Oligonucleotide composition and method therefor

Abstract

The present invention provides designed oligonucleotides, compositions, and methods thereof (e.g., altering transcriptional splicing, lower toxicity, providing an improved protein binding profile, improved delivery, improved uptake, etc.). [Solution] A chiral-controlled oligonucleotide composition comprising a plurality of oligonucleotides of a specific oligonucleotide type defined by (1) base sequence; (2) crosslinking pattern of the skeleton; (3) chiral center pattern of the skeleton; and (4) phosphorus modification pattern of the skeleton.

Description

Cross-reference of related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 195,779 filed on 22 July 2015, No. 62 / 236,847 filed on 2 October 2015, and No. 62 / 331,960 filed on 4 May 2016, the full texts of each application being 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 purposes may be limited due to their instability to extracellular and intracellular nucleases, toxicity, and / or poor cellular penetration and distribution. Novel and improved oligonucleotides and oligonucleotide compositions, such as new antisense and siRNA oligonucleotides and oligonucleotide compositions, are needed. [Overview of the project] [Problems that the invention aims to solve]

[0003] In particular, this disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequences, chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide crosslinks, and their patterns), and / or stereochemistry (e.g., stereochemistry of chiral centers in the backbone (chiral internucleotide crosslinks), and / or their patterns), can significantly influence oligonucleotide properties, such as activity and toxicity, which may be mediated by, for example, protein binding characteristics and stability. In some embodiments, this disclosure shows that oligonucleotide compositions comprising oligonucleotides having controlled structural elements (e.g., controlled chemical modifications and / or controlled backbone stereochemical patterns) impart, but not limited to, unexpected properties, including specific activity and toxicity. In some embodiments, this disclosure shows that oligonucleotide properties, such as activity and toxicity, can be tuned by chemical modifications (e.g., modifications of sugars, bases, internucleotide crosslinks, etc.), chiral structures (e.g., stereochemistry of chiral internucleotide crosslinks, and their patterns), and / or combinations thereof. [Means for solving the problem]

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

[0005] In some embodiments, the disclosure shows that oligonucleotide properties, such as activity and toxicity, can be tuned by chemical modification. In some embodiments, the disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, each having a common base sequence and comprising one or more modified sugar moieties, one or more natural phosphate crosslinks, or a combination thereof. In some embodiments, the disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, each having a common base sequence and comprising one or more modified internucleotide crosslinks, one or more modified sugar moieties, one or more natural phosphate crosslinks, or a combination thereof. In some embodiments, the first plurality of oligonucleotides have a wing-core-wing structure. In some embodiments, each wing region independently comprises one or more natural phosphate crosslinks and optionally one or more modified internucleotide crosslinks, and the core comprises one or more modified internucleotide crosslinks and optionally one or more natural phosphate crosslinks. In some embodiments, each wing region independently comprises one or more native phosphate crosslinks and one or more modified internucleotide crosslinks, and the core comprises one or more modified internucleotide crosslinks and does not contain native phosphate crosslinks. In some embodiments, the wings comprise a modified sugar moiety. In some embodiments, the modified internucleotide crosslink is a phosphorothioate. In some embodiments, the modified internucleotide crosslink is a substituted phosphorothioate. In some embodiments, the modified internucleotide crosslink has the structure of formula I as described in this disclosure. In some embodiments, the modified sugar moiety is 2'-modified. In some embodiments, the 2'-modification is 2'-OR 1 In some embodiments, the provided composition has low toxicity. In some embodiments, the provided composition has low complement activation.

[0006] In some embodiments, the Disclosure provides oligonucleotide compositions with improved protein-binding profiles, such as reduced harmful protein binding and / or increased beneficial protein binding. In some embodiments, the Disclosure provides a method for improved delivery of oligonucleotide compositions, including providing an oligonucleotide composition having an improved protein-binding profile. In some embodiments, the Disclosure shows that protein binding by an oligonucleotide composition can be tuned by chemical modification, stereochemistry, or a combination thereof. In some embodiments, protein binding by an oligonucleotide composition can be tuned by incorporating modified internucleotide crosslinks. In some embodiments, increasing the proportion of modified internucleotide crosslinks increases the binding of the oligonucleotide to a particular protein. In some embodiments, replacing one or more modified internucleotide crosslinks with native phosphate crosslinks reduces binding to a particular protein. In some embodiments, replacing one or more native phosphate crosslinks with modified internucleotide crosslinks increases binding to a particular protein. In some embodiments, certain chemical modifications increase protein binding to a particular protein. In some embodiments, certain chemical modifications decrease protein binding to a particular protein. In some embodiments, different chemical modifications of the same kind give different protein binding. For example, in some embodiments, 2'-MOE exhibits reduced protein binding compared to 2'-OMe (e.g., in certain aspects such as sequence and stereochemistry).

[0007] In particular, this disclosure includes the recognition that sterically random oligonucleotide preparations contain multiple distinct chemical moies that differ from one another, for example, in the stereochemical structure of the individual chiral centers in the oligonucleotide chain. Without controlling the stereochemistry of the chiral centers in the chiral centers, sterically random oligonucleotide preparations give an uncontrolled composition containing an undetermined amount of oligonucleotide stereoisomers. These stereoisomers may have the same base sequence and / or chemical modifications, but these stereoisomers are at least different chemical moies due to their different skeletal stereochemistry and may have different properties (e.g., activity, toxicity, etc.) as shown herein. In particular, this disclosure provides chirality-controlled compositions that are or contain specific stereoisomers of an oligonucleotide of interest. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, its length, the crosslinking pattern of its skeleton, and the pattern of chiral centers in its skeleton. As understood in the art, in some embodiments, the nucleotide sequence may refer to the identity and / or modified state of a nucleoside residue in an oligonucleotide (e.g., the identity and / or modified state of sugar and / or base elements 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 specific complementary residues). In some embodiments, the disclosure shows that improvements in properties achieved by including and / or positioning a particular chiral structure in an oligonucleotide (e.g., improved activity, lower toxicity) may be comparable to or better than those achieved by using chemical modifications, e.g., crosslinking of a particular backbone, modification of residues, etc. (e.g., by using certain 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, this disclosure shows that the toxicity of oligonucleotide compositions can be adjusted using stereochemistry. In some embodiments, this disclosure provides chirality-controlled oligonucleotide compositions that are less toxic compared to corresponding sterically random (or uncontrolled chirality) oligonucleotide compositions having the same nucleotide sequence and chemical modifications. In some embodiments, the more Rp-chiral internucleotide crosslinks an oligonucleotide contains, the less toxic an oligonucleotide composition is. In some embodiments, a chirality-controlled oligonucleotide composition having one Rp-chiral internucleotide crosslink is more toxic than other chirality-controlled oligonucleotide compositions and / or corresponding sterically random oligonucleotide compositions having the same nucleotide sequence and chemical modifications. In some embodiments, the one Rp-chiral internucleotide crosslink is located in the middle of the sequence. In some embodiments, a chirality-controlled oligonucleotide composition having one or more Rp-chiral internucleotide crosslinks at the 5'-terminus and / or 3'-terminus is less toxic. In some embodiments, oligonucleotide compositions with controlled chirality of oligonucleotides containing one or more natural phosphate crosslinks at the 5'-terminus and / or 3'-terminus have low toxicity. In some embodiments, the chiral internucleotide crosslink has the structure of formula I. In some embodiments, the chiral internucleotide crosslink is a phosphorothioate crosslink. In some embodiments, the chiral internucleotide crosslink is a substituted phosphorothioate crosslink.

