridge.

[0010] In some embodiments, the present disclosure provides oligonucleotide compositions with reduced toxicity. In some embodiments, the present disclosure provides oligonucleotide compositions with improved protein binding profiles. In some embodiments, the present disclosure provides oligonucleotide compositions with improved binding to albumin. In some embodiments, the provided compositions have reduced toxicity and improved binding to specific, desirable proteins. In some embodiments, the provided compositions have reduced toxicity and improved binding to specific, desirable proteins. In some embodiments, the provided oligonucleotide compositions simultaneously provide the same level of stability and / or activity, or significantly improved stability and / or activity, e.g., better target cleavage patterns, better targeting efficiency, better target specificity, etc.

[0011] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently comprises one or more modified internucleotide bridges and optionally one or more natural phosphate bridges, and the core region independently comprises one or more modified internucleotide bridges; or Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified sugar moieties and the core region comprises one or more unmodified sugar moieties.

[0012] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and optionally one or more natural phosphate bridges; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0013] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0014] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising two or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0015] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising two wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; the wing region relative to the 5'-end of the core region comprises at least one modified internucleotide bridge followed by a natural phosphate bridge in the wing; and the wing region relative to the 3'-end of the core region comprises at least one modified internucleotide bridge followed by a natural phosphate bridge in the wing; Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0016] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one wing region and one core region; the first plurality of oligonucleotides have the same base sequence; the wing region has a length of at least two bases and comprises one or more modified internucleotide bridges and one or more natural phosphate bridges; the wing region is to the 5'-end of the core region and includes a natural phosphate bridge between two nucleosides at its 3'-end, or the wing region is to the 3'-end of the core region and includes a natural phosphate bridge between two nucleosides at its 5'-end; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0017] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising two wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; The wing region relative to the 5'-end of the core region contains a natural phosphate bridge between two nucleosides at its 3'-end; the wing region relative to the 3'-end of the core region contains a natural phosphate bridge between two nucleosides at its 5'-end; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0018] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; Each wing region independently comprises one or more modified internucleotide bridges and optionally one or more natural phosphate bridges, and the core region independently comprises one or more modified internucleotide bridges; and Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified sugar moieties and the core region comprises one or more unmodified sugar moieties.

[0019] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; Each wing region independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges, and each core region independently comprises one or more modified internucleotide bridges; and Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified sugar moieties and the core region comprises one or more unmodified sugar moieties.

[0020] In some embodiments, the present disclosure provides an oligonucleotide composition, comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides comprises: (1) have a common base sequence; (2) includes one or more wing regions and one core region; each wing region comprises at least one modified sugar moiety; and Oligonucleotide compositions are provided in which each core region comprises at least one unmodified sugar moiety.

[0021] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; wherein the composition is chirally controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0022] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers The present invention provides a chirality-controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type, characterized by: wherein the composition is a substantially pure preparation of a single oligonucleotide, wherein at least about 10% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0023] Among other things, the present disclosure recognizes that combinations of oligonucleotide building blocks (e.g., patterns of chemical modifications, backbone linkages, backbone chiral centers, and / or backbone phosphorus modifications) can confer properties such as surprisingly improved biological activity. In some embodiments, the present disclosure provides oligonucleotide compositions comprising predetermined levels of oligonucleotides comprising one or more wing regions and a common core region, wherein: each wing region independently has a length of two or more bases and independently optionally contains one or more chiral internucleotide linkages; The core regions independently have a length of two or more bases, and independently contain one or more chiral internucleotide linkages, and the common core region comprises: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers It has.

[0024] In particular, the present disclosure recognizes that combinations of oligonucleotide structural elements (e.g., patterns of chemical modifications, backbone crosslinks, backbone chiral centers, and / or backbone phosphorus modifications) can confer surprising and improved properties, such as low toxicity and / or desirable protein binding. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a predetermined amount of an oligonucleotide, the oligonucleotide comprising one or more wing regions and a common core region, where: each wing region independently has a length of two or more bases and independently optionally contains one or more chiral internucleotide bridges; The core regions independently have a length of 2 or more bases and independently contain one or more chiral internucleotide bridges, and the common core region is (1) Common base sequence and length; (2) a common pattern of skeletal cross-linking; and (3) Oligonucleotide compositions are provided that contain a common pattern of backbone chiral centers.

[0025] The wings and core can be defined by any structural element. In some embodiments, the wings and core are defined by nucleoside modifications, with the wings including nucleoside modifications that are not present in the core region. In some embodiments, the oligonucleotides in the provided compositions have a wing-core structure of nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a core-wing structure of nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a wing-core-wing structure of nucleoside modifications. In some embodiments, the wings and core are defined by modifications of the sugar moieties. In some embodiments, the wings and core are defined by modifications of the base moieties. In some embodiments, each sugar moiety in the wing region has the same 2'-modification that is not found in the core region. In some embodiments, each sugar moiety in the wing region has the same 2'-modification that is different from the sugar modification in the core region. In some embodiments, each sugar moiety in the wing region has the same 2'-modification, and the core region has no 2'-modification. In some embodiments, when two or more wings are present, each sugar moiety in one wing region has the same 2'-modification, but the common 2'-modification in a first wing region can be the same or different from the common 2'-modification in a second wing region. In some embodiments, the wings and core are defined by the pattern of internucleotide bridges in the backbone. In some embodiments, the wings contain certain internucleotide bridges and / or patterns of internucleotide bridges that are not found in the core. In some embodiments, the wing regions contain both modified internucleotide bridges and natural phosphate bridges. In some embodiments, the internucleotide bridge at the 5'-end of the wing relative to the 5'-end of the core region is a modified internucleotide bridge. In some embodiments, the internucleotide bridge at the 3'-end of the wing relative to the 3'-end of the core region is a modified internucleotide bridge. In some embodiments, the modified internucleotide bridge is a chiral internucleotide bridge.

[0026] In some embodiments, each wing comprises at least one chiral internucleotide linkage and at least one natural phosphate linkage. In some embodiments, each wing comprises at least one modified sugar moiety. In some embodiments, each wing sugar moiety is modified. In some embodiments, the wing sugar moiety is modified with a modification that is not present in the core region. In some embodiments, only the wing region has a modified internucleotide linkage at one or both of its termini. In some embodiments, only the wing region has a modified internucleotide linkage at its 5'-terminus. In some embodiments, only the wing region has a modified internucleotide linkage at its 3'-terminus. In some embodiments, only the wing region has modified internucleotide linkages at its 5'-terminus and 3'-terminus. In some embodiments, the wing is to the 5'-terminus of the core and only the wing has a modified internucleotide linkage at its 5'-terminus. In some embodiments, the wing is to the 5'-terminus of the core and only the wing has a modified internucleotide linkage at its 3'-terminus. In some embodiments, the wings are to the 5'-end of the core and only the wings have modified internucleotide linkages at both their 5'-end and 3'-end. In some embodiments, the wings are to the 3'-end of the core and only the wings have modified internucleotide linkages at their 5'-end. In some embodiments, the wings are to the 3'-end of the core and only the wings have modified internucleotide linkages at their 3'-end. In some embodiments, the wings are to the 3'-end of the core and only the wings have modified internucleotide linkages at both their 5'-end and 3'-end. In some embodiments, the modification(s) to the sugar moiety or internucleotide linkage or other modification in one wing can differ from the modifications in another wing.

[0027] In some embodiments, each internucleotide bridge in the core region is modified. In some embodiments, each internucleotide bridge in the core region is chiral. In some embodiments, the core region comprises a pattern of backbone chiral centers of (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, the pattern of backbone chiral centers in the core region is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, the core region comprises a pattern of backbone chiral centers of (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, where m>2. In some embodiments, the pattern of chiral centers in the backbone of the core region is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, where m > 2. In particular, in some embodiments, such patterns can provide or enhance controlled cleavage of target sequences (e.g., RNA sequences).

[0028] In some embodiments, the oligonucleotides in the provided compositions have a common pattern of backbone phosphorus modifications. In some embodiments, the provided compositions are chiral controlled oligonucleotide compositions in that they comprise predetermined levels of oligonucleotides of individual oligonucleotide types, where the oligonucleotide types are: 1) Nucleotide sequence; 2) pattern of skeletal bonding; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification is defined as:

[0029] As noted above and understood in the art, in some embodiments, the base sequence of an oligonucleotide may refer to the identity and / or modification state of the nucleoside residues (e.g., sugar and / or base moieties, relative to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) within the oligonucleotide, and / or the hybridization properties of such residues (i.e., the ability to hybridize with specific complementary residues).

[0030] In some embodiments, a particular oligonucleotide type is 1A) base identity; 1B) base modification patterns; 1C) Glycosylation patterns; 2) pattern of skeletal bonding; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification It may be defined by: Thus, in some embodiments, oligonucleotides of a particular type may share the same bases but differ in their patterns of base and / or sugar modifications, hi some embodiments, oligonucleotides of a particular type may share the same bases and patterns of base modifications (including, for example, the absence of base modifications), but differ in their patterns of sugar modifications.

[0031] In some embodiments, oligonucleotides of a particular type have the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same pattern of backbone linkages (e.g., natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., the pattern of chiral internucleotide linkage stereochemistry (Rp / Sp)), and the same pattern of backbone phosphorus modifications (e.g., -S - and -LR of formula I 1They are chemically identical in that they have the same pattern of modifications to the phosphorus atom of an internucleotide such as ribonucleotides.

[0032] In some embodiments, the present disclosure provides oligonucleotide compositions with controlled oligonucleotide chirality, including oligonucleotides containing multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotide bridges, particularly oligonucleotides containing multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotide bridges. In some embodiments, in stereorandom or racemic preparations of oligonucleotides, at least one chiral internucleotide linkage is formed with a diastereoselectivity of less than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral-controlled preparations of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral-controlled preparations of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 95:5. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 96:4. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 97:3. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 98:2. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 99:1.In some embodiments, the diastereoselectivity of a chiral internucleotide linkage within an oligonucleotide may be measured by a model reaction, e.g., the formation of a dimer under substantially the same or equivalent conditions, in which the dimer has the same internucleotide linkage as the chiral internucleotide linkage, the 5'-nucleoside of the dimer is the same as the nucleoside to the 5'-terminus of the chiral internucleotide linkage, and the 3'-nucleoside of the dimer is the same as the nucleoside to the 3'-terminus of the chiral internucleotide linkage.

[0033] In particular, the present disclosure provides oligonucleotide compositions and techniques for optimizing properties such as activity, toxicity, and the like. In some embodiments, the present disclosure provides methods for reducing the toxicity of oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for reducing immune responses associated with the administration of oligonucleotides and compositions thereof (i.e., methods of administering oligonucleotide compositions whereby an undesirable immune response to the oligonucleotides in the composition is reduced relative to that observed, e.g., with a reference composition of nucleotides of comparable or identical nucleotide sequences). In some embodiments, the present disclosure provides methods for reducing complement activation associated with the administration of oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for improving the protein binding profile of oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for increasing binding to specific proteins by oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for increasing binding to specific proteins by oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for increasing delivery of oligonucleotides and compositions thereof. In particular, the present disclosure embraces the recognition that optimal delivery of oligonucleotides to their targets involves, in some embodiments, a balance between binding of the oligonucleotide to specific proteins that can deliver the oligonucleotide to a desired location, and release of the oligonucleotide from the specific proteins that can appropriately release the oligonucleotide to perform a desired function, such as, for example, hybridization with these targets, cleavage of these targets, inhibition of translation, or modulation of the transcription process. As exemplified herein, the present disclosure recognizes, among other things, that improvements in oligonucleotide properties can be achieved through chemical modification and / or stereochemistry.

[0034] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, the method comprising: The present invention provides a method comprising administering an oligonucleotide composition comprising a first plurality of oligonucleotides having controlled chirality, wherein the first plurality of oligonucleotides is an improvement characterized by reduced toxicity compared to a reference oligonucleotide composition of the same common nucleotide sequence.

[0035] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, the method comprising: The present invention provides a method for administering an oligonucleotide composition, wherein each oligonucleotide in a plurality of oligonucleotides comprises one or more modified sugar moieties, wherein the composition is an improvement characterized by reduced toxicity compared to a reference oligonucleotide composition having the same common nucleotide sequence but lacking at least one of the one or more modified sugar moieties.

[0036] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, the method comprising: administering an oligonucleotide composition, wherein each oligonucleotide in the plurality of oligonucleotides comprises one or more natural phosphate bridges and one or more modified phosphate bridges; The method provides an improvement in which the oligonucleotide composition is characterized by reduced toxicity when tested in at least one assay measured using a corresponding reference composition except that the oligonucleotide does not contain a native phosphate bridge.

[0037] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, the method comprising: The present invention provides a method for administering an oligonucleotide composition, wherein each oligonucleotide in a plurality of oligonucleotides comprises one or more modified sugar moieties, wherein the composition is an improvement characterized by reduced toxicity compared to a reference oligonucleotide composition having the same common nucleotide sequence but lacking at least one of the one or more modified sugar moieties.

[0038] In some embodiments, the disclosure provides methods comprising administering to a subject an oligonucleotide composition comprising a first plurality of oligonucleotides, each having a common base sequence and comprising a modified sugar moiety, wherein the oligonucleotide composition is characterized by low toxicity when tested in at least one assay using an otherwise corresponding reference composition comprising a reference plurality of oligonucleotides having the same common base sequence but which do not comprise a modified sugar moiety.

[0039] In some embodiments, the present disclosure provides methods comprising administering to a subject an oligonucleotide composition comprising a first plurality of oligonucleotides, each having a common base sequence and comprising one or more natural phosphate bridges and one or more modified phosphate bridges, wherein the oligonucleotide composition is characterized by low toxicity when tested in at least one assay measured using an otherwise corresponding reference composition comprising a reference plurality of oligonucleotides having the same common base sequence but not comprising the natural phosphate bridges.

[0040] In some embodiments, the present disclosure provides methods comprising administering to a subject a chirality-controlled oligonucleotide composition, wherein the chirality-controlled oligonucleotide composition is characterized by low toxicity when tested in at least one assay measured using an otherwise corresponding reference oligonucleotide composition comprising an oligonucleotide having the same base sequence and comprising a different chirality-controlled or stereo-random oligonucleotide composition.

[0041] In some embodiments, the reduced toxicity is or comprises reduced complement activation. In some embodiments, the reduced toxicity comprises reduced complement activation. In some embodiments, the reduced toxicity is or comprises reduced complement activation. In some embodiments, the reduced toxicity comprises reduced complement activation via an alternative pathway.

[0042] In some embodiments, the oligonucleotides can induce a pro-inflammatory response. In some embodiments, the present disclosure provides compositions and methods for reducing inflammation. In some embodiments, the present disclosure provides compositions and methods for reducing a pro-inflammatory response. In some embodiments, the present disclosure provides methods for reducing inflammation at an injection site using provided compositions. In some embodiments, the present disclosure provides methods for reducing drug-induced vascular damage using provided compositions.

[0043] In some embodiments, the disclosure provides a method comprising administering a composition comprising a first plurality of oligonucleotides, wherein the composition exhibits reduced inflammation at the injection site compared to a reference composition comprising a plurality of oligonucleotides, wherein each of the reference plurality of oligonucleotides also share a common base sequence but differs from the first plurality of oligonucleotides by: the individual oligonucleotides in the reference oligonucleotide plurality differ from each other in stereochemical configuration; and / or At least some of the oligonucleotides in the reference plurality have a structure that is different from the structure represented by the oligonucleotides of the composition plurality; and / or The method is provided wherein at least some of the oligonucleotides in the reference plurality of oligonucleotides are structurally distinct in that they do not contain wing and core regions.

[0044] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, the method comprising: The improvement includes administering an oligonucleotide comprising a first plurality of oligonucleotides characterized by reduced inflammation at the injection site compared to a reference oligonucleotide composition of the same common nucleotide sequence.

[0045] In some embodiments, the disclosure provides a method comprising administering a composition comprising a first plurality of oligonucleotides, wherein the composition exhibits altered protein binding compared to a reference composition comprising a plurality of oligonucleotides, each of the reference plurality of oligonucleotides also having a common base sequence but different from the first plurality of oligonucleotides, wherein: the individual oligonucleotides in the reference oligonucleotide plurality differ from each other in stereochemical configuration; and / or At least some of the oligonucleotides in the reference plurality have a structure that is different from the structure represented by the oligonucleotides of the composition plurality; and / or The method is provided wherein at least some of the oligonucleotides in the reference plurality of oligonucleotides are structurally distinct in that they do not contain wing and core regions.

[0046] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, the method comprising: The improvement includes administering an oligonucleotide composition comprising a first plurality of oligonucleotides characterized by altered protein binding compared to a reference oligonucleotide composition of the same common nucleotide sequence.

[0047] In some embodiments, the disclosure provides a method comprising administering a composition comprising a first plurality of oligonucleotides, wherein the composition exhibits improved delivery compared to a reference composition comprising a plurality of oligonucleotides, wherein each of the reference plurality of oligonucleotides also share a common base sequence, but the first plurality of oligonucleotides share a common base sequence: the individual oligonucleotides in the reference oligonucleotide plurality differ from each other in stereochemical configuration; and / or At least some of the oligonucleotides in the reference plurality have a structure that is different from the structure represented by the oligonucleotides of the composition plurality; and / or The method is provided wherein at least some of the oligonucleotides in the reference plurality of oligonucleotides are structurally distinct in that they do not contain wing and core regions.