[0009] In particular, the Disclosure recognizes that in some embodiments, the properties of an oligonucleotide (e.g., activity, toxicity, etc.) can be modulated by optimizing the pattern of chiral centers in the backbone, in combination with the modulation / optimization of one or more other features of the oligonucleotide (e.g., chemical modification, modification pattern, e.g., crosslinking pattern, nucleoside modification pattern, etc.). In some embodiments, the Disclosure recognizes and shows that chemical modification, e.g., modification of nucleosides and internucleotide crosslinks, can give enhanced properties. In some embodiments, the Disclosure shows that a combination of chemical modification and stereochemistry can give unexpectedly and significantly improved properties (e.g., activity, toxicity, etc.). In some embodiments, a chemical combination (e.g., modification of sugars, bases and / or internucleotide crosslinks) is combined with a stereochemical pattern to give oligonucleotides and compositions thereof having remarkably enhanced properties, including lower toxicity and a better protein-binding profile. In some embodiments, an oligonucleotide composition provided comprising a first plurality of oligonucleotides has controlled chirality, and the first plurality of oligonucleotides comprises a combination of 2'-modification of one or more sugar moieties, one or more native phosphate crosslinks and one or more chiral internucleotide crosslinks. In some embodiments, the provided oligonucleotide composition comprising a first plurality of oligonucleotides has controlled chirality, and the first plurality of oligonucleotides comprises a combination of one or more 2'-modifications of sugar moieties, one or more native phosphate crosslinks, and one or more chiral internucleotide crosslinks, wherein the 5'-terminal and / or 3'-terminal internucleotide crosslinks are chiral. In some embodiments, both the 5'-terminal and 3'-terminal internucleotide crosslinks are chiral. In some embodiments, both the 5'-terminal and 3'-terminal internucleotide crosslinks are chiral and Sp.In some embodiments, the provided oligonucleotide composition comprising a first plurality of oligonucleotides has controlled chirality, and the first plurality of oligonucleotides comprises a 2'-modification of one or more sugar moieties, one or more native phosphate crosslinks and one or more chiral internucleotide crosslinks and a combination of stereochemical patterns 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 crosslink has a structure of formula I. In some embodiments, the chiral internucleotide crosslink is a phosphorothioate crosslink. In some embodiments, the chiral internucleotide crosslink is a substituted phosphorothioate crosslink.

[0010] In some embodiments, the disclosure provides oligonucleotide compositions with low toxicity. In some embodiments, the disclosure provides oligonucleotide compositions having an improved protein binding profile. In some embodiments, the disclosure provides oligonucleotide compositions having improved binding to albumin. In some embodiments, the provided compositions have low toxicity and improved binding to a particular desired protein. In some embodiments, the provided compositions have low toxicity and improved binding to a particular desired protein. In some embodiments, the provided oligonucleotide compositions simultaneously provide the same level, or significantly improved, of stability and / or activity, e.g., better target cleavage pattern, better target efficiency, better target specificity, etc.

[0011] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently contains one or more modified internucleotide crosslinks and optionally one or more native phosphate crosslinks, and the core region independently contains one or more modified internucleotide crosslinks; or The present invention provides an oligonucleotide composition in which each wing region independently contains one or more modified sugar moieties, and the core region contains one or more unmodified sugar moieties.

[0012] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and optionally one or more native phosphate crosslinks; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0013] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0014] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising two or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0015] In some embodiments, the present disclosure relates to oligonucleotide compositions, It comprises a first plurality of oligonucleotides, each containing two wing regions and one core region. The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; The wing region relative to the 5'-terminus of the core region contains, within the wing, at least one modified internucleotide crosslink followed by a native phosphate crosslink; and The wing region relative to the 3'-terminus of the core region contains, within the wing, at least one modified internucleotide crosslink followed by a native phosphate crosslink; The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0016] In some embodiments, the present disclosure relates to oligonucleotide compositions, It comprises a first plurality of oligonucleotides, each containing one wing region and one core region. The first group of oligonucleotides have the same base sequence; The wing region has a length of 2 nucleotides or more and contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; The wing region is relative to the 5'-terminus of the core region and contains a natural phosphate crosslink between two nucleosides at its 3'-terminus, or the wing region is relative to the 3'-terminus of the core region and contains a natural phosphate crosslink between two nucleosides at its 5'-terminus; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0017] In some embodiments, the present disclosure relates to oligonucleotide compositions, It comprises a first plurality of oligonucleotides, each containing two wing regions and one core region. The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; The wing region relative to the 5'-terminus of the core region contains a natural phosphate crosslink between two nucleosides at its 3'-terminus; The wing region relative to the 3'-terminus of the core region contains a natural phosphate crosslink between two nucleosides at its 5'-terminus; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0018] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently comprises one or more modified internucleotide crosslinks and optionally one or more native phosphate crosslinks, and the core region independently comprises one or more modified internucleotide crosslinks; and The present invention provides an oligonucleotide composition in which each wing region independently contains one or more modified sugar moieties, and the core region contains one or more unmodified sugar moieties.

[0019] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks, and the core region independently contains one or more modified internucleotide crosslinks; and The present invention provides an oligonucleotide composition in which each wing region independently contains one or more modified sugar moieties, and the core region contains one or more unmodified sugar moieties.

[0020] In some embodiments, the Disclosure relates to an oligonucleotide composition comprising a first plurality of oligonucleotides, where the first plurality of oligonucleotides (1) Having a common base sequence; (2) comprising one or more wing regions and one core region; Each wing region contains at least one modified sugar moiety; and The present invention provides an oligonucleotide composition in which each core region contains at least one unmodified sugar moiety.

[0021] In some embodiments, this disclosure is: 1) Common base sequence and length; 2) Common patterns of skeletal connections; and 3) Common patterns of chiral centers in the skeleton; The present invention provides a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type characterized by the present invention, which is chiral-controlled in that it is concentrated with respect to the oligonucleotide of a specific oligonucleotide type compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0022] In some embodiments, this disclosure is: 1) Common base sequence and length; 2) Common patterns of skeletal connections; and 3) Common patterns of chiral centers in the skeleton The present invention provides a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type characterized by the present invention, wherein at least about 10% of the oligonucleotides in the composition are substantially pure preparations of a single oligonucleotide having a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers.

[0023] In particular, this disclosure acknowledges that combinations of oligonucleotide components (e.g., patterns of chemical modifications, skeletal bonding, skeletal chiral centers and / or skeletal phosphorus modifications) can confer properties such as remarkably improved biological activity. In some embodiments, this disclosure provides oligonucleotide compositions comprising a predetermined level of oligonucleotides including 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 bonds; Each core region is independently two bases or longer and independently contains one or more chiral internucleotide bonds, and the common core region is: 1) Common base sequence and length; 2) Common patterns of skeletal connections; and 3) Common patterns of chiral centers in the skeleton It has.

[0024] In particular, this disclosure recognizes that combinations of structural elements of oligonucleotides (e.g., patterns of chemical modifications, bridging of the skeleton, chiral centers of the skeleton and / or phosphorus modifications of the skeleton) can give remarkable improved properties such as low toxicity and / or desirable protein binding. In some embodiments, this disclosure provides an oligonucleotide composition comprising a predetermined amount of oligonucleotide, wherein the oligonucleotide comprises one or more wing regions and a common core region, Here, Each wing region independently has a length of 2 nucleotides or more and independently contains, optionally, one or more chiral internucleotide bridges; The core region independently has a length of 2 nucleotides or more, independently contains one or more chiral internucleotide crosslinks, and the common core region is (1) Common base sequence and length; (2) Common pattern skeletal crosslinking; and (3) Provide an oligonucleotide composition containing a common pattern of skeletal 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, and the wings include nucleoside modifications that are not present in the core region. In some embodiments, the oligonucleotides in the provided composition have a nucleoside-modified wing-core structure. In some embodiments, the oligonucleotides in the provided composition have a nucleoside-modified core-wing structure. In some embodiments, the oligonucleotides in the provided composition have a nucleoside-modified wing-core-wing structure. In some embodiments, the wings and core are defined by modifications of the sugar moiety. In some embodiments, the wings and core are defined by modifications of the base moiety. 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 modifications 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, if there are two or more wings, each sugar moiety in one wing region has the same 2'-modification, however, a common 2'-modification in the first wing region may be the same as or different from a common 2'-modification in the second wing region. In some embodiments, the wings and core are defined by the pattern of internucleotide crosslinks in the backbone. In some embodiments, the wings include certain internucleotide crosslinks and / or patterns of internucleotide crosslinks that are not found in the core. In some embodiments, the wing regions include both modified internucleotide crosslinks and native phosphate crosslinks. In some embodiments, the internucleotide crosslinks at the 5'-terminus of the wing relative to the 5'-terminus of the core region are modified internucleotide crosslinks. In some embodiments, the internucleotide crosslinks at the 3'-terminus of the wing relative to the 3'-terminus of the core region are modified internucleotide crosslinks. In some embodiments, the modified internucleotide crosslinks are chiral internucleotide crosslinks.

[0026] In some embodiments, each wing contains at least one chiral internucleotide bond and at least one native phosphate bond. In some embodiments, each wing contains at least one modified sugar moiety. In some embodiments, each wing sugar moiety is modified. In some embodiments, the wing sugar moiety is modified by a modification not present in the core region. In some embodiments, only the wing region has a modified internucleotide bond at one or both of its ends. In some embodiments, only the wing region has a modified internucleotide bond at its 5'-end. In some embodiments, only the wing region has a modified internucleotide bond at its 3'-end. In some embodiments, only the wing region has modified internucleotide bonds at both its 5'-end and 3'-end. In some embodiments, the wing is connected to the 5'-end of the core, and only the wing has a modified internucleotide bond at its 5'-end. In some embodiments, the wing is connected to the 5'-end of the core, and only the wing has a modified internucleotide bond at its 3'-end. In some embodiments, the wings are connected to the 5'-terminus of the core, and only the wings have modified internucleotide bonds at both their 5'-terminus and 3'-terminus. In some embodiments, the wings are connected to the 3'-terminus of the core, and only the wings have modified internucleotide bonds at their 5'-terminus. In some embodiments, the wings are connected to the 3'-terminus of the core, and only the wings have modified internucleotide bonds at their 3'-terminus. In some embodiments, the wings are connected to the 3'-terminus of the core, and only the wings have modified internucleotide bonds at both their 5'-terminus and 3'-terminus. In some embodiments, one or more modifications or other modifications to the sugar moiety or internucleotide bond within one wing may differ from modifications within 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 includes a pattern of chiral centers in the (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m or (Sp)t(Rp)n(Sp)m skeleton. In some embodiments, the pattern of chiral centers in the core region skeleton 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 includes a pattern of chiral centers in the (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m or (Sp)t(Rp)n(Sp)m skeleton, where m>2. In some embodiments, the chiral center pattern of the core region skeleton 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 give or enhance controlled cleavage of a target sequence (e.g., an RNA sequence).

[0028] In some embodiments, the oligonucleotides in the provided composition have a common pattern of phosphate modification of the skeleton. In some embodiments, the provided composition is a chiral-controlled oligonucleotide composition in that it contains a predetermined level of oligonucleotides of individual oligonucleotide types, where the oligonucleotide types are: 1) Base sequence; 2) Patterns of skeletal connections; 3) Patterns of skeletal chiral centers; and 4) Patterns of skeletal phosphorus modification Defined by:

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

[0030] In some embodiments, a particular type of oligonucleotide is 1A) Base identity; 1B) Patterns of base modification; 1C) Patterns of sugar modification; 2) Patterns of skeletal connections; 3) Patterns of skeletal chiral centers; and 4) Patterns of skeletal phosphorus modification It may also be defined by: Therefore, in some embodiments, certain types of oligonucleotides may share the same base, but their patterns of base modification and / or sugar modification may differ.

[0031] In some embodiments, certain types of oligonucleotides have the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same pattern of skeletal bonding (e.g., patterns of native phosphate bonds, phosphorothioate bonds, phosphorothioate triester bonds, and combinations thereof), the same pattern of skeletal chiral centers (e.g., patterns of the stereochemistry (Rp / Sp) of chiral internucleotide bonds), and the same pattern of skeletal phosphorus modifications (e.g., -S - , and -LR of formula I 1They are chemically identical in that they have patterns of modification to the internucleotide phosphate atom (such as the internucleotide phosphate atom).

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

[0033] In particular, this disclosure provides oligonucleotide compositions and techniques for optimizing their properties, such as activity and toxicity. In some embodiments, this disclosure provides methods for reducing the toxicity of oligonucleotides and their compositions. In some embodiments, this disclosure provides methods for reducing immune responses associated with the administration of oligonucleotides and their compositions (i.e., methods for administering oligonucleotide compositions such that an undesirable immune response to oligonucleotides in the composition is reduced compared to, for example, one observed using a reference composition of nucleotides with a comparable or identical nucleotide sequence). In some embodiments, this disclosure provides methods for reducing complement activation associated with the administration of oligonucleotides and their compositions. In some embodiments, this disclosure provides methods for improving the protein binding profile of oligonucleotides and their compositions. In some embodiments, this disclosure provides methods for increasing binding to specific proteins by oligonucleotides and their compositions. In some embodiments, this disclosure provides methods for increasing binding to specific proteins by oligonucleotides and their compositions. In some embodiments, this disclosure provides methods for increasing the delivery of oligonucleotides and their compositions. In particular, this disclosure includes the recognition that, in some embodiments, optimal delivery of oligonucleotides to a target involves a balance between the binding of the oligonucleotide to a specific protein that can deliver the oligonucleotide to a desired position, and the release of the oligonucleotide that can appropriately release the oligonucleotide from the specific protein to exert a desired function, such as hybridization with these targets, cleavage of these targets, inhibition of translation, or modulation of the transcription process. As illustrated in this disclosure, this disclosure recognizes, in particular, that improvements to the properties of oligonucleotides can be achieved by chemical modification and / or stereochemistry.

[0034] In some embodiments, the present disclosure provides a method for administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, The present invention provides an improved method comprising administering an oligonucleotide composition comprising a first plurality of oligonucleotides with controlled chirality, wherein the first plurality of oligonucleotides are less toxic compared to a reference oligonucleotide composition having the same common nucleotide sequence.

[0035] In some embodiments, the present disclosure provides a method for administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, The present invention provides an improved method comprising administering an oligonucleotide composition in which each oligonucleotide in a plurality of oligonucleotides contains one or more modified sugar moieties, characterized in that this composition has lower 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 for administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, The method involves administering an oligonucleotide composition in which each oligonucleotide in a plurality of oligonucleotides contains one or more natural phosphate crosslinks and one or more modified phosphate crosslinks. The present invention provides an improved method characterized by low toxicity when the oligonucleotide composition is tested in at least one assay measured using a corresponding reference composition, except that the oligonucleotide does not contain natural phosphate crosslinks.

[0037] In some embodiments, the present disclosure provides a method for administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, The present invention provides an improved method comprising administering an oligonucleotide composition in which each oligonucleotide in a plurality of oligonucleotides contains one or more modified sugar moieties, characterized in that this composition has lower 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 a method comprising administering to a subject an oligonucleotide composition comprising a plurality of first oligonucleotides, each having a common base sequence and containing a modified sugar moiety, wherein the oligonucleotide composition is low toxicity when tested in at least one assay measured using a corresponding reference composition comprising a plurality of reference oligonucleotides having the same common base sequence but without a modified sugar moiety.