[0048] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, the method comprising: The method is an improvement comprising administering an oligonucleotide comprising a first plurality of oligonucleotides characterized by improved delivery compared to a reference oligonucleotide composition of the same common nucleotide sequence.

[0049] Generally, the properties of the oligonucleotide compositions described herein can be evaluated using any suitable assay. The relative toxicity and / or protein binding properties of different compositions (e.g., stereocontrolled and non-stereocontrolled, and / or different stereocontrolled compositions) are typically determined, preferably in the same assay, and in some embodiments, substantially simultaneously, and in some embodiments, with reference to historical results.

[0050] Those skilled in the art will be aware of and / or can readily develop assays suitable for particular oligonucleotide compositions. The present disclosure provides descriptions of particular assays, e.g., assays that may be useful for assessing one or more characteristics of the behavior of an oligonucleotide composition, e.g., complement activation, injection site inflammation, protein binding, etc.

[0051] For example, particular assays that may be useful in assessing the toxicity and / or protein binding properties of oligonucleotide compositions may include any of the assays described and / or exemplified herein.

[0052] definition Aliphatic: As used herein, the terms "aliphatic" or "aliphatic group" refer to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or polycyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbocycle," "alicyclic," or "cycloalkyl"). In some embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. Unless otherwise specified, an aliphatic group contains 1-10 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-4 aliphatic carbon atoms. In still other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in yet other embodiments, an aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic or bicyclic C3-C6 ring that is fully saturated or contains one or more unsaturated units, but is not aromatic, with one point of attachment to another molecule. 10refers to a hydrocarbon. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, with one point of attachment to another molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, and composites thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0053] Alkylene: The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n -, where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0054] Alkenylene: The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene group is a polymethylene group containing at least one double bond in which one or more hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0055] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human, at any stage of development. In some embodiments, "animal" refers to a non-human animal, 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 some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0056] Approximately: As used herein, the word "approximately" or "about" when describing a number is generally intended to include numbers within a range of 5%, 10%, 15%, or 20% in either direction (greater or less) of that number, unless otherwise stated or otherwise apparent from the context (unless such number is less than 0% or more than 100% of its possible values). In some embodiments, the use of the word "about" when describing dosage means ±5 mg / kg / day.

[0057] Aryl: The term "aryl," used alone or as part of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring structures in which at least one ring of the structure is aromatic and each ring of the structure contains 3 to 7 ring members, with a total of 5 to 14 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present invention, "aryl" refers to aromatic ring structures, including but not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, are also included within the scope of the term "aryl" herein.

[0058] Characteristic portion: As used herein, the phrase "characteristic portion" of a protein or polypeptide includes a stretch of amino acids, or a set of stretches of amino acids, that, taken together, are characteristic of the protein or polypeptide. Each such stretch will generally contain at least two amino acids. Moreover, one of skill in the art will recognize that at least 5, 10, 15, 20, or more amino acids are usually required to be characteristic of a protein. Generally, a characteristic portion shares at least one functional characteristic with the related intact protein, in addition to the specific sequence homology described above.

[0059] Signature Sequence: A "signature sequence" is a sequence that is found in all members of a family of polypeptides or nucleic acids and therefore can be used by those of skill in the art to define the members of that family.

[0060] Characteristic structural element: The term "characteristic structural element" refers to a distinct structural element (e.g., backbone structure, collection of pendant moieties, sequence element, etc.) found in all members of a family of polypeptides, small molecules, or nucleic acids, and thus can be used by those skilled in the art to define the members of that family.

[0061] 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 some embodiments, equivalent sets of conditions or circumstances are characterized by a plurality of substantially the same characteristics and one or a few altered characteristics. Those skilled in the art will recognize that sets of conditions are equivalent to one another when they are characterized by a sufficient number and type of substantially the same characteristics such that the differences in the results obtained or the phenomena observed under the different sets of conditions or circumstances can justify a reasonable conclusion that the sets of conditions are caused by or exhibit differences in those altered characteristics.

[0062] Dosage regimen: As used herein, a "dosage regimen" or "treatment regimen" refers to a set of unit doses (usually one or more) administered individually to a subject, usually separated by a period of time. In some embodiments, a given therapeutic agent has a required dosing regimen that may include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated from the other by a period of equal length; in some embodiments, a dosing regimen includes multiple doses and at least two different periods that separate the separate doses. In some embodiments, all doses within a dosing regimen are the same unit dosage. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is different from the first dosage amount. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is the same as the first dosage amount.

[0063] Equivalent Agents: Upon reading this disclosure, those skilled in the art will recognize that the scope of agents useful in the context of the present invention is not limited to those specifically mentioned or exemplified herein. Specifically, those skilled in the art will recognize that active agents typically have a structure consisting of a backbone and attached pendant moieties, and will therefore understand that simple modifications of such backbone and / or pendant moieties do not significantly alter the activity of the agent. For example, in some embodiments, replacement of one or more pendant moieties with groups of equivalent three-dimensional structure and / or chemical reactivity properties can produce substituted compounds or moieties equivalent to the parent reference compound or moiety. In some embodiments, addition or removal of one or more pendant moieties can produce substituted compounds equivalent to the parent reference compound. In some embodiments, for example, alteration of the backbone structure by addition or removal of a small number of bonds (usually no more than 5, 4, 3, 2, or 1 bond, and often only a single bond) can produce substituted compounds equivalent to the parent reference compound. In many embodiments, equivalent compounds can be synthesized, for example, by the methods shown in the following general reaction schemes, or modifications thereof, using readily available materials, reagents, and conventional or provided synthetic procedures. Modifications to these reactions that are known per se but not mentioned here can also be employed.

[0064] Equivalent Dosage: The term "equivalent dosage" is used herein to compare dosages of different pharmaceutically active agents that produce the same biological result. Doses of two different agents are considered "equivalent" to one another according to the present invention if they achieve the same level or degree of biological result. In some embodiments, equivalent dosages of different pharmaceuticals used according to the present invention are determined using the in vitro and / or in vivo assays described herein. In some embodiments, one or more lysosomotropic agents used according to the present invention are utilized at a dose equivalent to that of a reference lysosomotropic agent; in some embodiments, such a reference lysosomotropic agent is selected from the group consisting of small molecule allosteric activators (e.g., pyrazolpyrimidines), iminosugars (e.g., isofagomine), antioxidants (e.g., n-acetylcysteine), and regulators of cellular trafficking (e.g., Rab1a polypeptides).

[0065] Heteroaliphatic: The term "heteroaliphatic" refers to an aliphatic group in which one or more units selected from C, CH, CH, or CH are independently replaced by a heteroatom. In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0066] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl" or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 pi electrons shared in the cyclic arrangement; and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and the quaternized form of any basic nitrogen. 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. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the aromatic 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 or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "aromatic heterocycle," any of which includes optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently may be optionally substituted.

[0067] Heteroatom: The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, boron, selenium, or silicon (any oxidized form of nitrogen, boron, selenium, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or a heterocycle, such as N (in 3,4-dihydro-2H-pyrrolyl), NH (in pyrrolidinyl), or NR + (including the substitutable nitrogen of) in N-substituted pyrrolidinyl).

[0068] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used to refer to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, wherein the nitrogen is N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + It can be NR (as in N-substituted pyrrolidinyl).

[0069] A heterocycle can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, 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 radical" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl groups, heteroaryl groups, or aliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. The heterocyclyl ring can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions are independently optionally substituted.

[0070] Intraperitoneal: As used herein, the phrases "intraperitoneal administration" and "administered intraperitoneally" have their art-recognized meaning to refer to administration of a compound or composition into the peritoneal membrane of a subject.

[0071] In vitro: As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal, plant, and / or microorganism) but in an artificial environment, e.g., in a test tube or reactor, in cell culture, etc.

[0072] In vivo: As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, a plant, and / or a microorganism).

[0073] Lower alkyl: The term "lower alkyl" refers to a C 1~4It represents a straight or branched chain alkyl group. Illustrative lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0074] Lower haloalkyl: The term "lower haloalkyl" refers to a C alkyl group substituted with one or more halogen atoms. 1~4 It represents a straight or branched chain alkyl group.

[0075] Optionally substituted: As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted," with or without the word "may," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, where one or more positions in any given structure may be the same or different at each position. Combinations of substituents contemplated by the present invention are preferably those that result in the production of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are substantially unchanged when in a condition that allows for their preparation, detection, and, in certain embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0076] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group 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;-(CH2) 0~4 SR ○ ;R ○ may be substituted with (CH2) 0~4 Ph;R ○ may be substituted with (CH2) 0~4O(CH2) 0~1 Ph;R ○ CH=CHPh optionally substituted with R ○ may be substituted with (CH2) 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) 0~4 N(R ○ )C(O)NR ○ 2;N(R ○ )C(S)NR ○ 2;-(CH2) 0~4 N(R ○ )C(O)OR ○ ;-N(R ○ )N(R ○ )C(O)R ○ ;N(R ○ )N(R ○ )C(O)NR ○ 2;N(R ○ )N(R ○ )C(O)OR ○ ;-(CH2) 0~4 C(O)R ○ ;-C(S)R ○ ;-(CH2) 0~4 C(O)OR ○ ;-(CH2) 0~4 C(O)SR ○ ;(CH2) 0~4 C(O)OSiR ○ 3;-(CH2) 0~4 OC(O)R ○ ;-OC(O)(CH2) 0~4 SR-, SC(S)SR ○ ;-(CH2) 0~4 SC(O)R ○ ;-(CH2) 0~4 C(O)NR ○ 2;-C(S)NR ○ 2;-C(S)SR ○ ;-SC(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)NR ○ 2;-N(R ○ )S(O)2R ○ ;-N(OR ○ )R ○ ;-C(NH)NR ○ 2;-P(O)2R ○ ;P(O)R ○ 2;OP(O)R ○ 2;-OP(O)(OR ○ )2;-SiR○3;-(C 1~4 Linear or branched alkylene)ON(R ○ )2; or -(C 1~4 Linear or branched alkylene)C(O)ON(R ○ )2(in the formula, each R ○ are optionally substituted as described below and independently represent hydrogen, C 1~6 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, -CH2- (5- to 6-membered heteroaryl ring) or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two independently occurring R ○taken together with their intervening atoms form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as described below.

[0077] R ○ The appropriate monovalent substituent (or two independently occurring R ○ together with the intervening atoms to form a ring) are independently halogen, -(CH2) 0~2 R ● ,-(Halo R ● ), -(CH2) 0~2 OH, -(CH2) 0~2 OR ● , -(CH2) 0~2 CH(OR ● )2;O(Halo R ● ), -CN, -N3, -(CH2) 0~2 C(O)R ● , -(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR ● , -(CH2) 0~2 SR ● , -(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR ● , -(CH2) 0~2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. C(O)SR ● , -(C 1~4 Straight or branched chain alkylene)C(O)OR ● , or -SSR ● (In the formula, each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and, independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. ○ Suitable divalent substituents on a saturated carbon atom of include ═O and ═S.

[0078] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include: =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-(wherein each R * can be substituted with hydrogen, 1~6 aliphatic, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to adjacent substitutable carbon atoms of an "optionally substituted" group include: -O(CR * 2) 2~3 O-(wherein each R * can be substituted with hydrogen, 1~6 aliphatic, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0079] Suitable substituents on the aliphatic group of R* include halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ●2, or -NO2 (wherein each R ● is unsubstituted or, if preceded by "halo", is 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, or sulfur.

[0080] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -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 † ;(In the formula, each R † are independently selected from halogen, and the following may be substituted C 1~6 an aliphatic, unsubstituted -OPh, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, notwithstanding the above definition, two independently occurring R † together with their intervening atoms form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0081] Suitable substituents on an aliphatic group of R† are independently halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2 (wherein each R● is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and is independently a C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0082] Oral: As used herein, the phrases "oral administration" and "orally administered" refer to administration of a compound or composition by mouth and have their art-recognized meaning.

[0083] Parenteral: As used herein, the phrases "parenteral administration" and "parenterally administered" have their art-recognized meaning to refer to modes of administration that are not enteral or topical, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0084] 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.

[0085] As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., for buccal, sublingual, and systemic absorption, boluses, powders, granules, and pastes to be applied to the tongue; parenteral administration, e.g., as a sterile solution or suspension, or sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray to be applied to the skin, lungs, or oral cavity; intravaginally or rectally, e.g., as a pessary, cream, or foam; sublingually; ophthalmically; transdermally; or intranasally to the lungs and other mucosal surfaces.

[0086] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that, within sound medical judgment, are 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.

[0087] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the transport or transfer of a substance from one organ or body part 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, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; 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.

[0088] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to a salt of such a compound that is suitable for use in a pharmaceutical context, i.e., a salt that is suitable for use in contact with the tissues of humans and lower animals, within the scope of sound medical judgment, at a reasonable benefit / risk ratio, and without undue harmful effects, irritation, allergic response, etc. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxypropyl methylcellulose ... Examples of suitable salts include, but are not limited to, sulfonate, 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. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates and arylsulfonates having 1 to 6 carbon atoms, as appropriate.

[0089] Prodrug: Generally, a "prodrug," as the term is used herein, and as understood in the art, is an entity that, when administered to an organism, is metabolized in the body to deliver an active (e.g., therapeutic or diagnostic) agent of interest. Typically, such metabolism results in the removal of at least one "prodrug moiety," so that the active agent is produced. Various forms of "prodrugs" are well known in the art. Examples of such prodrug moieties include: a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, 42:309-396, edited by K. Widder, et al. (Academic Press, 1985); b)Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley, 2011); c)Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley, 2011); d)A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen; e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H. Bundgaard, p. 113-191 (1991); f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992); g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988); and h)Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984) reference.

[0090] Like other compounds described herein, prodrugs can be provided in any of a variety of forms, e.g., crystalline forms, salt forms, etc. In some embodiments, the prodrug is provided as a pharmaceutically acceptable salt thereof.

[0091] Protecting group: As used herein, the term "protecting group" is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd edition, John Wiley & Sons, 1999 (the entire text of which is incorporated herein by reference). It also includes protecting groups specifically adapted to nucleotides and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012 (the entire text of Chapter 2 is incorporated herein by reference). Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenanthyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 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 carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitro p-Toluenesulfonylmethylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethylcarbamate, diphenylmethylcarbamate, 2-methylthioethylcarbamate, 2-methylsulfonylethylcarbamate, 2-(p-toluenesulfonyl)ethylcarbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthio Phenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acryloxybenzyl 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 carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-Dimethoxycarbonylvinylcarbamate, 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'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methyl 1-methyl-1-cyclohexyl 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 carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamates, 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-nitrophenoxy)acetamide o-(methyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, 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-pyrrolin-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-ferrocenylmethylamino (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'-isopropylideneamine, 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-diphenyl Carbolic acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, 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), benzenesulfonamide, 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.

[0092] Suitable protected carboxylic acids 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.

[0093] Suitable hydroxyl protecting groups include methyl, methoxymethyl (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 (ME), and the like. M), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl] -4-Methoxypiperazin-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-fluoroethyl, 2,2,2-trimethyl- methylethyl, 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(benzoyloxyphenyl)methyl, 3-(imidazolinone) (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), dimethylthiazolyl Silyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate ester, benzoylformate ester, acetate ester, chloroacetate ester, dichloroacetate ester, trichloroacetate ester, trifluoroacetate ester, methoxyacetate ester, triphenylmethoxyacetate ester, phenoxyacetate ester, p-chlorophenoxyacetate Acid esters, 3-phenylpropionate esters, 4-oxopentanoate esters (levulinate esters), 4,4-(ethylenedithio)pentanoate esters (levulinoyl dithioacetal), pivalate esters, adamantate, crotonate esters, 4-methoxycrotonate esters, benzoate esters, p-phenylbenzoate esters, 2,4,6-trimethylbenzoate esters (mesitoate), alkyl methyl carbonates, 9-fluorenylmethyl carbonates (Fmoc), alkyl ethyl carbonates, 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-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoic acid ester, 4-azidobutyric acid ester, 4-nitro-4-methylpentanoic acid ester, o-(dibromomethyl)benzoic acid ester, 2-formylbenzenesulfonic acid ester, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyric acid Acid esters, 2-(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-naphthoic acid, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothionyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the 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 suitable boronic acid derivatives 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.

[0094] In some 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, benzoylformate, 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 some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting groups are selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.

[0095] In some embodiments, the phosphite protecting group is a group attached to an internucleotide phosphite bond throughout oligonucleotide synthesis. In some embodiments, the phosphite protecting group is attached to the sulfur atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is attached to the oxygen atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is attached to the oxygen atom of an internucleotide phosphate bond. In some embodiments, the phosphite 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, 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0096] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked to one another by peptide bonds). In some embodiments, a protein contains only naturally occurring amino acids. In some embodiments, a protein contains one or more non-naturally occurring amino acids (e.g., a moiety that forms one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues of a protein chain contain a non-amino acid moiety (e.g., a glycan, etc.). In some embodiments, a protein contains more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means. In some embodiments, a protein contains L-amino acids, D-amino acids, or both; in some embodiments, a protein contains one or more amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 20 amino acids, or less than about 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0097] Sample: As used herein, a "sample" refers to a specific organism or material obtained from. In some embodiments, a sample is a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, a biological sample includes biological tissue or biological fluid. In some embodiments, a biological sample is, or includes any one or more of, bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; suspended nucleic acid; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural fluid; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washings or lavage fluids, such as ductal lavage or bronchoalveolar lavage; aspirates; scrapings; bone marrow specimen; tissue biopsy specimen; surgical specimen; feces, other body fluids, secretions, and / or cells therefrom, etc. In some embodiments, a biological sample is, or includes, cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), etc. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), for example, by filtration through a semipermeable membrane. Such a "processed sample" can include, for example, nucleic acids or proteins obtained by extraction from a sample or processing a primary sample by techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components, etc. In some embodiments, the sample is an organism. In some embodiments, the sample is a plant. In some embodiments, the sample is an animal. In some embodiments, the sample is a human. In some embodiments, the sample is a non-human organism.