[0039] In some embodiments, the disclosure provides a method comprising administering to a subject an oligonucleotide composition comprising a first plurality of oligonucleotides, each having a common nucleotide sequence and comprising one or more natural phosphate crosslinks and one or more modified phosphate crosslinks, wherein the oligonucleotide composition is low toxicity when tested in at least one assay measured using a corresponding reference composition comprising a plurality of reference oligonucleotides having the same common nucleotide sequence but without natural phosphate crosslinks.

[0040] In some embodiments, the disclosure provides a method comprising the step of administering a chirality-controlled oligonucleotide composition to a subject, wherein the chirality-controlled oligonucleotide composition comprises oligonucleotides having the same base sequence and comprises different chirality-controlled oligonucleotide compositions or sterically random oligonucleotide compositions, otherwise characterized by low toxicity when tested in at least one assay measured using a corresponding reference oligo composition.

[0041] In some embodiments, the reduction in toxicity is a reduction in complement activation or includes a reduction in complement activation. In some embodiments, the reduction in toxicity includes a reduction in complement activation. In some embodiments, the reduction in toxicity is a reduction in complement activation or includes a reduction in complement activation. In some embodiments, the reduction in toxicity includes a reduction in complement activation via an alternative pathway.

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

[0043] In some embodiments, the present disclosure relates to 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, and each of the reference plurality of oligonucleotides also has a common base sequence, but is different from the first plurality of oligonucleotides. The individual oligonucleotides in the reference oligonucleotides differ from each other in stereochemical structure; and / or At least some of the oligonucleotides in the reference oligonucleotides have a different structure from the structure represented by the oligonucleotides of the composition; and / or The method provides a structure in which at least some of the reference oligonucleotides in a set of oligonucleotides differ in that they do not contain wing and core regions.

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

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

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

[0047] In some embodiments, the present disclosure relates to 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, and each of the reference plurality of oligonucleotides also has a common base sequence, but is different from the first plurality of oligonucleotides. The individual oligonucleotides in the reference oligonucleotides differ from each other in stereochemical structure; and / or At least some of the oligonucleotides in the reference oligonucleotides have a different structure from the structure represented by the oligonucleotides of the composition; and / or The method provides a structure in which at least some of the reference oligonucleotides in a set of oligonucleotides differ in that they do not contain wing and core regions.

[0048] In some embodiments, the present disclosure provides a method for administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence, The present invention provides an improved method 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 uncontrolled, and / or different stereocontrolled compositions) are typically, preferably, determined using the same assay, substantially simultaneously in some embodiments, and in some embodiments, with reference to historical results.

[0050] Those skilled in the art will recognize and / or be able to easily develop assays suitable for specific oligonucleotide compositions. This disclosure provides a description of an assay that may be useful for evaluating one or more characteristics of the behavior of an oligonucleotide composition, such as complement activation, injection site inflammation, protein binding, etc.

[0051] For example, certain assays that may be useful for evaluating the toxicity and / or protein-binding properties of oligonucleotide compositions may include any assay described and / or illustrated herein.

[0052] definition Aliphatic: As used herein, the terms “aliphatic” or “aliphatic group” mean a straight (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain, or a monocyclic or polycyclic hydrocarbon (also referred herein as “carbocyclic,” “alicyclic,” or “cycloalkyl”) that is fully saturated or contains one or more unsaturated units and has one bond site to another molecule. In some embodiments, an aliphatic group contains 1 to 50 aliphatic carbon atoms. Unless otherwise specified, an aliphatic group contains 1 to 10 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1 to 6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1 to 5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 4 aliphatic carbon atoms. In yet another embodiment, an aliphatic group contains 1 to 3 aliphatic carbon atoms, and in yet another embodiment, an aliphatic group contains 1 to 2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocyclic" or "cycloalkyl") refers to a monocyclic or bicyclic C3-C3 compound that contains one bond site to another molecule, is fully saturated or contains one or more unsaturated units, but is not aromatic. 10This term represents a hydrocarbon. In some embodiments, "alicyclic" (or "carbocyclic" or "cycloalkyl") refers to a non-aromatic monocyclic C3-C6 hydrocarbon containing one bond site to another molecule, and containing one or more fully saturated or unsaturated units. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl groups, alkenyl groups, alkynyl groups, and complexes thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

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

[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 are substituted by substituents. Suitable substituents include the substituted aliphatic groups listed below.

[0055] Animals: 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, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals may be mammals, birds, reptiles, amphibians, fish, and / or helminths. In some embodiments, an animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0056] Approximately: As used herein, the words “approximately” or “about” when referring to a number generally mean that, unless otherwise specified or particularly evident from the context (unless such number is a possible value less than 0% or greater than 100%), the number falls within the range of 5%, 10%, 15%, or 20% in either direction (greater or less). In some embodiments, the use of the word “about” when referring to a dosage means ±5 mg / kg / day.

[0057] The term "aryl," used alone or as part of a larger phrase such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring structures having a total of 5 to 14 ring members, where at least one ring of the structure is aromatic and each ring of the structure contains 3 to 7 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, which may have one or more substituents. Groups in which an aromatic ring is fused with one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenantridinyl, or tetrahydronaphthyl, are also included herein in the scope of the term "aryl."

[0058] Characteristic segment: As used herein, the phrase “characteristic segment” of a protein or polypeptide, as a whole, includes a sequence of amino acids, or a set of amino acids, that are characteristic of the protein or polypeptide. Each such sequence will generally contain at least two amino acids. Furthermore, those skilled in the art will recognize that typically at least 5, 10, 15, 20, or more amino acids are required to be characteristic of a protein. Generally, in addition to the specific sequence homology described above, a characteristic segment shares at least one functional feature with the relevant intact protein.

[0059] Characteristic sequence: A "characteristic sequence" is a sequence found in all members of a family of polypeptides or nucleic acids, and can therefore be used by those skilled in the art to define members of the family.

[0060] Characteristic structural elements: The term “characteristic structural elements” refers to clearly distinguishable structural elements (e.g., skeletal structures, sets of suspensions, sequence elements, etc.) found in all members of a family of polypeptides, small molecules, or nucleic acids, and can therefore be used by those skilled in the art to define members of such families.

[0061] Equivalent: The term “equivalent” is used herein to describe two (or more) sets of states or environments that are sufficiently similar to each other to enable comparison of the results obtained or the phenomena observed. In some embodiments, equivalent sets of states or environments are characterized by several substantially identical features and one or a few modified features. Those skilled in the art will recognize that they are equivalent when they are characterized by a sufficient number and types of substantially identical features, and when differences in the results obtained or phenomena observed under different sets of states or environments can justify a reasonable conclusion that the sets of states are caused by or indicate differences in those modified features.

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

[0063] Equivalent Agents: A person skilled in the art will recognize, upon reading this disclosure, that the range of useful agents in the context of the present invention is not limited to those specifically mentioned or illustrated herein. Specifically, a person skilled in the art will recognize that activators typically have a structure consisting of a skeleton and bonded suspension parts, and will therefore understand that simple modifications of such skeleton and / or suspension parts do not significantly alter the activity of the agent. For example, in some embodiments, the substitution of one or more suspension parts having equivalent three-dimensional structures and / or chemical reaction properties may produce a substituted compound or part equivalent to the parent reference compound or part. In some embodiments, the addition or separation of one or more suspension parts may produce a substituted compound equivalent to the parent reference compound. In some embodiments, for example, modification of the skeleton structure by the addition or separation of a small number of bonds (usually 5, 4, 3, 2 or fewer, or 1 bond, and often only single bonds) may produce a substituted compound equivalent to the parent reference compound. In many embodiments, equivalent compounds can be synthesized, for example, by the methods shown in the general reaction scheme below, or variations thereof, using readily available substances, reagents, and conventional or provided synthetic procedures. Modifications of these reactions, known in themselves but not mentioned herein, are also available.