[0098] Stereochemical isomers: As used herein, the phrase "stereochemical isomers" refers to different compounds constructed of the same atoms connected by the same series of bonds but having different, incompatible three-dimensional structures. In some embodiments of the present invention, the provided chemical compositions may be or contain a pure synthesis of an individual stereochemical isomer of a compound; in some embodiments, the provided chemical compositions may be or contain a mixture of two or more stereochemical isomers of the compound. In certain embodiments, such mixtures contain equal amounts of different stereochemical isomers; in certain embodiments, such mixtures contain unequal amounts of at least two different stereochemical isomers. In some embodiments, the chemical composition may contain all diastereomers and / or enantiomers of the compound. In some embodiments, the chemical composition may contain less than all diastereomers and / or enantiomers of the compound. In some embodiments, if a specific enantiomer of a compound of the present invention is desired, it may be synthesized, for example, by asymmetric synthesis or derivatization with a chiral auxiliary, and the resulting diastereomeric mixture separated and the auxiliary cleaved to yield the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as amino, diastereomeric salts can be formed with a suitable optically active acid and resolved, for example, by fractional crystallization.

[0099] Subject: As used herein, the term "subject" or "subject" refers to any organism to which provided compounds or compositions are administered in accordance with the present invention, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject is suffering from and / or susceptible to a disease, disorder, and / or condition.

[0100] Substantial: As used herein, the term "substantial" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property under consideration. Those skilled in the biological arts will understand that biological and chemical phenomena rarely eschew completeness and / or completion or accomplishment or absolute results. Thus, the term "substantial" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0101] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0102] Susceptible (to a disease): An individual who is "susceptible to" a disease, disorder, and / or condition is one who is at a higher risk of developing the disease, disorder, and / or condition than members of the general community. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not be diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0103] Systemic: As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" have their art-recognized meaning of referring to administering a compound or composition so that it enters the recipient's entire body.

[0104] Tautomers: As used herein, the phrase "tautomers" is used to describe different isomers of an organic compound that are readily interchangeable. Tautomers can be characterized by the formal migration of a hydrogen atom or a proton accompanied by the shifting of a single bond and an adjacent double bond. In some embodiments, tautomers can result from proton tautomerism (i.e., relocation of a proton). In some embodiments, tautomers can result from valence tautomerism (i.e., rapid relocation of bonding electrons). All such tautomers are intended to be included within the scope of the present invention. In some embodiments, tautomers of a compound exist in mobile equilibrium with one another such that attempts to synthesize the separate substances would result in mixtures. In some embodiments, tautomers of a compound are separable and isolatable compounds. In some embodiments of the present invention, chemical compositions can be provided that are or contain a pure composition of a single tautomer of a compound. In some embodiments of the present invention, chemical compositions can be provided as mixtures of two or more tautomers of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomers; in certain embodiments, such mixtures contain different amounts of at least two tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain all tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain fewer than all tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain one or more tautomers of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the present invention, the tautomer is keto-enol tautomerism. One skilled in the chemical arts can "capture" (i.e., chemically modify to retain the "enol" form) keto-enol tautomerism using any suitable reagent known in the chemical arts to obtain an enol derivative that can be subsequently isolated using one or more suitable techniques known in the art. Unless otherwise indicated, the present invention encompasses all tautomers of the relevant compounds, whether in pure form or in mixtures with each other.

[0105] Therapeutic Agent: As used herein, the term "therapeutic agent" refers to any agent that, when administered to a subject, induces a therapeutic effect and / or a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, suppress, 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.

[0106] 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, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be recognized by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and others. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, suppresses, 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 some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0107] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, suppress, prevent, delay onset, reduce severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows only early signs of the disease, disorder, and / or condition, for example, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0108] Unsaturated: As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0109] Unit dose: As used herein, the phrase "unit dose" refers to an amount administered as a single dose of a pharmaceutical composition and / or in a physically discrete unit. In many embodiments, a unit dose contains a predetermined amount of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, one or more unit doses are administered to achieve the entire single dose. In some embodiments, administration of multiple unit doses is necessary or expected to be necessary to achieve the intended effect. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of a solid form, sustained-release formulation, or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose can be in a formulation containing any variety of ingredients in addition to the therapeutic agent. For example, an acceptable carrier (e.g., a pharmaceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, etc., as described below, can be included. It will be understood by those skilled in the art that in many embodiments, an appropriate total daily dosage of a particular therapeutic agent may comprise a fraction or multiple unit doses, and may be determined, for example, by an attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dosage level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active compound used; the specific composition used; the age, weight, health, sex, and diet of the subject; the number of administrations and the excretion rate of the specific compound used; the duration of treatment; drugs and / or additional therapies used in combination or simultaneously with the specific compound used, and similar factors well known in the medical arts.

[0110] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning of referring to an entity having a structure and / or activity actually found in a "normal" (as opposed to mutant, diseased, altered, etc.) state or context. Those of skill in the art will recognize that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0111] Nucleic Acid: The term "nucleic acid" includes any nucleotide, modified variants thereof, analogs thereof, and polymers thereof. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), or modified variants or analogs 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 made from nucleotide analogs and 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- or C-glycosides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages"). The terms encompass nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. The prefix poly- refers to nucleic acids containing from 2 to about 10,000 nucleotide monomer units, while the prefix oligo- refers to nucleic acids containing from 2 to about 200 nucleotide monomer units.

[0112] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more phosphate groups or phosphorus-containing internucleotide linkages. Naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but natural and unnatural base analogs are also understood to be included. Natural sugars are pentoses (five-carbon sugars), deoxyribose (forming DNA), or ribose (forming RNA), but natural and unnatural sugar analogs are also understood to be included. Nucleotides are linked via internucleotide linkages to form nucleic acids or polynucleotides. Many internucleotide linkages are known in the art (e.g., but not limited to, phosphate, phosphorothioate, boranophosphate, etc.). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothioates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. Other analogs (e.g., artificial nucleic acids, or components that can be incorporated into nucleic acids or artificial nucleic acids) include boranophosphate RNA, FANA, locked nucleic acids (LNA), morpholinos, peptide nucleic acids (PNAs), threose nucleic acids (TNAs), and glycol nucleic acids (GNAs). Those skilled in the art will recognize a variety of modified nucleotides or nucleotide analogs, including, for example, those described in any of the following: Gryaznov, S; Chen, J.-KJ Am. Chem. Soc. 1994, 116, 3143; Hendrix et al., 1997 Chem. Eur. J. 3:110; Hyrup et al., 1996 Bioorg. Med. Chem. 4:5; Jepsen et al., 2004 Oligo. 14:130-146; Jones et al., J. Org. Chem. 1993, 58, 2983; Koizumi et al., 2003 Nuc. Acids Res.12:3267-3273; Koshkin et al., 1998 Tetrahedron 54:3607-3630; Kumar et al., 1998 Bioo.Med.Chem.Let. 8:2219-2222; Lauritsen et al., 2002 Chem.Comm. 5:530-531; Lauritsen et al., 2003 Bioo.Med.Chem.Lett. 13:253-256; Mesmaeker et al., Angew.Chem., Int. Ed. Engl. 1994, 33, 226; Morita et al., 2001 Nucl.Acids Res.Supp. 1:241-242; Morita et al., 2002 Bioo.Med.Chem.Lett. 12:73-76; Morita et al., 2003 Bioo.Med.Chem.Lett.2211-2226;Nielsen et al., 1997 Chem.Soc.Rev.73;Nielsen et al., 1997 J.Chem.Soc.PerkinsTransl.1:3423-3433;Obika et al., 1997 Tetrahedron Lett.38(50):8735-8;Obika et al., 1998 Tetrahedron Lett.39:5401-5404;Pallan et al., 2012 Chem.Comm.48:8195-8197;Petersen et al., 2003 TRENDS Biotech.21:74-81;Rajwanshi et al., 1999 Chem.Commun.1395-1396;Schultz et al., 1996 Nucleic Acids Res.24:2966;Seth et al., 2009 J. Med.Chem.52:10-13;Seth et al., 2010 J.Med.Chem.53:8309-8318;Seth et al., 2010 J.Org.Chem.75:1569-1581;Seth et al., 2012 Bioo.Med.Chem.Lett.22:296-299;Seth et al., 2012 Mol.Ther-Nuc.Acids.1,e47;Seth,Punit P;Siwkowski,Andrew;Allerson,Charles R;Vasquez,Guillermo;Lee,Sam;Prakash,Thazha P;Kinberger,Garth;Migawa,Michael T;Gaus,Hans;Bhat,Balkrishen;et al. From Nucleic Acids Symposium Series(2008),52(1),553-554;Singh et al., 1998 Chem.Comm.1247-1248;Singh et al., 1998 J.Org.Chem.63:10035-39;Singh et al., 1998 J.Org.Chem.63:6078-6079;Sorensen 2003 Chem.Comm. 2130-2131; Ts'o et al., Ann.NY Acad.Sci. 1988, 507, 220; Van Aerschot et al., 1995 Angew.Chem.Int.Ed.Engl. 34:1338; Vasseur et al., J.Am.Chem.Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; or WO 2016 / 079181.

[0113] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar.

[0114] Sugar: The term "sugar" refers to a monosaccharide in closed and / or open form. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term also encompasses structural analogs used in place of normal sugar molecules, such as glycols, whose polymers form the backbone of nucleic acid analogs, glycol nucleic acids ("GNAs").

[0115] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar, such that the modified sugar mimics the spatial arrangement, electronic state, or some other physicochemical property of a sugar.

[0116] Nucleobase: The term "nucleobase" refers to a nucleic acid moiety that participates in hydrogen bonds to bind one nucleic acid strand to another complementary strand in a sequence-specific manner. Most naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase," e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobases mimic the spatial arrangement, electronic state, or some other physicochemical property of nucleobases and retain the hydrogen-bonding properties that allow one nucleic acid strand to bind to another complementary strand in a sequence-specific manner. In some embodiments, the modified nucleobases can pair with all five natural bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex.

[0117] Chiral Ligand: The term "chiral ligand" or "chiral auxiliary" refers to a moiety that is chiral and can be incorporated into a reactant so that a reaction can be carried out with a particular stereoselectivity.

[0118] Condensing Reagent: In a condensation reaction, the term "condensing reagent" refers to a reagent that activates a less reactive site, making it more susceptible to action with another reagent. In some embodiments, such another reagent is a nucleophile.

[0119] Blocking Group: The term "blocking group" refers to a group that masks the reactivity of a functional group that can be subsequently unmasked by removal of the blocking group. In some embodiments, a blocking group is a protecting group.

[0120] Moiety: The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often recognized as a chemical entity incorporated into or added to a molecule.

[0121] Solid support: The term "solid support" refers to any support that allows for the synthesis of nucleic acids. In some embodiments, the term refers to glass or polymer that is insoluble in the medium used in the reaction step to carry out nucleic acid synthesis and derivatize to introduce reactive groups. In some embodiments, the solid support is highly cross-linked polystyrene (HCP) or controlled pore glass (CPG). In some embodiments, the solid support is controlled pore glass (CPG). In some embodiments, the solid support is a composite support of controlled pore glass (CPG) and highly cross-linked polystyrene (HCP).

[0122] Linking moiety: The term "linking moiety" refers to any moiety that may be located between the terminal nucleotide and the solid support or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0123] DNA molecule: The term "DNA molecule" refers to either its single-stranded form or the polymeric form of double-helical deoxyribonucleotides (adenine, guanine, thymine, or cytosine). The term refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found in, inter alia, linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing specific double-stranded DNA molecule structures, the sequence may be described herein according to the convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA).

[0124] Coding sequence: A DNA "coding sequence" or "coding region" is a double-stranded DNA that is transcribed and translated into an in vivo polypeptide when placed under the control of appropriate expression control sequences. The boundaries of the coding sequence ("open reading frame" or "ORF") are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxylic) terminus. Coding sequences include, but are not limited to, prokaryotic sequences, cDNA from prokaryotic mRNA, genomic DNA sequences from prokaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence. The terms "non-coding sequence" or "non-coding region" refer to regions of a polynucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0125] Reading Frame: The term "reading frame" refers to one of six possible reading frames, three in each direction of a double-stranded DNA molecule. The reading frame used determines which codons are used to code for amino acids within the coding sequence of the DNA molecule.

[0126] Antisense: As used herein, an "antisense" nucleic acid molecule comprises a nucleotide sequence that is complementary to a protein-encoding "sense" nucleic acid, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence, or complementary to the coding strand of a gene. Thus, an antisense nucleic acid molecule can associate with a sense nucleic acid molecule through hydrogen bonds. In some embodiments, the antisense oligonucleotide is an oligonucleotide involved in RNase H-mediated cleavage; for example, the antisense oligonucleotide sequence-specifically hybridizes to a portion of the target mRNA, thus targeting the mRNA for cleavage by RNase H. In some embodiments, the antisense oligonucleotide can distinguish between wild-type and targeted mutant alleles. In some embodiments, the antisense oligonucleotide significantly participates in RNase H-mediated cleavage of the mutant allele, but to a much lesser extent in RNase H-mediated cleavage of the wild-type allele (e.g., does not significantly participate in RNase H-mediated cleavage of the targeted wild-type allele).

[0127] Wobble position: As used herein, "wobble position" refers to the third position of a codon. In some embodiments, a mutation in a DNA molecule within the wobble position of a codon results in a silent or conservative mutation at the amino acid level. For example, there are four codons that code for glycine, namely, GGU, GGC, GGA, and GGG; therefore, mutation of any nucleotide at any wobble position to another nucleotide selected from A, U, C, and G will not result in a change at the amino acid level of the encoded protein, and is therefore a silent substitution.

[0128] Silent Substitution: A "silent substitution" or "silent mutation" is one in which a nucleotide in a codon is changed but does not result in a change in the amino acid residue encoded by the codon. Examples include mutations in the first position of a particular codon, such as the codon "CGG," which when mutated to AGG still encodes Arg, as well as mutations in the third position of the codon.

[0129] Gene: As used herein, the terms "gene," "recombinant gene," and "gene construct" refer to a DNA molecule, or portion of a DNA molecule, that encodes a protein or portion thereof. The DNA molecule may include an open reading frame that encodes the protein (as an exon sequence) and may further include intron sequences. As used herein, the term "intron" refers to a DNA sequence present in a given gene that is not translated into protein, and in some, but not all, cases, found between exons. As is well known in the art, it may be desirable for a gene to be operably associated with (or may include) one or more promoters, enhancers, repressors, and / or other control sequences that regulate the activity or expression of the gene.

[0130] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes recombinant polynucleotides synthesized by reverse transcription of mRNA, from which intervening sequences (introns) have been removed.

[0131] Homology: "Homology" or "identity" or "similarity" refers to the sequence similarity between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence that can be positioned for comparison purposes. When the same position in the compared sequences is occupied by the same base, then the molecules are identical at that position; when the same site is occupied by the same or similar nucleic acid residue (e.g., similar in steric and / or electronic state), then the molecules can be said to be homologous (similar) at that position. Expression of percentage homology / similarity or identity represents a function of the number of identical or similar nucleic acids at a position shared by the compared sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with a sequence described herein. When comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of extra residues also reduces the identity and homology / similarity.

[0132] In some embodiments, the term "homology" describes a mathematically based comparison of sequence similarity used for identical genes with similar functions or motifs. The nucleic acid sequences described herein can be used as "query sequences" to perform searches against public databases, for example, to identify other family members, related sequences, or homologs. In some embodiments, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments, BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. In some embodiments, gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, to obtain gapped alignments for comparison purposes. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).

[0133] Identity: As used herein, "identity" means the percentage of identical nucleotide residues at corresponding positions in two or more sequences when the sequences are aligned for maximum sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to maximize the match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The well known Smith-Waterman algorithm may also be used to determine identity.

[0134] Heterologous: A "heterologous" region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not found in relation to the larger sequence. Thus, when a heterologous region encodes a mammalian gene, the gene may be located on a side of DNA that is not normally adjacent to the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a sequence in which the coding sequence itself is not found at all (e.g., a cDNA containing introns or synthetic sequences in which the genomic coding sequence has codons or motifs that differ from the native gene). Allelic variation or natural mutational events do not give rise to a non-homologous region of DNA as defined herein.

[0135] Transition Mutation: The term "transition mutation" refers to a base change in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T)) is replaced by another pyrimidine, or a purine (adenosine (A) or guanosine (G)) is replaced by another purine.

[0136] Transversion Mutation: The term "transversion mutation" refers to a base change in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T)) is replaced by a purine, or a purine (adenosine (A) or guanosine (G)) is replaced by a pyrimidine.

[0137] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotide monomers containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages," as further defined herein).

[0138] Oligonucleotides can be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" encompasses single-stranded oligonucleotides. Single-stranded oligonucleotides can have double-stranded regions, and double-stranded oligonucleotides can have single-stranded regions. Illustrative oligonucleotides include, but are not limited to, structural genes, genes including regulatory and terminal regions, viral or plasmid DNA, self-replicating systems such as single-stranded and double-stranded siRNAs and other RNA interference agents (RNAi agents or iRNA agents), shRNAs, 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.