[0064] Equivalent Doses: The term “equivalent doses” is used herein to compare doses of different pharmacoactively active drugs that produce the same biological results. Doses of two different drugs are considered “equivalent” to each other according to the present invention if they achieve equivalent levels or degrees of biological results. In some embodiments, equivalent doses of different pharmaceuticals used according to the present invention are determined using in vitro and / or in vivo assays described herein. In some embodiments, one or more lysosomal activators used according to the present invention are utilized in doses equivalent to the dose of a reference lysosomal activator; in some embodiments, such reference lysosomal activators are selected from the group consisting of small molecule allosteric activators (e.g., pyrazolpyrimidines), iminosaccharides (e.g., isofagomine), antioxidants (e.g., n-acetylcysteine), and regulatory factors of cell transport (e.g., Rab1a polypeptide).

[0065] Heteroaliphatic: The term "heteroaliphatic" refers to an aliphatic group in which one or more units selected from C, CH, CH2, or CH3 are independently substituted by a heteroatom. In some embodiments, the heteroaliphatic group is a heteroalkyl. In some embodiments, the heteroaliphatic group is a heteroalkenyl.

[0066] Heteroaryl: The terms "heteroaryl" and "heteroaryl-", used alone or as part of a larger group, e.g., "heteroaryl" or "heteroarylcosi", represent a group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; 6, 10, or 14 π electrons shared in a cyclic arrangement; and 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 any quaternized form of basic nitrogen. Examples of heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridadinyl, pyrimidinyl, pyrazinyl, indolidinyl, prinyl, naphthilidinyl, and pteridinyl. In this specification, the terms "heteroaryl" and "hetero-" also include groups in which the heteroaromatic ring is condensed with one or more aryl, alicyclic, or heterocyclyl rings whose radical or bond site lies on the aromatic ring. Examples that are not limited include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, sinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazine-3(4H)-one. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "aromatic heterocycle," all of which may include a substituted ring. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, and the alkyl and heteroaryl portions may be substituted independently.

[0067] Heteroatom: The term "heteroatom" refers to one or more oxygen, sulfur, nitrogen, phosphorus, boron, selenium, or silicon (oxidized forms of any of nitrogen, boron, selenium, sulfur, phosphorus, or silicon; quaternized forms of any basic nitrogen or; heterocycles, e.g., N (in 3,4-dihydro-2H-pyrrolyl), NH (in pyrrolidinyl), or NR + This means (containing the substitutable nitrogen in N-substituted pyrrolidinyl).

[0068] Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are interchangeable and represent a stable 3-7 member monocyclic or 7-10 member bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more, preferably 1-4, of the above heteroatoms in addition to the carbon atom. When used to represent a ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. For example, a saturated or partially unsaturated ring having 0-3 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 (like in N-substituted pyrrolidinyl).

[0069] The heterocyclic ring may be bonded to its side groups on any heteroatom or carbon atom that provides a stable structure, and any ring atom may 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 “heterocyclic,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical” are used interchangeably herein and include groups such as indolinyl, 3H-indolyl, chromanyl, phenantridinyl, or tetrahydroquinolinyl, in which a heterocyclyl ring is condensed to one or more aryl groups, heteroaryl groups, or aliphatic rings, wherein the radical or bond site is located on the heterocyclyl ring. The heterocyclyl ring may be monocyclic or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted with a heterocyclyl, wherein the alkyl and heterocyclyl moieties may be independently substituted.

[0070] Intraperitoneal: As used herein, the phrases “intraperitoneal administration” and “administered intraperitoneally” have the meanings understood in the art to refer to the administration of a compound or composition into the peritoneum of the subject.

[0071] In vitro: As used herein, “in vitro” refers to events occurring in an artificial environment, such as in a test tube or reactor, in a cell culture medium, or otherwise, rather than within a living organism (e.g., an animal, plant, and / or microorganism).

[0072] In vivo: In this specification, the term “in vivo” refers to events occurring within living organisms (e.g., animals, plants, and / or microorganisms).

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

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

[0075] Optionally substituted: When described herein, the compounds of the present invention may contain a portion that is "optionally substituted". Generally, the term "substituted" means that one or more hydrogens of the designated portion are substituted with a suitable substituent, whether or not the phrase "may be" is present. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group when one or more positions in any given structure may be the same or different for each position. The combinations of substituents contemplated by the present invention preferably result in the formation of stable or chemically viable compounds. As used herein, the term "stable" refers to compounds that do not substantially change when in a state that enables their manufacture, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0076] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are, independently, halogen; -(CH2) 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 ○ Optionally substituted (CH2) 0~4 Ph; R ○ Optionally substituted (CH2) 0~4O(CH2) 0~1 Ph;R ○ CH=CHPh;R ○ It may be replaced 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)2NR ○ 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)O-N(R ○ )2; or -(C 1~4 linear or branched alkylene)C(O)O-N(R ○ )2(where each R ○ may be substituted as follows and independently is hydrogen, C 1~6 aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, -CH2-(5-6 member heteroaryl ring) or a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two R's that appear independently ○However, these intervening atoms may be substituted together as shown below, and independently form a 3-12 member saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms selected from nitrogen, oxygen, or sulfur.

[0077] R ○ The appropriate monovalent substituent above (or two independently occurring R ○ However, the rings formed by these intervening atoms are independently halogens, -(CH2) 0~2 R ● ,-(HaroR ● ), -(CH2) 0~2 OH, -(CH2) 0~2 Ure ● ,-(CH2) 0~2 CH(OR ● )2;O(HaroR ● ), -CN, -N3, -(CH2) 0~2 C(O)R ● ,-(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR ● ,-(CH2) 0~2 SR ● ,-(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR ● ,-(CH2) 0~2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. C(O)SR ● ,-(C 1~4 Straight-chain or branched-chain alkylene)C(O)OR ● , or -SSR ● (In the formula, each R ● If it is unsubstituted or preceded by "halo", it is substituted by only one or more halogens, and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1(Ph is selected from aryl rings having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which are saturated, partially unsaturated, or have 5 to 6 members.) ○ Suitable divalent substituents on the saturated carbon atom include =O and =S.

[0078] Suitable divalent substituents on saturated carbon atoms of the "may be substituted" groups 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-(Each R that appears independently in the formula) * C is a hydrogen atom that can be substituted as follows: 1~6 Examples include aliphatic, or unsubstituted 5-6 member saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. A suitable divalent substituent bonded to an adjacent substituted carbon of the "may be substituted" group is: -O(CR * 2) 2~3 O-(Each R that appears independently in the formula) * C is a hydrogen atom that can be substituted as follows: 1~6 Examples include aliphatic, or unsubstituted 5-6 member saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0079] Suitable substituents on the aliphatic group R* include halogens, -R ● ,-(HaroR ● ), -OH, -OR ● ,-O(HaroR ● ), -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● , -NR ●2, or -NO2 (wherein each R ● If unsubstituted or preceded by "halo", it is substituted by only one or more halogens, and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Examples include a pH (or an aryl ring that is 5-6 member saturated, partially unsaturated, or has 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0080] A suitable substitutionable substituent on the nitrogen of a group that "may be substituted" is -R † , -NR † 2, -C(O)R † , -C(O)OR † ,-C(O)C(O)R † -C(O)CH2C(O)R † -S(O)2R † , S(O)2NR † 2, -C(S)NR † 2, -C(NH)NR † 2, or -N(R † )S(O)2R † ;(In the formula, each R † Independently, halogens, the following C can be substituted. 1~6 Aliphatic, unsubstituted-OPh, or unsubstituted 5-6 member 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 † However, these intervening atoms, together with each other, independently form unsubstituted 3-12 member saturated, partially unsaturated, or aryl monocyclic or bicyclic rings having 0-4 heteroatoms selected from nitrogen, oxygen, or sulfur.

[0081] Suitable substituents on the aliphatic group of R† are, independently, halogens, -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, if preceded by "halo", substituted with only one or more halogens, and independently, C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6 member 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 “administered orally” refer to the administration of a compound or composition by mouth and have the meanings understood in the art.

[0083] Parenteral: As used herein, the terms “parenteral administration” and “administered parenterally” have the meaning as understood in the art, usually by injection, and not by intestinal or topical administration, including, but not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0084] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring portion containing at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple unsaturated parts, but not to include aryl or heteroaryl parts as defined herein.

[0085] When used herein, the term “pharmaceutical composition” refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose appropriate for administration in a therapeutic 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 for the following uses: oral administration, e.g., liquid drugs (aqueous or non-aqueous solutions or suspensions), tablets, e.g., for oral, sublingual, and systemic absorption uses, pills, powders, granules, pastes applied to the tongue; parenteral administration, e.g., sterile solutions or suspensions, or as sustained-release formulations, e.g., by subcutaneous, intramuscular, intravenous or epidural injection; topical administration, e.g., as creams, ointments, or sustained-release patches or sprays applied to the skin, lungs, or oral cavity; e.g., as pessaries, creams, or foams administered intravaginally or rectally; sublingually; into the eyes; percutaneously; or nasally, into the lungs, and other mucous membranes.

[0086] Pharmacopoeia-acceptable: As used herein, the phrase “pharmacopoeia-acceptable” means a compound, substance, composition, and / or dosage form that, within sound medical judgment, is appropriate for use in contact with human and animal tissues, with a reasonable benefit / risk ratio, without excessive harm, irritation, allergic response, or other problems or complications.

[0087] Pharmacopoeia-acceptable carrier: As used herein, the term “pharmacopoeia-acceptable carrier” means a pharmacopoeia-acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulant, involved in the transport or delivery of a substance from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; 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 polyacid anhydrides; and other harmless and suitable substances used in pharmaceutical formulations.

[0088] Pharmacopoeia-acceptable salts: As used herein, the term “pharmacopoeia-acceptable salt” means a salt of such compound that is appropriate for use in a pharmaceutical context, i.e., a salt that, within sound medical judgment, is commensurate with a reasonable benefit / risk ratio, is free from excessive harm, irritation, allergic reactions, etc., and is appropriate for use in contact with human and lower animal tissues. Pharmacopoeia-acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmacopoeia-acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanate, glycerophosphate, gluconate, hemisulfate, heptanate, hexanoate, hydroiodide, and 2-hydroxyethane. Examples of pharmaceutically acceptable salts include, but are not limited to, sulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonic acids, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, and valersates. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. In some embodiments, pharmaceutically acceptable salts may include, where appropriate, harmless ammonium, quaternary ammonium, and amine cations produced using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates and arylsulfonates having 1 to 6 carbon atoms.

[0089] Prodrug: Generally, a “prodrug,” as used herein and understood in the art, is an entity that, when administered to a living organism, is metabolized in the body to deliver an active agent of interest (e.g., therapeutic or diagnostic). 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 may be provided in various forms, such as crystalline, salt, and others. In some embodiments, prodrugs are provided as pharmaceutically acceptable salts thereof.

[0091] Protecting groups: As used herein, the term “protecting groups” includes those well known in the art, including those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999 (the entire text of which is incorporated herein by reference). It also includes protecting groups particularly applicable to nucleotides and nucleotide chemistry, as 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-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfo)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluorenyl methyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxantyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenanthyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2- Phenylethylcarbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethylcarbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2'-and 4'-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamide) ethylcarbamate, t-butylcarbamate (BOC), 1-adamantylcarbamate (Adoc), vinylcarbamate (Voc), allylcarbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamylcarbamate (Coc), 4-nitrocinnamylcarbamate (Noc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, alkyldithiocarbamate, benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), p- Trobenzylcarbamate, 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 Phenylcarbamate (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acryloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenz Carbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, t-amylcarbamate, S-benzylthiocarbamate, p-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate, 2,2-Dimethoxycarbonylvinylcarbamate, o-(N,N-dimethylcarboxamide)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamide)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methyl Lucyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, 2,4,6-trimethylbenzyl Lucarbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenoxy (L)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazofenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, 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-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, Quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosberylamine, 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 Rubonic 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), dialkylphosphoramidates, dibenzylphosphoramidate, diphenylphosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (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- Examples include 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] Appropriately protected carboxylic acids include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and triisopropylsilyl. Suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and tetrahydropyran-2-yl. Suitable alkenyl groups include allyl. Suitable aryl groups include phenyl, biphenyl, or naphthyl, which may be substituted. Suitable arylalkyl groups include benzyl, which may be substituted (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, (phenyldimethylyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, and 2-methoxyethoxymethyl (ME). 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-methoxypiperazine-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-tri Loroethyl, 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-Dibenzosberyl, 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(Lebrinoyloxyphenyl)methyl, 4,4',4''-Tris(Benzoyloxyphenyl)methyl, 3-(Imidal Zole-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltex Lucilyl, 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 ester Acid esters, 3-phenylpropionic acid ester, 4-oxopentanoic acid ester (levulinic acid ester), 4,4-(ethylenedithio)pentanoic acid ester (levulinoyl dithioacetal), pivalic acid ester, adamantate, crotonic acid ester, 4-methoxycrotonic acid ester, benzoic acid ester, p-phenylbenzoic acid ester, 2,4,6-trimethylbenzoic acid ester (mesitoate), alkylmethyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkylethyl carbonate, alkyl 2,2,2-Trichloroethyl (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkylisobutyl carbonate, alkylvinyl carbonate, alkylallyl carbonate, alkylp-nitrophenyl carbonate, alkylbenzyl carbonate, alkylp-methoxybenzyl carbonate, alkyl3,4-dimethoxybenzyl carbonate, alkylo-nitrobenzyl carbonate, alkylp-nitrobenzyl carbonate, alkylsodium-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, dithiomethyl carbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonic acid, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate Examples include acid esters, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid, chlorodiphenyl acetate, isobutyric acid, monosuccinate, (E)-2-methyl-2-butenoic acid, o-(methoxycarbonyl)benzoate, α-naphthoic acid, nitrate, alkyl N,N,N',N'-tetramethylphosphodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothonyl, alkyl 2,4-dinitrophenyl sulfenate, sulfate, methanesulfonic acid (mesylate), benzylsulfonic acid, and tosylate (Ts). To protect 1,2- or 1,3-diols, the protecting group may be 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 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 derivative, α-(N,N'-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonate esters, cyclic boronic acid esters, ethyl boronic acid, and phenyl boronic acid.

[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, benzoyl ester, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl, ( These include 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, levlinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthene-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 group is selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.

[0095] In some embodiments, the phosphite protecting group is a group bonded to the internucleotide phosphite bond throughout the oligonucleotide synthesis. In some embodiments, the phosphite protecting group is bonded to the sulfur atom of the internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is bonded to the oxygen atom of the internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is bonded to the oxygen atom of the 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-butylcarboxamide)-1-propyl, 4-oxopentyl, 4-methylthio-l-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0096] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked together 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 portion that forms one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues of the protein chain contain non-amino acid portions (e.g., glycans, etc.). In some embodiments, a protein contains one or more polypeptide chains linked, for example, 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 well known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. The term "peptide" is generally used to refer to a polypeptide 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, its biologically active portion, and / or its characteristic portion.