[0139] The double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference are also referred to herein as siRNA, RNAi agent or iRNA agent.In some embodiments, these RNA interference-inducing oligonucleotides are associated with the cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC).In many embodiments, single-stranded and double-stranded RNAi agents are long enough that they can be cut by endogenous molecules, such as Dicer, to produce smaller oligonucleotides that can enter RISC mechanism and participate in the RISC-mediated cleavage of target sequence, such as target mRNA.

[0140] Oligonucleotides of the present invention can vary in length. In certain embodiments, oligonucleotides can range from about 2 to about 200 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides can range in length from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides. In some embodiments, the oligonucleotides are about 9 to about 39 nucleotides in length. In some embodiments, the oligonucleotides are at least 4 nucleotides in length. In some embodiments, the oligonucleotides are at least 5 nucleotides in length. In some embodiments, the oligonucleotides are at least 6 nucleotides in length. In some embodiments, the oligonucleotides are at least 7 nucleotides in length. In some embodiments, the oligonucleotides are at least 8 nucleotides in length. In some embodiments, the oligonucleotides are at least 9 nucleotides in length. In some embodiments, the oligonucleotides are at least 10 nucleotides in length. In some embodiments, the oligonucleotides are at least 11 nucleotides in length. In some embodiments, the oligonucleotides are at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a double-stranded complementary strand at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a double-stranded complementary strand at least 21 nucleotides in length.

[0141] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to a phosphorus-containing bond between nucleotide units of an oligonucleotide, and is synonymous above and herein with "intersugar linkage" and "phosphorus atom bridge." In some embodiments, the internucleotide linkage is a phosphodiester linkage found in natural DNA and RNA molecules. In some embodiments, the internucleotide linkage is a "modified internucleotide linkage" in which each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are selected from, but not limited to, =S, =Se, =NR', -SR', -SeR', -N(R')2, B(R')3, -S-, -Se-, and -N(R')- (wherein each R' is independently as defined and described below). In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a phosphodiester diester linkage. [ka] or a modified phosphorothioate triester bond. Those skilled in the art will appreciate that the internucleotide linkage may exist as an anion or cation at a given pH depending on the presence of an acid or base moiety in the linkage.

[0142] Unless otherwise specified, when used with an oligonucleotide sequence, each s, s1, s2, s3, s4, s5, s6 and s7 independently represents the following modified internucleotide linkages shown in Table 1 below:

[0143] Table 1. Illustrative modified internucleotide linkages [Table 1] TIFF0007818563000003.tif224166TIFF0007818563000004.tif96166

[0144] For example, (Rp,Sp)-ATsCs1GA has 1) a phosphorothioate internucleotide bond between T and C; ( [ka] ) and 2) between C and G [ka] Unless otherwise specified, the Rp / Sp designation preceding an oligonucleotide sequence refers to the configuration of the chiral phosphorus atoms of the internucleotide linkages sequentially from 5' to 3' of the oligonucleotide sequence. For example, in (Rp,Sp)-ATsCs1GA, the phosphorus of the "s" bond between T and C has the Rp configuration, and the phosphorus of the "s1" bond between C and G has the Sp configuration. In some embodiments, "all (Rp)" or "all (Sp)" is used to indicate that all chiral phosphorus atoms of the oligonucleotide have the same Rp or Sp configuration, respectively. For example, all (Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linking phosphorus atoms of the oligonucleotide have the Rp configuration; all (Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linking phosphorus atoms of the oligonucleotide have the Sp configuration.

[0145] Oligonucleotide Type: As used herein, the phrase "oligonucleotide type" refers to an oligonucleotide having a particular base sequence, backbone linkage pattern (i.e., internucleotide linkage pattern, e.g., phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., linked phosphorus stereochemistry pattern (Rp / Sp)), and backbone phosphorus modification pattern (e.g., "-XLR" in Formula I). 1 "Type" is used to define oligonucleotides having a pattern of "groups." Oligonucleotides of a commonly designated "type" are structurally identical to each other.

[0146] Those skilled in the art will recognize that the synthesis methods of the present invention provide a degree of control during the synthesis of an oligonucleotide chain, such that each nucleotide unit of the oligonucleotide chain can be designed and / or preselected 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 some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the linking phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the above structural features. The present invention provides compositions (e.g., chiral-controlled oligonucleotide compositions) comprising or consisting of a plurality of oligonucleotide molecules. In some embodiments, all such molecules are of the same type (i.e., structurally identical to each other). However, in many embodiments, the provided compositions typically contain multiple oligonucleotides of different types in predetermined relative amounts.

[0147] Chiral control: As used herein, "chiral control" refers to the ability to control the stereochemical representation of any chiral linking phosphorus within an oligonucleotide chain. The phrase "chiral controlled oligonucleotide" refers to an oligonucleotide that exists in a single diastereomeric form with respect to the chiral linking phosphorus. Chiral controlled oligonucleotides are prepared by chiral controlled oligonucleotide synthesis.

[0148] Chiral control oligonucleotide composition: As used herein, the phrase "chiral control oligonucleotide composition" refers to an oligonucleotide composition that contains a predetermined level of an individual oligonucleotide type. For example, in some embodiments, the chiral control oligonucleotide composition contains one oligonucleotide type. In some embodiments, the chiral control oligonucleotide composition contains a mixture of multiple oligonucleotide types. Illustrative chiral control oligonucleotide compositions are further described herein.

[0149] Chiral pure: The phrase "chiral pure" is used herein to describe chiral controlled oligonucleotide compositions in which the entire oligonucleotide exists in a single diastereoisomer with respect to the attached phosphorus.

[0150] Chiral homogeneous: As used herein, the phrase "chiral homogeneous" is used to describe an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the junction phosphorus. For example, an oligonucleotide in which all nucleotide units have Rp stereochemistry at the junction phosphorus is chiral homogeneous. Similarly, an oligonucleotide in which all nucleotide units have Sp stereochemistry at the junction phosphorus is chiral homogeneous.

[0151] Predetermined: Predetermined means deliberately selected, e.g., as opposed to randomly occurring or achieved. Those skilled in the art will understand upon reading this specification that the present invention provides new and surprising technology that allows for the selection of specific oligonucleotide types for formulation and / or inclusion in provided compositions, and further allows for the controlled formulation of the selected specific types, optionally in selected specific relative amounts, precisely so that provided compositions are formulated. Such provided compositions are "predetermined" as described herein. Compositions that may contain specific individual oligonucleotide types are not "predetermined" compositions because they were created through a process that, by chance, does not control the intentional creation of specific oligonucleotide types. In some embodiments, a predetermined composition is one that can be intentionally replicated (e.g., through the repetition of a controlled process).

[0152] Bound phosphorus: As defined herein, the phrase "bound phosphorus" is used to indicate that the particular phosphorus atom depicted is present in an internucleotide linkage, and that the phosphorus atom corresponds to the phosphorus atom of the phosphodiester internucleotide linkage that occurs in natural DNA and RNA. In some embodiments, the bound phosphorus atom is 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 some embodiments, the bound phosphorus atom is represented by P * In some embodiments, the linking phosphorus atom is chiral. In some embodiments, the chiral linking phosphorus atom is P of Formula I * is.

[0153] P modification: As used herein, the term "P modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In some embodiments, a P modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus. In some embodiments, the "P modification" ... 1 (Wherein X, L and R 1 are independently as defined and described herein and below).

[0154] Blockmir: As used herein, the term "blockmir" refers to an oligonucleotide chain in which the pattern of structural features that characterize each individual nucleotide unit is characterized by the presence of at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus linkage. A common structural feature refers to a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus. In some embodiments, the at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus linkage are referred to as a "block."

[0155] In some embodiments, the blockmirror is a "stereoblockmirror," e.g., at least two consecutive nucleotide units have the same stereochemistry at the linked phosphorus. Such at least two consecutive nucleotide units form a "stereoblockmirror." For example, (Rp,Sp)-ATsCs1GA is a stereoblockmirror because at least two consecutive nucleotide units, Ts and Cs1, have the same stereochemistry at the linked phosphorus (both Sp). In the same oligonucleotide, (Rp,Sp)-ATsCs1 forms a block, which is a stereoblock.

[0156] In some embodiments, a blockmir is a "P-modified blockmir," e.g., at least two consecutive nucleotide units have the same modification at the linked phosphorus. Such at least two consecutive nucleotide units form a "P-modified block." For example, (Rp,Sp)-ATsCsGA is a P-modified blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same P modification (i.e., both are phosphorothioate diester). In the same oligonucleotide of (Rp,Sp)-ATsCsGA, Ts and Cs form a block, which is a P-modified block.

[0157] In some embodiments, a blockmir is a "linked blockmir," e.g., at least two consecutive nucleotide units have the same stereochemistry and the same modification at the linking phosphorus. At least two consecutive nucleotide units form a "linked block." For example, (Rp,Rp)-ATsCsGA is a linked blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same stereochemistry (both Rp) and P modification (both phosphorothioate). In the same oligonucleotide of (Rp,Rp)-ATsCsGA, TsCs forms a block and is a linked block.

[0158] In some embodiments, the blockmir comprises one or more blocks independently selected from a stereoblock, a P-modified block, and a linking block, hi some embodiments, the blockmir is a stereoblockmir to one block, and / or a P-modified blockmir to another block, and / or a linking blockmir to yet another block. For example, (Rp,Rp,Rp,Rp,Rp,Sp,Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG is a stereoblockmir with respect to the stereoblock AsTsCsGsAs1 (all Rp's at the bound phosphorus) or Ts1Cs1Gs1 (all Sp's at the bound phosphorus), a P-modified blockmir with respect to the P-modified block AsTsCsGs (all s-bonds) or As1Ts1Cs1Gs1 (all s1-bonds), or a bonded blockmir with respect to the bonded block AsTsCsGs (all Rp's and all s-bonds at the bound phosphorus) or Ts1Cs1Gs1 (all Sp's and all s1-bonds at the bound phosphorus).

[0159] Altmer: As used herein, the term "altmer" refers to an oligonucleotide chain whose structural feature pattern characterizing each individual nucleotide unit is characterized by the absence of two consecutive nucleotide units in the oligonucleotide chain that share a particular structural feature in the internucleotide phosphorus bond. In some embodiments, an altmer is designed so that it contains a repeating pattern. In some embodiments, an altmer is designed so that it does not contain a repeating pattern.

[0160] In some embodiments, the altomer is a "stereoaltomer," e.g., no two consecutive nucleotide units have the same stereochemistry at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

[0161] In some embodiments, the altmer is a "P-modified altmer," e.g., no two consecutive nucleotide units have the same modification at the linked phosphorus, e.g., all (Sp)CAs1GsT, where each linked phosphorus has a different P modification than the others.

[0162] In some embodiments, the altmer is a "linked altmer," e.g., no two consecutive nucleotide units have the same stereochemistry or the same modification at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC.

[0163] Unimer: As used herein, the term "unimer" refers to an oligonucleotide chain whose structural feature pattern characterizes each individual nucleotide unit, in which all nucleotide units in the chain share at least one common structural feature at the internucleotide phosphorus linkage. A common structural feature refers to a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus.

[0164] In some embodiments, the unimer is a "stereounimer," e.g., all nucleotide units have the same stereochemistry at the linkage phosphorus, e.g., all (Sp)-CsAs1GsT, where all the linkages have Sp phosphorus.

[0165] In some embodiments, the unimer is a "P-modified unimer," e.g., all nucleotide units have the same modification at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, where all the internucleotide linkages are phosphorothioate diesters.

[0166] In some embodiments, the unimer is a "linked unimer," e.g., all nucleotide units have the same stereochemistry and the same modification at the linked phosphorus, e.g., all (Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, where all the internucleotide linkages are phosphorothioate diesters with an Sp linked phosphorus.

[0167] Gapmer: As used herein, the term "gapmer" refers to an oligonucleotide chain characterized in that at least one internucleotide phosphorus bond of the oligonucleotide chain is a phosphodiester bond, such as that found in natural DNA or RNA. In some embodiments, one or more internucleotide phosphorus bonds of the oligonucleotide chain are phosphodiester bonds, such as those found in natural DNA or RNA. For example, all (Sp)-CAs1GsT, in which the internucleotide bond between C and A is a phosphodiester bond.

[0168] Skipmer: As used herein, the term "skipmer" refers to a type of gapmer in which every other internucleotide phosphorus bond of the oligonucleotide strand is a phosphodiester bond, such as found in natural DNA or RNA, and every other internucleotide phosphorus bond of the oligonucleotide strand is a modified internucleotide bond, e.g., all (Sp)-AsTCs1GAs2TCs3G.

[0169] For purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, edited by CAS, Handbook of Chemistry and Physics, 67th Edition, 1986-87, inside cover.

[0170] The methods and structures described herein relating to the compounds and compositions of the present invention also apply to pharmaceutically acceptable acid or base addition forms and all stereoisomers of these compounds and compositions. [Brief explanation of the drawings]

[0171] [Figure 1] Example of dose response of C3a complement activation (measured by C3a) by oligonucleotides targeting human SOD1 in pooled serum (3 individual cynomolgus monkeys). 40 min incubation, 37°C.

[0172] [Figure 2]Example time course of 3Ca complement activation (measured by C3a) by SOD1 oligonucleotide in pooled serum (3 individual cynomolgus monkeys). Oligonucleotide concentration: 330 μg / mL; 37° C.

[0173] [Figure 3] Example time course of 3Ca complement activation (measured by C3a) by oligonucleotides targeting mouse ApoB in pooled serum (3 individual cynomolgus monkeys). Oligonucleotide concentration: 330 μg / mL; 37° C.

[0174] [Figure 4] Example time course of 3Ca complement activation (measured by C3a) by oligonucleotides targeting human HTT in pooled serum (3 individual cynomolgus monkeys). Oligonucleotide concentration: 330 μg / mL; 37° C.

[0175] [Figure 5] Example time course of Bb complement activation (measured by Bb) by oligonucleotides targeting human HTT in pooled serum (3 individual cynomolgus monkeys). Oligonucleotide concentration: 330 μg / mL; 37° C.

[0176] [Figure 6] Example of albumin binding by oligonucleotides targeting human HTT.

[0177] [Figure 7] Example of albumin binding by oligonucleotides targeting mouse ApoB.

[0178] [Figure 8] Example of albumin binding by oligonucleotides targeting human SOD1. DETAILED DESCRIPTION OF THE INVENTION

[0179] Synthetic oligonucleotides provide useful molecular tools in a variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research, and novel nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endonucleases and exonucleases. Thus, to circumvent these drawbacks, various synthetic counterparts have been developed. These synthetic counterparts include synthetic oligonucleotides containing chemical modifications, such as base modifications, sugar modifications, and backbone modifications, that render these molecules less susceptible to denaturation and improve other properties of the oligonucleotides. Chemical modifications can also have certain undesirable effects, such as increased toxicity. From a structural perspective, modifications to the internucleotide phosphate bridges can introduce chirality, and certain properties of oligonucleotides can be influenced by the conformation of the phosphorus atoms that form the oligonucleotide backbone. For example, in vitro studies have shown that the properties of antisense nucleotides, such as binding affinity, the sequence that specifically binds to complementary RNA, and stability against nucleases, are affected, inter alia, by the chirality of the backbone (e.g., the conformation of the phosphorus atom).

[0180] In particular, the present disclosure encompasses the recognition that structural elements of oligonucleotides, e.g., base sequence, chemical modifications (e.g., sugar, base, and / or internucleotide bridge modifications and their pattern), and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide bridges) and / or their pattern), can significantly affect properties such as, for example, activity and toxicity of the oligonucleotide, and can be modulated to tailor the properties of the oligonucleotide. In some embodiments, oligonucleotide properties can be modulated by optimizing chemical modifications (base, sugar, and / or internucleotide bridge modifications) and / or stereochemistry (pattern of backbone chiral centers).

[0181] In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements (e.g., controlled chemical modifications and / or controlled backbone stereochemical patterns) confer unexpected properties, including, but not limited to, those described herein. In some embodiments, provided compositions comprising oligonucleotides with chemical modifications (e.g., base modifications, sugar modifications, internucleotide bridge modifications, etc.) have improved properties, such as lower toxicity, improved protein binding profiles, or improved delivery. In some embodiments, provided oligonucleotides (e.g., a first plurality of oligonucleotides) in a provided composition comprise a base modification, a sugar modification, and / or an internucleotide bridge modification. In some embodiments, provided oligonucleotides comprise a base modification and a sugar modification. In some embodiments, provided oligonucleotides comprise a base modification and an internucleotide bridge modification. In some embodiments, provided oligonucleotides comprise a sugar modification and an internucleotide modification. In some embodiments, provided compositions comprise a base modification, a sugar modification, or an internucleotide bridge modification. Examples of chemical modifications (e.g., base modifications, sugar modifications, internucleotide bridge modifications, etc.) are widely known in the art, including, but not limited to, those described in this disclosure. In some embodiments, the modified base is a substituted A, T, C, G, or U. In some embodiments, the sugar modification is a 2'-modification. In some embodiments, the 2'-modification is a 2-F modification. In some embodiments, the 2'-modification is a 2'-OR 1 In some embodiments, the 2'-modification is 2'-OR 1 where R 1is an optionally substituted alkyl. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring containing 5-20 ring atoms, wherein one or more ring atoms are optionally and independently a heteroatom. Examples of ring structures are widely known in the art and are found, for example, in BNAs, LNAs, etc. In some embodiments, the provided oligonucleotides contain both one or more modified internucleotide bridges and one or more native phosphate bridges. In some embodiments, oligonucleotides and compositions thereof containing both modified internucleotide bridges and native phosphate bridges exhibit improved properties, such as activity and toxicity. In some embodiments, the modified internucleotide bridge is a chiral internucleotide bridge. In some embodiments, the modified internucleotide bridge is a phosphorothioate bridge. In some embodiments, the modified internucleotide bridge is a substituted phosphorothioate bridge. In particular, the present disclosure embraces the recognition that stereo-randomized oligonucleotide preparations contain multiple distinct chemical moieties that differ from one another, for example, in the stereochemical configuration of individual backbone chiral centers in the oligonucleotide chain. Without controlling the stereochemistry of the backbone chiral centers, stereo-randomized oligonucleotide preparations result in uncontrolled compositions containing undetermined quantities of oligonucleotide stereoisomers. While these stereoisomers may have the same base sequence, they are at least different chemical moieties due to their different backbone stereochemistry and may have different properties (e.g., activity, toxicity, etc.), as demonstrated herein. In particular, the present disclosure provides new compositions that are or contain specific stereoisomers of a given oligonucleotide. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, its length, its backbone crosslinking pattern, and its backbone chiral center pattern. As understood in the art, in some embodiments, base sequence may refer to the identity and / or modification state of the nucleoside residues in an oligonucleotide (e.g., the identity and / or modification state of the sugar and / or base elements relative to standard naturally occurring nucleotides, e.g., adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize with a specific complementary residue). In some embodiments, the oligonucleotides in the provided compositions contain sugar modifications, e.g., 2'-modifications, in the wing regions. In some embodiments, the oligonucleotides in the provided compositions contain a central (e.g., core) region that does not have sugar modifications. In some embodiments, the present disclosure provides oligonucleotide compositions comprising a predetermined level of individual types of oligonucleotides, where each type of oligonucleotide is chemically identical, e.g., they have the same base sequence, the same pattern of nucleoside modifications (modifications to the sugar and base moieties, if any), the same pattern of backbone chiral centers, and the same pattern of backbone phosphorus modifications.The present disclosure demonstrates, in particular, that individual stereoisomers of a particular oligonucleotide can exhibit different stability and / or activity (e.g., functional and / or toxicological properties) from one another. In some embodiments, the relevant improvements achieved by including and / or arranging specific chiral structures in an oligonucleotide can be comparable to or better than those achieved by using specific backbone crosslinks, residue modifications, etc. (e.g., by using specific types of modified phosphates (e.g., phosphorothioates, substituted phosphorothioates, etc.), sugar modifications (e.g., 2'-modifications, etc.), and / or base modifications (e.g., methylation, etc.). In particular, the present disclosure recognizes that, in some embodiments, the properties (e.g., activity, toxicity, etc.) of an oligonucleotide can be adjusted by optimizing the pattern of chiral centers in the backbone, optionally in combination with adjusting / optimizing one or more other features of the oligonucleotide (e.g., crosslinking pattern, nucleoside modification pattern, etc.). As illustrated by the various examples of the present disclosure, the provided controlled chirality oligonucleotide compositions can exhibit improved properties, such as lower toxicity, improved protein binding profiles, improved delivery, and the like.

[0182] In some embodiments, oligonucleotide properties can be adjusted by optimizing stereochemistry (the pattern of chiral centers in the backbone) and chemical modifications (modifications of bases, sugars, and / or internucleotide bridges). In particular, the present disclosure demonstrates that stereochemistry can further improve the properties of oligonucleotides containing chemical modifications. In some embodiments, the present disclosure provides oligonucleotide compositions in which the oligonucleotides contain nucleoside modifications, chiral internucleotide bridges, and natural phosphate bridges. For example, WV-1092 contains 2'-OMe modifications, phosphate, and phosphorothioate bridges in its 5'- and 3'-wing regions, and phosphorothioate bridges in its core region.

[0183] In some embodiments, the present disclosure provides oligonucleotide compositions with unexpectedly significant improvements in oligonucleotide properties. In some embodiments, the provided oligonucleotide compositions provide surprisingly low toxicity. In some embodiments, the provided oligonucleotide compositions provide surprisingly improved protein binding profiles. In some embodiments, the provided oligonucleotide compositions provide surprisingly enhanced delivery. In some embodiments, improvements in certain properties, such as low toxicity, improved protein binding profiles, and / or enhanced delivery, are achieved without sacrificing other properties, such as activity, specificity, etc. In some embodiments, the provided compositions provide low toxicity, improved protein binding profiles, and / or enhanced delivery, and improved activity, stability, and / or specificity (e.g., target specificity, cleavage site specificity, etc.). Improved activity (e.g., increased cleavage rate, target specificity, increased cleavage site specificity, etc.) includes, but is not limited to, those described in WO / 2014 / 012081 and WO / 2015 / 107425.

[0184] In some embodiments, the pattern of chiral centers in the backbone provides increased stability. In some embodiments, the pattern of chiral centers in the backbone provides surprisingly increased activity. In some embodiments, the pattern of chiral centers in the backbone provides increased stability and activity. In some embodiments, the pattern of chiral centers in the backbone provides surprisingly low toxicity. In some embodiments, the pattern of chiral centers in the backbone provides surprisingly low immune response. In some embodiments, the pattern of chiral centers in the backbone provides surprisingly low complement activation. In some embodiments, the pattern of chiral centers in the backbone provides surprisingly low complement activation by an alternative pathway. In some embodiments, the pattern of chiral centers in the backbone provides a surprisingly improved protein binding profile. In some embodiments, the pattern of chiral centers in the backbone provides surprisingly increased binding to a specific protein. In some embodiments, the pattern of chiral centers in the backbone provides surprisingly enhanced delivery. In some embodiments, the pattern of chiral centers in the backbone comprises or is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, the pattern of chiral centers in the backbone comprises or is (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, where m > 2. In some embodiments, the pattern of chiral centers in the backbone comprises or is (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, where n is 1, t > 1, and m > 2. In some embodiments, m > 3. In some embodiments, m > 4. In some embodiments, the pattern of chiral centers in the backbone comprises one or more achiral natural phosphate bridges. In some embodiments, the pattern of chiral centers in the backbone comprises (Sp) m (Rp) n , (Rp) n (Sp) m , (Np) t(Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m In some embodiments described herein, m is 1 to 50; n is 1 to 10; and t is 1 to 50. In some embodiments, the pattern of chiral centers in the backbone includes or is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, the pattern of chiral centers in the backbone includes or is (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) mwhere m>2. In some embodiments, the pattern of chiral centers in the backbone is a sequence comprising at least 5, 6, 7, 8, 9, or 10 or more consecutive (Sp) positions. In some embodiments, the pattern of chiral centers in the backbone is a sequence comprising at least 5 consecutive (Sp) positions. In some embodiments, the pattern of chiral centers in the backbone is a sequence comprising at least 8 consecutive (Sp) positions. In some embodiments, the pattern of chiral centers in the backbone is a sequence comprising at least 10 consecutive (Sp) positions. In some embodiments, the pattern of chiral centers in the backbone is a sequence comprising one (Rp) and all (Sp). In some embodiments, the pattern of chiral centers in the backbone is a sequence comprising one (Rp) and all (Sp) at or adjacent to the SNP position. In some embodiments, the pattern of chiral centers in the backbone is a sequence comprising one (Rp) and all (Sp), wherein the molecule has a wing-core-wing configuration. In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all-(Sp) sequence, where the molecule has a wing-core-wing configuration, where the wings at the 5'-end are 1-9 nt long, the core is 1-15 nt long, and the wings at the 3'-end are 1-9 nt long. In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all-(Sp) sequence, where the molecule has a wing-core-wing configuration, where the wings at the 5'-end are 5 nt long, the core is 1-15 nt long, and the wings at the 3'-end are 5 nt long. In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all-(Sp) sequence, where the molecule has a wing-core-wing configuration, where the wings at the 5'-end are 1-9 nt long, the core is 10 nt long, and the wings at the 3'-end are 1-9 nt long. In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all (Sp) sequence, where the molecule has a wing-core-wing configuration, with the wings at the 5' end being 5 nt long, the core being 10 nt long, and the wings at the 3' end being 5 nt long.In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all-(Sp) sequence, where the molecule has a wing-core-wing configuration, where the wing at the 5' end is 5 nt long, the core is 10 nt long, and the wing at the 3' end is 5 nt long, and at least one wing includes a nucleotide with a 2'-OMe modification. In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all-(Sp) sequence, where the molecule has a wing-core-wing configuration, where each wing includes at least one nucleotide with a 2'-OMe modification. In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all-(Sp) sequence, where the molecule has a wing-core-wing configuration, where each wing in both wings includes a 2'-OMe modification. In some embodiments, the pattern of chiral centers in the backbone includes one (Rp) and an all (Sp) sequence, where the molecule has a wing-core-wing configuration, where the wings at the 5' end are 5 nt long, the core is 10 nt long, the wings at the 3' end are 5 nt long, and each nucleotide in each wing has a 2'-OMe modification. In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing configuration, where the 5'-terminal wings of the molecule contain 4 to 8 nt, each having a 2'-OMe modification, and the 5'-terminal nt of the molecule has phosphorothioate in the Sp conformation; the core contains 8 to 12 nt, each of which is DNA(2'-H), and each of which has phosphorothioate in the Sp position except for one nt that has phosphorothioate in the Rp position; and the 3'-terminal wings of the molecule contain 4 to 8 nt, each of which has a 2'-OMe modification, and the 3'-terminal nt of the molecule has phosphorothioate in the Sp conformation.In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing configuration, where the 5'-terminal wing of the molecule comprises 6 nt, each having a 2'-OMe modification, and the 5'-terminal nt of the molecule has phosphorothioate in the Sp conformation; the core comprises 10 nt, each of which is DNA(2'-H), and each of which has phosphorothioate in the Sp position except for one nt that has phosphorothioate in the Rp position; and the 3'-terminal wing of the molecule comprises 6 nt, each of which has a 2'-OMe modification, and the 3'-terminal nt of the molecule has phosphorothioate in the Sp conformation.

[0185] In some embodiments, the present disclosure recognizes that chemical modifications, such as modifications of nucleosides and internucleotide linkages, can improve properties. In some embodiments, the present disclosure demonstrates that a combination of chemical modifications and stereochemistry can result in unexpected and significantly improved properties (e.g., biological activity, selectivity, etc.). In some embodiments, the combination of chemical modifications, such as modifications of sugars, bases, and / or internucleotide linkages, can result in stereochemical patterns, e.g., (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m In some embodiments, the oligonucleotide compositions provided are chiral and comprise one or more 2'-modifications of the sugar moiety, one or more natural phosphate linkages, one or more phosphorothioate linkages, and (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m(wherein m>2). In some embodiments, n is 1, t>1 and m>2. In some embodiments, m>3. In some embodiments, m>4.

[0186] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides have the same base sequence; The first plurality of oligonucleotides comprises one or more modified sugar moieties, or comprises one or more natural phosphate bridges and one or more modified internucleotide bridges.

[0187] In some embodiments, the first plurality of oligonucleotides comprises one or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise one or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise two or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise three or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise four or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise five or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise six or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise seven or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise eight or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise nine or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise ten or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 15 or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 20 or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 25 or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 30 or more modified sugar moieties.

[0188] In some embodiments, 5% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 10% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 15% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 20% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 25% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 30% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 35% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 40% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 45% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 50% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 55% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 60% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 65% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 70% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 75% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 80% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 85% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 90% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, 95% or more of the sugar moieties of the provided oligonucleotides are modified. In some embodiments, each sugar moiety of the provided oligonucleotides is modified.

[0189] In some embodiments, the first plurality of oligonucleotides comprises one or more natural phosphate bridges and one or more modified internucleotide bridges.

[0190] The provided oligonucleotides may contain a varying number of natural phosphate bridges. In some embodiments, the provided oligonucleotides contain no natural phosphate bridges. In some embodiments, the provided oligonucleotides contain one natural phosphate bridge. In some embodiments, the provided oligonucleotides contain two or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain three or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain four or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain five or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain six or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain seven or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain eight or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain nine or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain ten or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain fifteen or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain twenty or more natural phosphate bridges. In some embodiments, the provided oligonucleotides contain twenty-five or more natural phosphate bridges. In some embodiments, provided oligonucleotides contain 30 or more natural phosphate bridges.

[0191] In some embodiments, 5% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 10% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 15% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 20% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 25% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 30% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 35% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 40% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 45% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 50% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 55% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 60% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 65% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 70% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 75% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 80% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 85% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges.In some embodiments, 90% or more of the internucleotide bridges of a provided oligonucleotide are native phosphate bridges. In some embodiments, 95% or more of the internucleotide bridges of a provided oligonucleotide are native phosphate bridges.

[0192] The provided oligonucleotides may contain varying numbers of modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain one modified internucleotide bridge. In some embodiments, the provided oligonucleotides contain two or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain three or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain four or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain five or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain six or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain seven or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain eight or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain nine or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain ten or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain fifteen or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain twenty or more modified internucleotide bridges. In some embodiments, the provided oligonucleotides contain twenty-five or more modified internucleotide bridges. In some embodiments, a provided oligonucleotide comprises 30 or more modified internucleotide bridges.

[0193] In some embodiments, 5% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 10% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 15% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 20% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 25% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 30% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 35% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 40% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 45% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 50% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 55% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 60% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 65% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 70% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 75% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges.In some embodiments, 80% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 85% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 90% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 95% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, each internucleotide bridge in the provided oligonucleotides is a modified internucleotide bridge.

[0194] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently comprises one or more modified internucleotide bridges and optionally one or more natural phosphate bridges, and the core region independently comprises one or more modified internucleotide bridges; or Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified sugar moieties and the core region comprises one or more unmodified sugar moieties.

[0195] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; and Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified internucleotide bridges and optionally one or more natural phosphate bridges, and the core region independently comprises one or more modified internucleotide bridges.

[0196] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; and Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified sugar moieties and the core region comprises one or more unmodified sugar moieties.

[0197] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; Each wing region independently comprises one or more modified internucleotide bridges and optionally one or more natural phosphate bridges, and the core region independently comprises one or more modified internucleotide bridges; and Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified sugar moieties and the core region comprises one or more unmodified sugar moieties.

[0198] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; Each wing region independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges, and each core region independently comprises one or more modified internucleotide bridges; and Oligonucleotide compositions are provided in which each wing region independently comprises one or more modified sugar moieties and the core region comprises one or more unmodified sugar moieties.

[0199] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and optionally one or more natural phosphate bridges; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0200] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one or more wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0201] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising two wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0202] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising two wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; the wing region relative to the 5'-end of the core region comprises at least one modified internucleotide bridge followed by a natural phosphate bridge in the wing; and the wing region relative to the 3'-end of the core region comprises at least one modified internucleotide bridge followed by a natural phosphate bridge in the wing; Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0203] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising one wing region and one core region; the first plurality of oligonucleotides have the same base sequence; the wing region has a length of at least two bases and comprises one or more modified internucleotide bridges and one or more natural phosphate bridges; the wing region is to the 5'-end of the core region and comprises a modified internucleotide bridge between two nucleosides at its 3'-end, or the wing region is to the 3'-end of the core region and comprises a modified internucleotide bridge between two nucleosides at its 5'-end; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0204] In some embodiments, the present disclosure provides an oligonucleotide composition comprising: a first plurality of oligonucleotides comprising two wing regions and a core region; the first plurality of oligonucleotides have the same base sequence; each wing region independently has a length of two or more bases and independently comprises one or more modified internucleotide bridges and one or more native phosphate bridges; The wing region relative to the 5'-end of the core region comprises a modified internucleotide bridge between two nucleosides at its 3'-end; the wing region relative to the 3'-end of the core region comprises a modified internucleotide bridge between two nucleosides at its 5'-end; and Oligonucleotide compositions are provided in which the core regions independently have a length of two or more bases and independently comprise one or more modified internucleotide bridges.

[0205] An example of a composition is WV-1497, in which the core region is * A * A * G * G * G * C * A * C * A * G * and the wing region to the 5'-end of the core region is mG * mGmCmAmC, and the wing region to the 3'-end of the core region is mAmCmUmU * In some embodiments, the wing region comprises a modified internucleotide bridge between two nucleosides at its 3'-end. In some embodiments, the wing region relative to the 5'-end of the core region comprises a modified internucleotide bridge between two nucleosides at its 3'-end. For example, in WV-1497, mG * mGmCmAmC is the wing to the 5'-end of the core region ( * A * A * G * G * G * C * A * C* A * G * ), which contains at its 3'-end a modified internucleotide bridge between two nucleosides (mG * mGmC mAmC In some embodiments, the wing region comprises a modified internucleotide bridge between two nucleosides at its 5'-end. In some embodiments, the wing region relative to the 3'-end of the core region comprises a modified internucleotide bridge between two nucleosides at its 5'-end. For example, in WV-1497, mAmCmUmU * mC is the wing to the 3'-end of the core region ( * A * A * G * G * G * C * A * C * A * G * ), which contains at its 5'-end a modified internucleotide bridge between two nucleosides ( mAmC mUmU * mC).