[0097] Sample: As used herein, “sample” refers to a specific organism or material obtained therefrom. In some embodiments, a sample is a biological sample obtained from or derived from a source of interest as described herein. In some embodiments, the source of interest includes an animal or a living organism such as a human. In some embodiments, a biological sample includes biological tissue or biological fluid. In some embodiments, a biological sample includes bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy specimens; cell-containing fluids; suspended nucleic acids; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washes or lavages such as duct lavage or bronchoalveolar lavage; aspirates; scrapes; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other bodily fluids, secretions, and / or cells therefrom, or any one or more of them. In some embodiments, a biological sample includes cells obtained from an individual, or cells therefrom. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any suitable means. For example, in some embodiments, the 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.), and others. In some embodiments, as will be apparent from the context, the term “sample” refers to a preparation obtained by processing the primary sample (e.g., by removing one or more of its components and / or adding one or more agents to it). For example, filtration using a semipermeable membrane. Such a “processed sample” may include nucleic acids or proteins obtained by extraction from the sample or by processing the primary sample by techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components, and others. In some embodiments, the sample is a living 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 term “stereochemical isomers” refers to different compounds that are assembled from the same atoms bonded together by the same set of bonds but have different, incompatible three-dimensional structures. In some embodiments of the present invention, the provided chemical composition may be, or may contain, pure synthesis of individual stereochemical isomers of a compound; in some embodiments, the provided chemical composition may be, or may contain, a mixture of two or more stereochemical isomers of the compound. In certain embodiments, such a mixture may contain equal amounts of different stereochemical isomers; in certain embodiments, such a mixture may contain different 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 fewer diastereomers and / or enantiomers than all of the compound. In some embodiments, if a particular enantiomer of a compound of the present invention is desired, it may be synthesized, for example, by asymmetric synthesis or by induction using chiral auxiliary groups, the resulting diastereomer mixture may be separated, and the auxiliary groups may be cleaved to obtain the pure, desired enantiomer. Alternatively, if the molecule contains basic functional groups such as amino acids, the diastereomer salt can be generated using an appropriate optically active acid and then separated, for example, by fractional recrystallization.

[0099] Subject: As used herein, the terms “subject” or “subject” refer to any organism to which the provided compound or composition is administered according to the invention, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mice, rats, rabbits, non-human primates, and humans; insects; worms; and other mammals) and plants. In some embodiments, the subject suffers from and / or is susceptible to disease, disorder, and / or symptoms.

[0100] Substantial: As used herein, the term “substantial” describes a qualitative state indicating the entire or nearly entire range or degree of the feature or characteristic under consideration. Those skilled in the field of biological technology will understand that biological and chemical phenomena rarely avoid completion and / or finish, achievement, or absolute results. Therefore, the term “substantial” is used herein to capture the inherent lack of completeness in many biological and / or chemical phenomena.

[0101] Afflicted: An individual “suffering” from a disease, disorder, and / or symptom has been diagnosed with and / or exhibits one or more symptoms of a disease, disorder, and / or symptom.

[0102] Susceptible to disease: Individuals who are "susceptible" to disease, disorder, and / or symptoms are at a higher risk of developing such disease, disorder, and / or symptoms than members of the general population. In some embodiments, individuals susceptible to disease, disorder, and / or symptoms may not be diagnosed with such disease, disorder, and / or symptoms. In some embodiments, individuals susceptible to disease, disorder, and / or symptoms may exhibit symptoms of such disease, disorder, and / or symptoms. In some embodiments, individuals susceptible to disease, disorder, and / or symptoms may not exhibit symptoms of such disease, disorder, and / or symptoms. In some embodiments, individuals susceptible to disease, disorder, and / or symptoms will develop such disease, disorder, and / or symptoms. In some embodiments, individuals susceptible to disease, disorder, and / or symptoms will not develop such disease, disorder, and / or symptoms.

[0103] Systemic: As used herein, the phrases “systemic administration,” “systemically administered,” “peripherally administered,” and “peripherally administered” have the meanings understood in the art to mean that the compound or composition is administered so that it enters the whole body of the recipient.

[0104] Tautomers: As used herein, the term “tautomer” is used to describe organic compounds that are readily convertible isomers of different isomers. Tautomers can be characterized by formal transfer of hydrogen atoms or protons, occurring simultaneously with the conversion of single bonds and adjacent double bonds. In some embodiments, tautomers can result from proton tautomerism (i.e., rearrangement of protons). In some embodiments, tautomers can result from valence tautomerism (i.e., rapid rearrangement of bond electrons). All such tautomers are included within the scope of the invention. In some embodiments, tautomers of a compound exist in a movable equilibrium with respect to each other, such that an attempt to synthesize separate substances results in the production of a mixture. In some embodiments, tautomers of a compound are separable and isolateable compounds. In some embodiments of the invention, a chemical composition may be provided that is a pure synthesis of a single tautomer of a compound, or contains such a compound. In some embodiments of the invention, a chemical composition may be provided as a mixture 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 the compound. In some embodiments of the present invention, a chemical composition may contain all tautomers of the compound. In some embodiments of the present invention, a chemical composition may contain fewer tautomers than all of the compound. In some embodiments of the present invention, a chemical composition may contain one or more tautomers of the compound in amounts that change over time as a result of interconversion. In some embodiments of the present invention, the tautomerism is ketoenol tautomerism. Those skilled in the art of chemical art can “capture” (i.e., chemically modify to retain the “enol” form) ketoenol tautomerism using any suitable reagent known in the art, and subsequently obtain an enol derivative that can be isolated using one or more suitable techniques known in the art. Unless otherwise specified, the present invention encompasses all tautomers of the relevant compounds, whether in pure form or mixtures thereof.

[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, relieve, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or decrease the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0106] Therapeutic dose: As used herein, the term “therapeutic dose” means the amount of a substance (e.g., a therapeutic agent, composition, and / or prescription) administered as part of a therapeutic regimen that elicits a desired biological response. In some embodiments, the therapeutic dose of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or symptom when administered to a subject suffering from or susceptible to said disease, disorder, and / or symptom. As will be recognized by those skilled in the art, the effective dose of a substance may vary depending on factors such as the desired biological endpoint, the substance being delivered, the target cells or tissues, and others. For example, the effective dose of a compound in a prescription for treating a disease, disorder, and / or symptom is an amount that reduces, remits, relieves, suppresses, prevents, delays the onset, reduces the severity, and / or lowers the incidence of one or more symptoms or characteristics of said disease, disorder, and / or symptom. In some embodiments, the therapeutic dose is administered in a single dose; in some embodiments, multiple unit doses are required to deliver the therapeutic dose.

[0107] Treatment: As used herein, the terms “to treat,” “to treat,” or “to treat” describe any method used to partially or completely alleviate, remit, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or decrease the incidence of one or more symptoms or features of a disease, disorder, and / or symptom. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or symptom. In some embodiments, for example, for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and / or symptom, treatment may be administered to subjects who show only the initial signs of said disease, disorder, and / or symptom.

[0108] Unsaturated: As used herein, the term “unsaturated” means that a part has one or more unsaturated units.