[0206] In some embodiments, the first plurality of oligonucleotides comprises two wing regions and one core region. In some embodiments, the two wing regions are identical. In some embodiments, the two wing regions are different.

[0207] In some embodiments, the wing region comprises two or more modified internucleotide bridges. In some embodiments, the wing region comprises three or more modified internucleotide bridges. In some embodiments, the wing region comprises four or more modified internucleotide bridges. In some embodiments, the wing region comprises five or more modified internucleotide bridges. In some embodiments, the wing region comprises six or more modified internucleotide bridges. In some embodiments, the wing region comprises seven or more modified internucleotide bridges. In some embodiments, the wing region comprises eight or more modified internucleotide bridges. In some embodiments, the wing region comprises nine or more modified internucleotide bridges. In some embodiments, the wing region comprises ten or more modified internucleotide bridges. In some embodiments, the wing region comprises eleven or more modified internucleotide bridges. In some embodiments, the wing region comprises twelve or more modified internucleotide bridges. In some embodiments, the wing region comprises thirteen or more modified internucleotide bridges. In some embodiments, the wing region comprises fourteen or more modified internucleotide bridges. In some embodiments, the wing region comprises fifteen or more modified internucleotide bridges. In some embodiments, the wing region comprises two or consecutive modified internucleotide bridges. In some embodiments, the wing region comprises three or consecutive modified internucleotide bridges. In some embodiments, the wing region comprises four or consecutive modified internucleotide bridges. In some embodiments, the wing region comprises five or consecutive modified internucleotide bridges. In some embodiments, the wing region comprises six or consecutive modified internucleotide bridges. In some embodiments, the wing region comprises seven or consecutive modified internucleotide bridges. In some embodiments, the wing region comprises eight or consecutive modified internucleotide bridges. In some embodiments, the wing region comprises nine or consecutive modified internucleotide bridges.In some embodiments, the wing region comprises 10 or more consecutive modified internucleotide bridges. In some embodiments, the wing region comprises 11 or more consecutive modified internucleotide bridges. In some embodiments, the wing region comprises 12 or more consecutive modified internucleotide bridges. In some embodiments, the wing region comprises 13 or more consecutive modified internucleotide bridges. In some embodiments, the wing region comprises 14 or more consecutive modified internucleotide bridges. In some embodiments, the wing region comprises 15 or more consecutive modified internucleotide bridges. In some embodiments, each internucleotide bridge in the wing region is independently a modified internucleotide bridge.

[0208] In some embodiments, 5% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 10% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 15% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 20% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 25% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 30% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 35% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 40% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 45% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 50% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 55% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 60% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 65% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 70% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 75% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges.In some embodiments, 80% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 85% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 90% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 95% or more of the internucleotide bridges in the wing regions are modified internucleotide bridges. In some embodiments, each internucleotide bridge in the wing regions is a modified internucleotide bridge.

[0209] In some embodiments, the wing region comprises two or more native phosphate bridges. In some embodiments, the wing region comprises three or more native phosphate bridges. In some embodiments, the wing region comprises four or more native phosphate bridges. In some embodiments, the wing region comprises five or more native phosphate bridges. In some embodiments, the wing region comprises six or more native phosphate bridges. In some embodiments, the wing region comprises seven or more native phosphate bridges. In some embodiments, the wing region comprises eight or more native phosphate bridges. In some embodiments, the wing region comprises nine or more native phosphate bridges. In some embodiments, the wing region comprises ten or more native phosphate bridges. In some embodiments, the wing region comprises eleven or more native phosphate bridges. In some embodiments, the wing region comprises twelve or more native phosphate bridges. In some embodiments, the wing region comprises thirteen or more native phosphate bridges. In some embodiments, the wing region comprises fourteen or more native phosphate bridges. In some embodiments, the wing region comprises fifteen or more native phosphate bridges. In some embodiments, the wing region comprises two or consecutive native phosphate bridges. In some embodiments, the wing region comprises three or consecutive native phosphate bridges. In some embodiments, the wing region comprises four or more consecutive native phosphate bridges. In some embodiments, the wing region comprises five or more consecutive native phosphate bridges. In some embodiments, the wing region comprises six or more consecutive native phosphate bridges. In some embodiments, the wing region comprises seven or more consecutive native phosphate bridges. In some embodiments, the wing region comprises eight or more consecutive native phosphate bridges. In some embodiments, the wing region comprises nine or more consecutive native phosphate bridges. In some embodiments, the wing region comprises ten or more consecutive native phosphate bridges. In some embodiments, the wing region comprises eleven or more consecutive native phosphate bridges. In some embodiments, the wing region comprises twelve or more consecutive native phosphate bridges. In some embodiments, the wing region comprises thirteen or more consecutive native phosphate bridges. In some embodiments, the wing region comprises fourteen or more consecutive native phosphate bridges. In some embodiments, the wing region comprises fifteen or more consecutive native phosphate bridges. In some embodiments, each internucleotide bridge in the wing region is independently a native phosphate bridge.

[0210] In some embodiments, 5% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 10% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 15% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 20% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 25% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 30% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 35% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 40% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 45% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 50% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 55% or more of the internucleotide crosslinks in the provided oligonucleotides are naturally occurring phosphate bridges. In some embodiments, 60% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 65% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 70% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 75% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 80% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges. In some embodiments, 85% or more of the internucleotide bridges in the provided oligonucleotides are natural phosphate bridges.In some embodiments, 90% or more of the internucleotide bridges in the provided oligonucleotides are native phosphate bridges. In some embodiments, 95% or more of the internucleotide bridges in the wing regions are native phosphate bridges. In some embodiments, each internucleotide bridge in the wing regions is a native phosphate bridge.

[0211] In some embodiments, the core region comprises two or more modified internucleotide bridges. In some embodiments, the core region comprises three or more modified internucleotide bridges. In some embodiments, the core region comprises four or more modified internucleotide bridges. In some embodiments, the core region comprises five or more modified internucleotide bridges. In some embodiments, the core region comprises six or more modified internucleotide bridges. In some embodiments, the core region comprises seven or more modified internucleotide bridges. In some embodiments, the core region comprises eight or more modified internucleotide bridges. In some embodiments, the core region comprises nine or more modified internucleotide bridges. In some embodiments, the core region comprises ten or more modified internucleotide bridges. In some embodiments, the core region comprises eleven or more modified internucleotide bridges. In some embodiments, the core region comprises twelve or more modified internucleotide bridges. In some embodiments, the core region comprises thirteen or more modified internucleotide bridges. In some embodiments, the core region comprises fourteen or more modified internucleotide bridges. In some embodiments, the core region comprises fifteen or more modified internucleotide bridges. In some embodiments, the core region comprises two or consecutive modified internucleotide bridges. In some embodiments, the core region comprises three or consecutive modified internucleotide bridges. In some embodiments, the core region comprises 15 or consecutive modified internucleotide bridges. In some embodiments, each internucleotide bridge in the core region is independently a modified internucleotide bridge.

[0212] In some embodiments, 5% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 10% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 15% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 20% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 25% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 30% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 35% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 40% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 45% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 50% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 55% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 60% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 65% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 70% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 75% or more of the internucleotide bridges in the provided oligonucleotides are modified internucleotide bridges.In some embodiments, 80% or more of the internucleotide bridges of the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 85% or more of the internucleotide bridges of the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 90% or more of the internucleotide bridges of the provided oligonucleotides are modified internucleotide bridges. In some embodiments, 95% or more of the internucleotide bridges of the core region are modified internucleotide bridges. In some embodiments, each internucleotide bridge of the core region is a modified internucleotide bridge.

[0213] In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide composition comprising: (1) Common base sequence and length; (2) common backbone cross-linking patterns; and (3) Pattern of chiral centers in a common skeleton a first plurality of oligonucleotides defined by having The compositions provide chirality-controlled oligonucleotide compositions that are substantially pure preparations of single oligonucleotides, in that a given level of oligonucleotides in the composition have a common base sequence and length, a common backbone crosslinking pattern, and a common pattern of backbone chiral centers.

[0214] In some embodiments, the common base sequence and length may be referred to as the common base sequence. In some embodiments, oligonucleotides with a common base sequence may have the same pattern of nucleoside modifications, such as sugar modifications, base modifications, etc. In some embodiments, the nucleoside modification pattern may be represented by a combination of position and modification. For example, for WV-1092, the nucleoside bridge pattern, from the 5'-end to the 3'-end, is 5x2'-OMe (2'-OMe modification on the sugar moiety)-DNA (no 2'-modification on the sugar moiety)-5x2'-OMe. In some embodiments, the backbone bridge pattern includes the position and type (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.) of each internucleotide bridge. For example, for WV-1092, the backbone bridge pattern is 1xPS (phosphorothioate)-3xPO (phosphate)-11xPS-3xPO-1xPS. The chiral center pattern of the backbone of an oligonucleotide can be indicated by the combination of the stereochemistry (Rp / Sp) of the bridge phosphorus from 5' to 3'. For example, WV-1092 has the pattern 1S-3PO(phosphate)-8S-1R-2S-3PO-1S. In some embodiments, all non-chiral bridges (e.g., PO) can be omitted. As exemplified above, the position of non-chiral bridges can be obtained, for example, from the bridge pattern of the backbone.

[0215] In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide composition comprising: (1) Common base sequence and length; (2) common backbone cross-linking patterns; and (3) Pattern of chiral centers in a common skeleton a first plurality of oligonucleotides of a particular oligonucleotide type characterized by: The compositions provide oligonucleotide compositions with controlled chirality in that oligonucleotides of a particular oligonucleotide type are enriched compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0216] As will be understood by those skilled in the art, stereorandom or racemic preparations of oligonucleotides are typically prepared by non-stereoselective and / or low-stereoselective coupling of nucleotide monomers without the use of any chiral auxiliary, chiral modifying reagent, and / or chiral catalyst. In some embodiments, in a substantially racemic (or non-chiral controlled) preparation of oligonucleotides, all or most of the coupling steps are not chirally controlled, in that the coupling steps are not specifically performed to enhance stereoselectivity. An example of a substantially racemic preparation of oligonucleotides is a phosphorothioate oligonucleotide prepared by a process well known in the art, in which a phosphite triester is sulfurized using either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) in commonly used phosphoramidite oligonucleotide synthesis. In some embodiments, a substantially racemic preparation of oligonucleotides results in a substantially racemic oligonucleotide composition (or a non-chirally controlled oligonucleotide composition). In some embodiments, at least one coupling of nucleotide monomers has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least two couplings of nucleotide monomers have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least three couplings of nucleotide monomers have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1.In some embodiments, the coupling of at least four of the nucleotide monomers has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, the coupling of at least five of the nucleotide monomers has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least one internucleotide linkage has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least two internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least three internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least four internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least five internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 60:40.In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 70:30. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 80:20. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 91:9. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 92:8. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 93:7. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 94:6. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 95:5. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 96:4. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 97:3. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 98:2. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 99:1. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 91:9. In some embodiments, at least one coupling has a diastereoselectivity of less than about 90:10. In some embodiments, at least two couplings have a diastereoselectivity of less than about 90:10. In some embodiments, at least three couplings have a diastereoselectivity of less than about 90:10. In some embodiments, at least four couplings have a diastereoselectivity of less than about 90:10.In some embodiments, at least five couplings have a diastereoselectivity of less than about 90:10. In some embodiments, at least one internucleotide linkage has a diastereoselectivity of less than about 90:10. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least three internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least four internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least five internucleotide linkages have a diastereoselectivity of less than about 90:10.

[0217] As will be appreciated by those skilled in the art, in some embodiments, the diastereoselectivity of coupling or conjugation can be assessed by the diastereoselectivity of dimer formation under the same or equivalent conditions, where the dimers have the same 5'- and 3'-nucleosides and internucleotide linkages. For example, WV-1092 mG * SmGmCmAmC * SA * SA * S G * SG * SG * SC * SA * SC * RA * SG * SmAmCmUmU * The diastereoselectivity of the underlined coupling or bond in SmC can be assessed by coupling two G moieties under the same or equivalent conditions (e.g., monomers, chiral auxiliaries, solvents, activators, temperature, etc.).

[0218] In some embodiments, the present disclosure provides chirality-controlled (and / or stereochemically pure) oligonucleotide compositions comprising: (1) Common base sequence and length; (2) common backbone cross-linking patterns; and (3) Pattern of chiral centers in a common skeleton a first plurality of oligonucleotides defined by having The compositions provide chirality-controlled oligonucleotide compositions that are substantially pure preparations of single oligonucleotides, in that at least about 10% of the oligonucleotides in the composition have a common base sequence and length, a common backbone crosslinking pattern, and a common pattern of backbone chiral centers.

[0219] In some embodiments, the present disclosure provides oligonucleotide compositions in which the chirality of a first plurality of oligonucleotides is controlled, in that the composition is enriched for one type of oligonucleotide compared to a substantially racemic preparation of the same oligonucleotide. (1) Common base sequence and length; (2) common backbone cross-linking patterns; and (3) Pattern of chiral centers in a common skeleton The present invention provides an oligonucleotide composition in which the chirality of a first plurality of oligonucleotides is controlled in that one type of oligonucleotide sharing the chirality is enriched relative to a substantially racemic preparation of the same oligonucleotide.

[0220] In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide composition comprising: (1) Common base sequence and length; (2) common backbone cross-linking patterns; and (3) Pattern of chiral centers in a common skeleton a first plurality of oligonucleotides of a particular oligonucleotide type characterized by: The compositions provide oligonucleotide compositions with controlled chirality in that oligonucleotides of a particular oligonucleotide type are enriched compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0221] In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have the same structure.

[0222] In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides of an oligonucleotide type are identical.

[0223] In some embodiments, the chiral controlled oligonucleotide composition is a substantially pure preparation of an oligonucleotide type, wherein any oligonucleotides in the composition that are not of the oligonucleotide type are impurities form the preparation process of said oligonucleotide type (optionally after certain purification procedures).

[0224] In some embodiments, at least about 20% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 25% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 30% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 35% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 40% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 45% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 50% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 55% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 60% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 65% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 70% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, at least about 75% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 80% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 85% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 90% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 92% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 94% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 95% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, more than about 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, the purity of the chiral-controlled oligonucleotide composition of oligonucleotides can be expressed as the percentage of oligonucleotides in the composition that have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0225] In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are identical.

[0226] In some embodiments, the oligonucleotides in the provided compositions share a common backbone phosphorus modification pattern. In some embodiments, the common base sequence is the base sequence of a certain type of oligonucleotide. In some embodiments, the provided compositions are chirality-controlled oligonucleotide compositions in that the compositions comprise a predetermined amount of a first plurality of oligonucleotides of each type of oligonucleotide, and the types of oligonucleotides are: (1) Nucleotide sequence; (2) skeletal cross-linking pattern; (3) the pattern of chiral centers in the backbone; and (4) Defined by the pattern of backbone phosphorus modifications.

[0227] As noted above and understood in the art, in some embodiments, the base sequence of an oligonucleotide may refer to the identity and / or modification state of the nucleoside residues (e.g., sugar and / or base moieties, relative to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) within the oligonucleotide, and / or the hybridization properties of such residues (i.e., the ability to hybridize with specific complementary residues).

[0228] In some embodiments, a particular oligonucleotide type is 1A) base identity; 1B) base modification patterns; 1C) Glycosylation patterns; 2) pattern of skeletal bonding; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification It may be defined by: Thus, in some embodiments, oligonucleotides of a particular type may share the same bases but differ in their patterns of base and / or sugar modifications, hi some embodiments, oligonucleotides of a particular type may share the same bases and patterns of base modifications (including, for example, the absence of base modifications), but differ in their patterns of sugar modifications.

[0229] In some embodiments, oligonucleotides of a particular type have the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same pattern of backbone linkages (e.g., natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., the pattern of chiral internucleotide linkage stereochemistry (Rp / Sp)), and the same pattern of backbone phosphorus modifications (e.g., -S - and -LR of formula I 1They are identical in that they have a pattern of internucleotide modifications to the phosphorus atom, such as α- and β-nucleotides.

[0230] In some embodiments, the purity of a chiral controlled oligonucleotide composition of an oligonucleotide type is expressed as the percentage of oligonucleotides in the composition that are of that oligonucleotide type. In some embodiments, at least about 10% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 20% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 30% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 40% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 50% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 60% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 70% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 80% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 90% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 92% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type, hi some embodiments, at least about 94% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type.In some embodiments, at least about 95% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 96% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 97% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 98% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 99% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type.

[0231] In some embodiments, the purity of a chiral controlled oligonucleotide composition can be controlled by the stereoselectivity of each coupling step in its preparation process. In some embodiments, the coupling step has 60% stereoselectivity (e.g., diastereoselectivity) (60% of the new internucleotide linkages formed by the coupling step have the desired stereochemistry). After such a coupling step, the new internucleotide linkages formed may be said to have 60% purity. In some embodiments, each coupling step has at least 60% stereoselectivity. In some embodiments, each coupling step has at least 70% stereoselectivity. In some embodiments, each coupling step has at least 80% stereoselectivity. In some embodiments, each coupling step has at least 85% stereoselectivity. In some embodiments, each coupling step has at least 90% stereoselectivity. In some embodiments, each coupling step has at least 91% stereoselectivity. In some embodiments, each coupling step has at least 92% stereoselectivity. In some embodiments, each coupling step has at least 93% stereoselectivity. In some embodiments, each coupling step has at least 94% stereoselectivity. In some embodiments, each coupling step has at least 95% stereoselectivity. In some embodiments, each coupling step has at least 96% stereoselectivity. In some embodiments, each coupling step has at least 97% stereoselectivity. In some embodiments, each coupling step has at least 98% stereoselectivity. In some embodiments, each coupling step has at least 99% stereoselectivity. In some embodiments, each coupling step has substantially 100% stereoselectivity. In some embodiments, the coupling step has substantially 100% stereoselectivity, wherein all products of the coupling step, detectable by analytical methods (e.g., NMR, HPLC, etc.), have the desired stereoselectivity.

[0232] Among other things, the present disclosure recognizes that combinations of oligonucleotide building blocks (e.g., patterns of chemical modifications, backbone linkages, backbone chiral centers and / or backbone phosphorus modifications) can confer properties such as surprisingly improved biological activity.

[0233] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a predetermined amount of a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising one or more wing regions and a common core region, wherein: each wing region independently has a length of two or more bases and independently optionally contains one or more chiral internucleotide bridges; The core regions independently have a length of 2 or more bases and independently contain one or more chiral internucleotide bridges, and the common core region is (1) Common base sequence and length; (2) a common pattern of skeletal cross-linking; and (3) Oligonucleotide compositions are provided that contain a common pattern of backbone chiral centers.

[0234] In some embodiments, the wing regions comprise structural features not present in the core region. In some embodiments, the wings and core can be defined by any component, such as base modification (e.g., methylated / unmethylated, 1-methylated / 2-methylated, etc.), sugar modification (e.g., modified / unmodified, 2'-modified / another type of modification, one type of 2'-modification / another type of 2'-modification, etc.), backbone linkage type (e.g., phosphate / phosphorothioate, phosphorothioate / substituted phosphorothioate, etc.), backbone chiral center stereochemistry (e.g., all Sp / all Rp, (SpRp) repeat / all Rp, etc.), backbone phosphorus modification type (e.g., s1 / s2, s1 / s3, etc.), etc.

[0235] In some embodiments, the wings and core are defined by nucleoside modifications, where the wings include nucleoside modifications that are not in the core region. In some embodiments, the wings and core are defined by sugar modifications, where the wings include sugar modifications that are not in the core region. In some embodiments, the sugar modifications are 2'-modifications. In some embodiments, the sugar modifications are 2'-OR 1 In some embodiments, the sugar modification is 2'-MOE. In some embodiments, the sugar modification is 2'-OMe. Further exemplary sugar modifications are described in this disclosure. In some embodiments, the wings and core are defined by internucleotide bridges, where the wings include types of internucleotide bridges (e.g., natural phosphate bridges, certain modified internucleotide bridges, etc.) that are not present in the core region. In some embodiments, the wings and core are defined by internucleotide bridges, where the wings have a pattern of backbone bridges that differs from the backbone bridges of the core.

[0236] In some embodiments, the oligonucleotides in the provided compositions have a wing-core structure (hemimer). In some embodiments, the oligonucleotides in the provided compositions have a wing-core structure with nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a core-wing structure (another type of hemimer). In some embodiments, the oligonucleotides in the provided compositions have a core-wing structure with nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a wing-core-wing structure (gapmer). In some embodiments, the oligonucleotides in the provided compositions have a wing-core-wing structure with nucleoside modifications. In some embodiments, the wings and core are defined by modifications of the sugar moieties. In some embodiments, the wings and core are defined by modifications of the base moieties. In some embodiments, each sugar moiety in the wing region has the same 2'-modification that is not found in the core region. In some embodiments, each sugar moiety in the wing region has the same 2'-modification that is different from any sugar modification in the core region. In some embodiments, the core region has no sugar modifications. In some embodiments, each sugar moiety in the wing region has the same 2'-modification, and the core region has no 2'-modification. In some embodiments, when more than one wing is present, each wing is defined by its own modification. In some embodiments, each wing has its own characteristic sugar modification. In some embodiments, each wing has the same characteristic sugar modification that distinguishes it from the core. In some embodiments, each wing sugar moiety has the same modification. In some embodiments, each wing sugar moiety has the same 2'-modification. In some embodiments, each sugar moiety within a wing region has the same 2'-modification, but the common 2'-modification within a first wing region can be the same or different from the common 2'-modification within a second wing region. In some embodiments, each sugar moiety within a wing region has the same 2'-modification, and the common 2'-modification within a first wing region is the same as the common 2'-modification within a second wing region.In some embodiments, each sugar moiety within a wing region has the same 2'-modification, and the common 2'-modification within a first wing region is different from the common 2'-modification within a second wing region.

[0237] In some embodiments, the chirality-controlled (and / or stereochemically pure) preparations provided are antisense oligonucleotides (e.g., chiromersen). In some embodiments, the chirality-controlled (and / or stereochemically pure) preparations provided are siRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions provided are of oligonucleotides that may be antisense oligonucleotides, antagomirs, microRNAs, pre-microRNs, anti-mirs, supermirs, ribozymes, Ul adapters, RNA activators, RNAi agents, decoy oligonucleotides, triplex-forming oligonucleotides, aptamers, or adjuvants. In some embodiments, the chirality-controlled oligonucleotide compositions are of antisense oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of antagomir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of microRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of pre-microRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of anti-mir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of supermir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide composition is a ribozyme oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is a Ul adaptor oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an RNA activator oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an RNAi agent oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is a decoy oligonucleotide.In some embodiments, the chirality-controlled oligonucleotide composition is a triplex-forming oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an aptamer oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an adjuvant oligonucleotide.

[0238] In some embodiments, chirally controlled (and / or stereochemically pure) preparations provided are of oligonucleotides containing one or more modified backbone linkages, bases, and / or sugars.

[0239] In some embodiments, provided oligonucleotides contain one or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain two or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain three or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain four or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain five or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain 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 chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain five or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain six or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 7 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 8 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 9 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 10 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 11 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 12 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 13 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 14 or more chiral modified phosphate linkages.In some embodiments, the oligonucleotide types provided contain 15 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 16 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 17 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 18 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 19 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 20 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 21 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 22 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 23 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 24 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 25 or more chiral modified phosphate linkages.

[0240] In some embodiments, provided oligonucleotides contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral modified phosphate linkages. Exemplary such chiral modified phosphate linkages are described above and herein. In some embodiments, provided oligonucleotides contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral modified phosphate linkages in the Sp configuration.

[0241] In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 80% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 85% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 90% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 91% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 92% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 93% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 94% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 95% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 96% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 97% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 98% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 99% stereochemical purity.

[0242] In some embodiments, the chiral modified phosphate linkage is a chiral phosphorothioate linkage, i.e., a phosphorothioate internucleotide linkage. In some embodiments, the provided oligonucleotide contains at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral phosphorothioate internucleotide linkages. In some embodiments, all chiral modified phosphate linkages are chiral phosphorothioate internucleotide linkages. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide linkages of the provided oligonucleotide are in the Sp configuration. In some embodiments, at least about 10% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 20% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 30% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 40% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 50% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 60% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 70% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 80% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Sp configuration.In some embodiments, at least about 90% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are in the Sp configuration. In some embodiments, at least about 95% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are in the Sp configuration.

[0243] In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 10% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 20% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 30% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 40% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 50% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 60% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 70% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 80% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 90% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation. In some embodiments, at least about 95% of the chiral phosphorothioate internucleotide crosslinks in the provided oligonucleotides are in the Rp conformation.

[0244] In some embodiments, less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide crosslinks in a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 10% of the chiral phosphorothioate internucleotide crosslinks in a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 20% of the chiral phosphorothioate internucleotide crosslinks in a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 30% of the chiral phosphorothioate internucleotide crosslinks in a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 40% of the chiral phosphorothioate internucleotide crosslinks in a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 50% of the chiral phosphorothioate internucleotide crosslinks in a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 60% of the chiral phosphorothioate internucleotide crosslinks of a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 70% of the chiral phosphorothioate internucleotide crosslinks of a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 80% of the chiral phosphorothioate internucleotide crosslinks of a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 90% of the chiral phosphorothioate internucleotide crosslinks of a provided oligonucleotide are in the Rp conformation. In some embodiments, less than about 95% of the chiral phosphorothioate internucleotide crosslinks of a provided oligonucleotide are in the Rp conformation. In some embodiments, a provided oligonucleotide has only one Rp chiral phosphorothioate internucleotide crosslink. In some embodiments, a provided oligonucleotide has only one Rp chiral phosphorothioate internucleotide crosslink, and all internucleotide crosslinks are chiral phosphorothioate internucleotide crosslinks.

[0245] In some embodiments, the chiral phosphorothioate internucleotide linkages are chiral phosphorothioate diester linkages. In some embodiments, the chiral phosphorothioate internucleotide linkages are each independently a chiral phosphorothioate diester linkage. In some embodiments, the internucleotide linkages are each independently a chiral phosphorothioate diester linkage. In some embodiments, the internucleotide linkages are each independently a chiral phosphorothioate diester linkage and only one linkage is Rp.

[0246] In some embodiments, the chiral (and / or stereochemically pure) preparations provided are of oligonucleotides containing one or more modified bases. In some embodiments, the chiral (and / or stereochemically pure) preparations provided are of oligonucleotides that do not contain modified bases. Exemplary such modified bases are described above and herein.

[0247] In some embodiments, the oligonucleotides of the provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 1 natural phosphate linkage. In some embodiments, the oligonucleotides of the provided compositions comprise at least 2 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 3 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 4 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 5 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 6 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 7 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 8 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 9 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions contain at least 10 natural phosphate linkages.

[0248] In some embodiments, the oligonucleotides of the provided compositions contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise one natural phosphate linkage. In some embodiments, the oligonucleotides of the provided compositions comprise two natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise three natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise four natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise five natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise six natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise seven natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise eight natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise nine natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise ten natural phosphate linkages.

[0249] In some embodiments, the oligonucleotides of the provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 2 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 3 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 4 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 5 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 6 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 7 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 8 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 9 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions contain at least 10 consecutive natural phosphate linkages.

[0250] In some embodiments, the oligonucleotides of the provided compositions comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 2 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 3 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 4 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 5 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 6 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 7 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 8 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 9 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 10 consecutive natural phosphate linkages.

[0251] In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 8 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 9 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 10 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 11 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 12 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 13 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 14 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 15 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 16 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 17 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 18 bases.In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 19 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 20 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 21 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 22 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 23 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 24 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 25 bases. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of oligonucleotides having a common base sequence of at least 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 bases.

[0252] In some embodiments, provided compositions comprise oligonucleotides comprising one or more residues modified at the sugar moiety. In some embodiments, provided compositions comprise oligonucleotides comprising one or more residues modified at the 2'-position of the sugar moiety (hereinafter referred to as "2'-modifications"). Examples of such modifications are described above and herein and include, but are not limited to, 2'-OMe, 2'-MOE, 2'-LNA, 2'-F, FRNA, FANA, S-cEt, etc. In some embodiments, provided compositions comprise oligonucleotides comprising one or more residues that are 2'-O-methoxyethyl (2'-MOE)-modified residues. In some embodiments, provided compositions comprise oligonucleotides that do not comprise a 2'-modification. In some embodiments, provided compositions are oligonucleotides that do not comprise a 2'-MOE residue. That is, in some embodiments, provided oligonucleotides are not MOE-modified. Further examples of sugar modifications are described in this disclosure.

[0253] In some embodiments, provided oligonucleotides have a wing-core or core-wing general motif (hemimers, also commonly designated XY or YX, respectively). In some embodiments, provided oligonucleotides have a wing-core-wing general motif (gapmers, also commonly designated XYX). ​​In some embodiments, each wing region independently comprises one or more residues with a particular modification, which is absent from the core "Y" portion. In some embodiments, each wing region independently comprises one or more residues with a particular nucleoside modification, which is absent from the core "Y" portion. In some embodiments, each wing region independently comprises one or more residues with a particular base modification, which is absent from the core "Y" portion. In some embodiments, each wing region independently comprises one or more residues with a particular sugar modification, which is absent from the core "Y" portion. Examples of sugar modifications are well known in the art. In some embodiments, the sugar modification is a modification selected from those described in US9006198, which sugar modifications are incorporated herein by reference. Further examples of sugar modifications are described in this disclosure. In some embodiments, each wing comprises one or more residues having a 2'-modification that is not present in the core moiety. In some embodiments, the 2'-modification is a 2'-OR 1 and R 1 are defined and described in this disclosure.

[0254] In some embodiments, provided oligonucleotides have a wing-core motif designated as XY, or a core-wing motif designated as YX, where the residues in the "X" portion are sugar-modified residues of a particular type and the residues in the core "Y" portion are not sugar-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core-wing motif designated as XYX, where the residues in each "X" portion are sugar-modified residues of a particular type and the residues in the core "Y" portion are not sugar-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core motif designated as XY, or a core-wing motif designated as YX, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core motif designated as XY, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a core-wing motif designated YX, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core-wing motif designated XYX, where the residues in each "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core motif designated XY, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are 2'-deoxyribonucleosides. In some embodiments, provided oligonucleotides have a core-wing motif designated YX, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are 2'-deoxyribonucleosides.In some embodiments, provided oligonucleotides have a wing-core-wing motif represented as XYX, where the residue of each "X" portion is a particular type of 2'-modified residue and the residue of the core "Y" portion is a 2'-deoxyribonucleoside. In some embodiments, provided oligonucleotides have a wing-core-wing motif represented as XYX, where the residue of each "X" portion is a particular type of 2'-modified residue and the residue of the core "Y" portion is a 2'-deoxyribonucleoside. For example, in some embodiments, provided oligonucleotides have a wing-core-wing motif represented as XYX, where the residue of each "X" portion is a 2'-MOE-modified residue and the residue of the core "Y" portion is not a 2'-MOE-modified residue. In some embodiments, provided oligonucleotides have a wing-core-wing motif designated XYX, where the residues of each "X" portion are 2'-MOE-modified residues and the residues of the core "Y" portion are 2'-deoxyribonucleosides. Those of skill in the art will understand that in the context of such XY, YX and / or XYX motifs, all such 2'-modifications described above and herein are contemplated.

[0255] In some embodiments, the wings are 1 base or more in length. In some embodiments, the wings are 2 bases or more in length. In some embodiments, the wings are 3 bases or more in length. In some embodiments, the wings are 4 bases or more in length. In some embodiments, the wings are 5 bases or more in length. In some embodiments, the wings are 6 bases or more in length. In some embodiments, the wings are 7 bases or more in length. In some embodiments, the wings are 8 bases or more in length. In some embodiments, the wings are 9 bases or more in length. In some embodiments, the wings are 10 bases or more in length. In some embodiments, the wings are 11 bases or more in length. In some embodiments, the wings are 12 bases or more in length. In some embodiments, the wings are 13 bases or more in length. In some embodiments, the wings are 14 bases or more in length. In some embodiments, the wings are 15 bases or more in length. In some embodiments, the wings are 16 bases or more in length. In some embodiments, the wings are 17 bases or more in length. In some embodiments, the wings are 18 bases or more in length. In some embodiments, the wings are 19 bases or more in length. In some embodiments, the wings have a length of 10 bases or more.

[0256] In some embodiments, the wings are 1 base long. In some embodiments, the wings are 2 base long. In some embodiments, the wings are 3 base long. In some embodiments, the wings are 4 base long. In some embodiments, the wings are 5 base long. In some embodiments, the wings are 6 base long. In some embodiments, the wings are 7 base long. In some embodiments, the wings are 8 base long. In some embodiments, the wings are 9 base long. In some embodiments, the wings are 10 base long. In some embodiments, the wings are 11 base long. In some embodiments, the wings are 12 base long. In some embodiments, the wings are 13 base long. In some embodiments, the wings are 14 base long. In some embodiments, the wings are 15 base long. In some embodiments, the wings are 16 base long. In some embodiments, the wings are 17 base long. In some embodiments, the wings are 18 base long. In some embodiments, the wings are 19 base long. In some embodiments, the wings are 10 base long.

[0257] In some embodiments, the wing comprises one or more chiral internucleotide linkages. In some embodiments, the wing comprises one or more natural phosphate linkages. In some embodiments, the wing comprises one or more chiral internucleotide linkages and one or more natural phosphate linkages. In some embodiments, the wing comprises one or more chiral internucleotide linkages and two or more natural phosphate linkages. In some embodiments, the wing comprises one or more chiral internucleotide linkages and two or more natural phosphate linkages, wherein the two or more natural phosphate linkages are contiguous. In some embodiments, the wing does not comprise a chiral internucleotide linkage. In some embodiments, each wing linkage is a natural phosphate linkage. In some embodiments, the wing does not comprise a phosphate linkage. In some embodiments, each wing is independently a chiral internucleotide linkage.

[0258] In some embodiments, each wing region independently comprises one or more chiral internucleotide bridges. In some embodiments, each wing region independently comprises one or more naturally occurring phosphate bridges. In some embodiments, each wing region independently comprises one or more chiral internucleotide bridges and one or more naturally occurring phosphate bridges. In some embodiments, each wing region independently comprises one or more chiral internucleotide bridges and two or more naturally occurring phosphate bridges. In some embodiments, each wing region independently comprises one or more chiral internucleotide bridges and two or more naturally occurring phosphate bridges, wherein the two or more naturally occurring phosphate bridges are contiguous.

[0259] In some embodiments, each wing region independently comprises at least one chiral internucleotide bridge. In some embodiments, each wing region independently comprises at least two chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least three chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least four chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least five chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least six chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least seven chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least eight chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least nine chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least ten chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least eleven chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 12 chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 13 chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 14 chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 15 chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 16 chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 17 chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 18 chiral internucleotide bridges.In some embodiments, each wing region independently comprises at least 19 chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 20 chiral internucleotide bridges.

[0260] In some embodiments, each wing region independently comprises one chiral internucleotide bridge. In some embodiments, each wing region independently comprises two chiral internucleotide bridges. In some embodiments, each wing region independently comprises three chiral internucleotide bridges. In some embodiments, each wing region independently comprises four chiral internucleotide bridges. In some embodiments, each wing region independently comprises five chiral internucleotide bridges. In some embodiments, each wing region independently comprises six chiral internucleotide bridges. In some embodiments, each wing region independently comprises seven chiral internucleotide bridges. In some embodiments, each wing region independently comprises eight chiral internucleotide bridges. In some embodiments, each wing region independently comprises nine chiral internucleotide bridges. In some embodiments, each wing region independently comprises ten chiral internucleotide bridges. In some embodiments, each wing region independently comprises eleven chiral internucleotide bridges. In some embodiments, each wing region independently comprises 12 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 13 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 14 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 15 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 16 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 17 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 18 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 19 chiral internucleotide bridges. In some embodiments, each wing region independently comprises 20 chiral internucleotide bridges.

[0261] In some embodiments, each wing region independently comprises at least one consecutive natural phosphate bridge. In some embodiments, each wing region independently comprises at least two consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least three consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least four consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least five consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least six consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least seven consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least eight consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least nine consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least ten consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 11 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 12 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 13 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 14 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 15 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 16 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 17 consecutive chiral internucleotide bridges.In some embodiments, each wing region independently comprises at least 18 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 19 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises at least 20 consecutive chiral internucleotide bridges.

[0262] In some embodiments, each wing region independently comprises one consecutive natural phosphate bridge. In some embodiments, each wing region independently comprises two consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises three consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises four consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises five consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises six consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises seven consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises eight consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises nine consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises ten consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 11 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 12 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 13 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 14 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 15 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 16 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 17 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 18 consecutive chiral internucleotide bridges.In some embodiments, each wing region independently comprises 19 consecutive chiral internucleotide bridges. In some embodiments, each wing region independently comprises 20 consecutive chiral internucleotide bridges.

[0263] In some embodiments, each wing region independently comprises at least one native phosphate bridge. In some embodiments, each wing region independently comprises at least two native phosphate bridges. In some embodiments, each wing region independently comprises at least three native phosphate bridges. In some embodiments, each wing region independently comprises at least four native phosphate bridges. In some embodiments, each wing region independently comprises at least five native phosphate bridges. In some embodiments, each wing region independently comprises at least six native phosphate bridges. In some embodiments, each wing region independently comprises at least seven native phosphate bridges. In some embodiments, each wing region independently comprises at least eight native phosphate bridges. In some embodiments, each wing region independently comprises at least nine native phosphate bridges. In some embodiments, each wing region independently comprises at least ten native phosphate bridges. In some embodiments, each wing region independently comprises at least eleven native phosphate bridges. In some embodiments, each wing region independently comprises at least twelve native phosphate bridges. In some embodiments, each wing region independently comprises at least 13 native phosphate bridges. In some embodiments, each wing region independently comprises at least 14 native phosphate bridges. In some embodiments, each wing region independently comprises at least 15 native phosphate bridges. In some embodiments, each wing region independently comprises at least 16 native phosphate bridges. In some embodiments, each wing region independently comprises at least 17 native phosphate bridges. In some embodiments, each wing region independently comprises at least 18 native phosphate bridges. In some embodiments, each wing region independently comprises at least 19 native phosphate bridges. In some embodiments, each wing region independently comprises at least 20 native phosphate bridges.

[0264] In some embodiments, each wing region independently comprises one native phosphate bridge. In some embodiments, each wing region independently comprises two native phosphate bridges. In some embodiments, each wing region independently comprises three native phosphate bridges. In some embodiments, each wing region independently comprises four native phosphate bridges. In some embodiments, each wing region independently comprises five native phosphate bridges. In some embodiments, each wing region independently comprises six native phosphate bridges. In some embodiments, each wing region independently comprises seven native phosphate bridges. In some embodiments, each wing region independently comprises eight native phosphate bridges. In some embodiments, each wing region independently comprises nine native phosphate bridges. In some embodiments, each wing region independently comprises ten native phosphate bridges. In some embodiments, each wing region independently comprises eleven native phosphate bridges. In some embodiments, each wing region independently comprises twelve native phosphate bridges. In some embodiments, each wing region independently comprises 13 native phosphate bridges. In some embodiments, each wing region independently comprises 14 native phosphate bridges. In some embodiments, each wing region independently comprises 15 native phosphate bridges. In some embodiments, each wing region independently comprises 16 native phosphate bridges. In some embodiments, each wing region independently comprises 17 native phosphate bridges. In some embodiments, each wing region independently comprises 18 native phosphate bridges. In some embodiments, each wing region independently comprises 19 native phosphate bridges. In some embodiments, each wing region independently comprises 20 native phosphate bridges.

[0265] In some embodiments, each wing region independently comprises at least 1 consecutive native phosphate bridge. In some embodiments, each wing region independently comprises at least 2 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 3 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 4 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 5 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 6 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 7 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 8 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 9 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 10 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 11 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 12 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 13 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 14 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 15 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 16 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 17 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 18 consecutive native phosphate bridges.In some embodiments, each wing region independently comprises at least 19 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises at least 20 consecutive native phosphate bridges.

[0266] In some embodiments, each wing region independently comprises one consecutive native phosphate bridge. In some embodiments, each wing region independently comprises two consecutive native phosphate bridges. In some embodiments, each wing region independently comprises three consecutive native phosphate bridges. In some embodiments, each wing region independently comprises four consecutive native phosphate bridges. In some embodiments, each wing region independently comprises five consecutive native phosphate bridges. In some embodiments, each wing region independently comprises six consecutive native phosphate bridges. In some embodiments, each wing region independently comprises seven consecutive native phosphate bridges. In some embodiments, each wing region independently comprises eight consecutive native phosphate bridges. In some embodiments, each wing region independently comprises nine consecutive native phosphate bridges. In some embodiments, each wing region independently comprises ten consecutive native phosphate bridges. In some embodiments, each wing region independently comprises eleven consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 12 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 13 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 14 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 15 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 16 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 17 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 18 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 19 consecutive native phosphate bridges. In some embodiments, each wing region independently comprises 20 consecutive native phosphate bridges.

[0267] In some embodiments, the wings are directed to the 5'-end of the core (5'-terminal wings). In some embodiments, the wings are directed to the 3'-end of the core (3'-terminal wings). For example, in WV-1092 (mG * SmGmCmAmC * SA * SA * SG * SG * SG * SC * SA * SC * RA * SG * SmAmCmUmU * SmC), mG * SmGmCmAmC is the 5'-terminal wing, * SA * SA * SG * SG * SG * SC * SA * SC * RA * SG * S is the core, mAmCmUmU * SmC is the 3'-terminal wing.

[0268] In some embodiments, the 5'-terminal wing comprises one or more modified internucleotide bridges and one or more natural phosphate internucleotide bridges. In some embodiments, the 3'-terminal wing comprises one or more modified internucleotide bridges and one or more natural phosphate internucleotide bridges. In some embodiments, each wing independently comprises one or more modified internucleotide bridges and one or more natural phosphate internucleotide bridges. For example, WV-1092 has a 5'-terminal wing, the 5'-terminal wing comprises one or more modified internucleotide bridges and one or more natural phosphate internucleotide bridges, and the 3'-terminal wing comprises one or more modified internucleotide bridges and one or more natural phosphate internucleotide bridges.

[0269] In some embodiments, the 5'-terminal wing comprises a modified internucleotide bridge in the 5'-terminal wing (relative to the 3'-terminus) followed by one or more natural phosphate bridges connecting two or more nucleosides. For example, the 5'-terminal wing mG * SmGmCmAmC is a modified internucleotide bridge (mG) with three natural phosphate bridges connecting four nucleosides (mGmCmAmC) after the modified internucleotide bridge in the 5'-terminal wing. * S In some embodiments, the 5'-terminal wing comprises a modified internucleotide bridge followed by one or more natural phosphate bridges and / or one or more modified internucleotide bridges followed by one or more natural phosphate bridges in the 5'-terminal wing (e.g., mG * SmG * mG in SmCmAmC * S mG and mG * S mC). In some embodiments, the 5'-terminal wing comprises one or more natural phosphate bridges in the 5'-terminal wing after a modified internucleotide bridge. In some embodiments, the 5'-terminal wing comprises one or more consecutive natural phosphate bridges in the 5'-terminal wing after a modified internucleotide bridge. In some embodiments, the 5'-terminal wing comprises a natural phosphate bridge between two nucleosides at its 3'-end. For example, the 5'-terminal wing mG * SmGmCmAmC contains a natural phosphate bridge between two nucleosides at its 3'-end (mG * SmGmC mAmC ).

[0270] In some embodiments, the 3'-terminal wing comprises a modified internucleotide bridge with one or more natural phosphate bridges connecting two or more nucleosides prior to the modified internucleotide bridge in the 3'-terminal wing (relative to the 5'-terminus). For example, the 3'-terminal wing mAmCmUmU *SmC is a modified internucleotide bridge (mU) with three natural phosphate bridges connecting four nucleosides (mAmCmUmU) before the modified internucleotide bridge in the 3'-terminal wing. * S In some embodiments, the 3'-terminal wing comprises one or more natural phosphate bridges and / or one or more modified internucleotide bridges, preceded by one or more natural phosphate bridges in the 3'-terminal wing (e.g., mAmCmU). * SmU * mU in SmC * S mU and mU * S mC). In some embodiments, the 3'-terminal wing comprises one or more natural phosphate bridges in the 3'-terminal wing before the modified internucleotide bridge. In some embodiments, the 3'-terminal wing comprises one or more consecutive natural phosphate bridges in the 3'-terminal wing before the modified internucleotide bridge. In some embodiments, the 3'-terminal wing comprises a natural phosphate bridge between two nucleosides at its 5'-end. For example, mAmCmUmU * The 3'-terminal wing of the SmC structure contains a natural phosphate bridge between two nucleosides at its 5'-end ( mAmC mUmU * SmC).

[0271] In some embodiments, one or more is 1. In some embodiments, one or more is 2. In some embodiments, one or more is 3. In some embodiments, one or more is 4. In some embodiments, one or more is 5. In some embodiments, one or more is 6. In some embodiments, one or more is 7. In some embodiments, one or more is 8. In some embodiments, one or more is 9. In some embodiments, one or more is 10. In some embodiments, one or more is at least 1. In some embodiments, one or more is at least 2. In some embodiments, one or more is at least 3. In some embodiments, one or more is at least 4. In some embodiments, one or more is at least 5. In some embodiments, one or more is at least 6. In some embodiments, one or more is at least 7. In some embodiments, one or more is at least 8. In some embodiments, one or more is at least 9. In some embodiments, one or more is at least 10.

[0272] In some embodiments, a wing contains only one chiral internucleotide linkage. In some embodiments, a 5'-terminal wing contains only one chiral internucleotide linkage. In some embodiments, a 5'-terminal wing contains only one chiral internucleotide linkage at the 5'-end of the wing. In some embodiments, a 5'-terminal wing contains only one chiral internucleotide linkage at the 5'-end of the wing, and the chiral internucleotide linkage is Rp. In some embodiments, a 5'-terminal wing contains only one chiral internucleotide linkage at the 5'-end of the wing, and the chiral internucleotide linkage is Sp. In some embodiments, a 3'-terminal wing contains only one chiral internucleotide linkage at the 3'-end of the wing. In some embodiments, a 3'-terminal wing contains only one chiral internucleotide linkage at the 3'-end of the wing, and the chiral internucleotide linkage is Rp. In some embodiments, the 3'-terminal wing contains only one chiral internucleotide linkage at the 3'-end of the wing, and the chiral internucleotide linkage is Sp.

[0273] In some embodiments, the wing contains two or more native phosphate linkages, hi some embodiments, all phosphate linkages within the wing are contiguous, and there are no non-phosphate linkages between any two phosphate linkages within the wing.

[0274] In some embodiments, when describing bonds, e.g., bond chemistry, bond stereochemistry, etc., the bond connecting the wing and core is considered to be part of the core. For example, WV-1092, mG * SmGmCmAmC * S A * SA * SG * SG * SG * SC * SA * SC * RA * SG * SmAmCmUmU * In SmC, the underlined bonds may be considered part of the core (bold), the 5′-wing (with 2′-OMe in the sugar moiety) has a single Sp phosphorothioate bond at its 5′-end, the 3′-wing (with 2′-OMe in the sugar moiety) has one Sp phosphorothioate bond at its 3′-end, and the core has no 2′-modifications in the sugar).

[0275] In some embodiments, the 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a modified linkage. In some embodiments, the 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a linkage having the structure of Formula I. In some embodiments, the 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a phosphorothioate linkage. In some embodiments, the 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a substituted phosphorothioate linkage. In some embodiments, the 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a phosphorothioate triester linkage. In some embodiments, each 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a modified linkage. In some embodiments, each 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification has the structure of Formula I. In some embodiments, each 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a phosphorothioate linkage. In some embodiments, each 5'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a substituted phosphorothioate linkage. In some embodiments, each 5'-internucleotide linkage attached to a sugar moiety that does not have a 2'-modification is a phosphorothioate triester linkage.

[0276] In some embodiments, the 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a modified linkage. In some embodiments, the 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a linkage having the structure of Formula I. In some embodiments, the 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a phosphorothioate linkage. In some embodiments, the 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a substituted phosphorothioate linkage. In some embodiments, the 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a phosphorothioate triester linkage. In some embodiments, each 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a modified linkage. In some embodiments, each 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification has the structure of Formula I. In some embodiments, each 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a phosphorothioate linkage. In some embodiments, each 3'-internucleotide linkage attached to the sugar moiety without a 2'-modification is a substituted phosphorothioate linkage. In some embodiments, each 3'-internucleotide linkage attached to a sugar moiety that does not have a 2'-modification is a phosphorothioate triester linkage.

[0277] In some embodiments, both internucleotide linkages attached to the sugar moiety without a 2'-modification are modified linkages. In some embodiments, both internucleotide linkages attached to the sugar moiety without a 2'-modification are linkages having the structure of Formula I. In some embodiments, both internucleotide linkages attached to the sugar moiety without a 2'-modification are phosphorothioate linkages. In some embodiments, both internucleotide linkages attached to the sugar moiety without a 2'-modification are substituted phosphorothioate linkages. In some embodiments, both internucleotide linkages attached to the sugar moiety without a 2'-modification are phosphorothioate triester linkages. In some embodiments, each internucleotide linkage attached to the sugar moiety without a 2'-modification is a modified linkage. In some embodiments, each internucleotide linkage attached to the sugar moiety without a 2'-modification has the structure of Formula I. In some embodiments, each internucleotide linkage attached to the sugar moiety without a 2'-modification is a phosphorothioate linkage. In some embodiments, each internucleotide linkage attached to the sugar moiety without a 2'-modification is a substituted phosphorothioate linkage. In some embodiments, each internucleotide linkage attached to a sugar moiety that does not have a 2'-modification is a phosphorothioate triester linkage.

[0278] In some embodiments, the sugar moiety without a 2'-modification is a sugar moiety found in naturally occurring DNA nucleosides.

[0279] In some embodiments, in a wing-core-wing structure, the 5'-terminal wing contains only one chiral internucleotide linkage. In some embodiments, in a wing-core-wing structure, the 5'-terminal wing contains only one chiral internucleotide linkage at the 5'-end of the wing. In some embodiments, in a wing-core-wing structure, the 3'-terminal wing contains only one chiral internucleotide linkage. In some embodiments, in a wing-core-wing structure, the 3'-terminal wing contains only one chiral internucleotide linkage at the 3'-end of the wing. In some embodiments, in a wing-core-wing structure, each wing contains only one chiral internucleotide linkage. In some embodime...

Claims

1. 1. A chiral controlled oligonucleotide composition comprising: (1) base sequence; (2) backbone crosslinking pattern; (3) the pattern of chiral centers in the backbone; and (4) Pattern of backbone phosphorus modification and a plurality of oligonucleotides of a particular oligonucleotide type defined by The composition is enriched for oligonucleotides of a particular oligonucleotide type relative to a racemic preparation of oligonucleotides having the same base sequence; the plurality of oligonucleotides have a wing-core-wing structure and have the same base sequence; each wing region independently having a length of 2 or more bases; the linkages connecting the sugars of the wing regions comprise one or more phosphate bridges, each independently a phosphate linkage or a phosphorothioate linkage; each sugar of the wing region is independently a modified sugar; each of the linkages connecting a sugar of the wing region to a sugar of the core region is independently a phosphorothioate linkage; each sugar of the core region is independently a natural DNA sugar; each of the linkages connecting the sugars of the core region is independently a phosphorothioate linkage; 60% or more of the interoligonucleotide bridges of said plurality of oligonucleotides are phosphorothioate internucleotide linkages; Oligonucleotide Compositions.

2. 10. The chiral controlled oligonucleotide composition of claim 1, A composition wherein the pattern of chiral centers in the backbone comprises at least one Rp internucleotide linkage and at least one Sp internucleotide linkage.

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