[0109] Unit Dose: As used herein, the expression “unit dose” refers to a single dose of a pharmaceutical composition and / or an amount administered in physically separate units. In many embodiments, a unit dose comprises a predetermined amount of the active agent. In some embodiments, a unit dose comprises the 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 may be, for example, a predetermined amount of one or more therapeutic agents, a predetermined amount of solid form, a sustained-release formulation or a drug delivery device containing a predetermined amount of one or more therapeutic agents, or a certain volume of liquid (e.g., an acceptable carrier). It will be recognized that a unit dose may be present in a formulation containing any of the various components in addition to the therapeutic agent. For example, an acceptable carrier (e.g., a pharmaceutically acceptable carrier), diluents, stabilizers, buffers, preservatives, etc., may be included, as described below. It will be understood by those skilled in the art that in many embodiments, the appropriate total daily dose of a particular therapeutic agent may consist of a portion or a number of unit doses, which may be determined, for example, by the attending physician within the bounds of sound medical judgment. In some embodiments, the specific effective dose level for any particular subject or organism may depend on a variety of factors, including the disorder being treated and its severity; the activity of the specific active compound used; the specific composition used; the subject's age, weight, health status, sex, and diet; the number of doses and the rate of excretion of the specific compound used; the duration of treatment; any drugs and / or additional therapies used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0110] Wild-type: As used herein, the term “wild-type” has the meaning understood in the art of representing an entity having the structure and / or activity actually found in a “normal” state or context (as opposed to mutated, diseased, altered, etc.). Those skilled in the art will recognize that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0111] Nucleic acids: The term “nucleic acid” includes any nucleotide, its modified variants, its analogues, and its polymers. As used herein, the term “polynucleotide” refers to any polymeric form of a nucleotide of any length, ribonucleotide (RNA), or deoxyribonucleotide (DNA), or any modified variant or analogue thereof. These terms refer to the primary structure of a molecule and therefore include double-stranded and single-stranded DNA and double-stranded and single-stranded RNA. These terms include, as equivalents, nucleotide analogues such as (but not limited to) methylated, protected, and / or capped nucleotides or polynucleotides, and analogues of RNA or DNA made from modified polynucleotides. These terms encompass nucleic acids that include poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleic acid bases and / or modified nucleic acid bases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate crosslinks and / or modified phosphorus atom crosslinks (also referred to herein as “internucleotide bonds”). These terms encompass nucleic acids that include any combination of nucleic acid bases, modified nucleic acid bases, sugars, modified sugars, phosphate crosslinks, or modified phosphorus atom crosslinks. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxy-ribose moiety, nucleic acids containing both a ribose moiety and a deoxyribose moiety, and nucleic acids containing a ribose moiety and a modified ribose moiety. The prefix poly- refers to nucleic acids containing 2 to about 10,000 nucleotide monomer units, where the prefix oligo- refers to nucleic acids containing 2 to about 200 nucleotide monomer units.

[0112] Nucleotides: As used herein, the term “nucleotide” refers to the monomeric unit of a polynucleotide, which consists of a heterocyclic base, a sugar, and one or more phosphate groups or phosphorus-containing internucleotide links. Naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but should be understood to include natural and unnatural base analogs. Natural sugars are pentoses (five-carbon sugars), deoxyribose (which forms DNA), or ribose (which forms RNA), but should be understood to include natural and unnatural sugar analogs. Nucleotides are linked via internucleotide links to form nucleic acids or polynucleotides. Many internucleotide links are known in the art (e.g., phosphates, phosphorothioates, boranophosphates, etc.). Artificial nucleic acids include PNA (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids, as described herein. Other analogues (e.g., artificial nucleic acids, or components that can be incorporated into nucleic acids or artificial nucleic acids) include: boranophosphate RNA, FANA, immobilized nucleic acids (LNA), morpholino, peptide nucleic acids (PNA), threose nucleic acids (TNA), and glycol nucleic acids (GNA). Those skilled in the art are aware of various modified nucleotides or nucleotide analogs, including, for example, those described in any of the following: Gryaznov, S; Chen, J.-K. J.A.M.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.AcidsRes.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.NYAcad.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; International Publication No. 20070900071; or International Publication No. 2016 / 079181.

[0113] Nucleoside: The term "nucleoside" refers to the portion of a molecule in which a nucleic acid base or modified nucleic acid base is covalently bonded to a sugar or modified sugar.

[0114] Sugars: The term “sugar” refers to monosaccharides in closed and / or open forms. Examples of sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. In this specification, the term also includes structural analogues in which their polymers are used in place of ordinary sugar molecules, such as nucleic acid analogues, glycols that form the backbone of glycol nucleic acids ("GNAs").

[0115] Modified sugar: The term "modified sugar" refers to a part of a sugar that can be replaced. These modified sugars mimic the spatial arrangement, electronic state, or some other physicochemical properties of the sugar.

[0116] Nucleic acid bases: The term “nucleic acid base” refers to a nucleic acid moiety associated with hydrogen bonding that links one nucleic acid chain to another complementary chain in a sequence-specific manner. Most natural nucleic acid bases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the natural nucleic acid base is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the natural nucleic acid base is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleic acid base is a “modified nucleic acid base,” e.g., adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleic acid base is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleic acid bases mimic the spatial arrangement, electronic state, or several other physicochemical properties of the nucleic acid bases and retain the properties of hydrogen bonding, which links one nucleic acid chain to another in a sequence-specific manner. In some embodiments, the modified nucleic acid bases can pair with the five all-natural bases (uracil, thymine, adenine, cytosine, or guanine) with substantially no effect on melting behavior, recognition by intercellular enzymes, or the activity of oligonucleotide doubles.

[0117] Chiral ligand: The term "chiral ligand" or "chiral auxiliary agent" refers to a part of a reactant that is chiral and can be incorporated into the reactants so that the reaction can be carried out with specific stereoselectivity.

[0118] Condensation reagent: In a condensation reaction, the term "condensation reagent" refers to a reagent that activates a less reactive site, thereby increasing its sensitivity to interaction with another reagent. In some embodiments, this other reagent is a nucleophile.

[0119] Blocking group: The term "blocking group" refers to a group that shields the reactivity of a functional group. The functional group may subsequently lose its shielding by removing the blocking group. In some embodiments, the blocking group is a protecting group.

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

[0121] Solid Carrier: The term “solid carrier” refers to any carrier that enables the synthesis of nucleic acids. In some embodiments, the term refers to a glass or polymer insoluble in the medium used in the reaction step that carries out nucleic acid synthesis and derivatizes to introduce reactive groups. In some embodiments, the solid carrier is highly crosslinked polystyrene (HCP) or controlled pore glass (CPG). In some embodiments, the solid carrier is controlled pore glass (CPG). In some embodiments, the solid carrier is a composite carrier of controlled pore glass (CPG) and highly crosslinked polystyrene (HCP).

[0122] Binding portion: The term “binding portion” refers to any portion that may be located between a terminal nucleotide and the solid carrier, or between a terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0123] DNA molecule: The term “DNA molecule” refers to its single-stranded form or the polymerized form of its double-helical deoxyribonucleotides (adenine, guanine, thymine, or cytosine). This term refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Accordingly, this term includes, among other things, linear DNA molecules (e.g., restriction enzyme fragments), viruses, plasmids, and double-stranded DNA found in chromosomes. In discussing a particular double-stranded DNA molecular structure, sequences may be described herein in accordance with the convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a homologous sequence to mRNA).

[0124] Coding Sequence: A "coding sequence" or "coding region" of DNA is double-stranded DNA that, when controlled by appropriate expression regulatory sequences, is transcribed and translated into endogenous polypeptides. The boundaries of a coding sequence ("open reading frame" or "ORF") are determined by a start codon at the 5' (amino) end and a translation termination codon at the 3' (carboxylic acid) end. Examples of coding sequences, but not limited to, include 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 on the 3' side of the coding sequence. The terms "non-coding sequence" or "non-coding region" refer to regions of polynucleotide sequences that are not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0125] Reading Frame: The term "reading frame" refers to one of six possible reading frames, three for each direction in a double-stranded DNA molecule. The reading frame used determines which codons to use to encode the amino acids in 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, for example, the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence, or complementary to the coding strand of a gene, or complementary to a protein-coding “sense” nucleic acid. Thus, an antisense nucleic acid molecule can associate with a sense nucleic acid molecule via hydrogen bonding. In some embodiments, an antisense oligonucleotide is an oligonucleotide involved in RNaseH-mediated cleavage; for example, an antisense oligonucleotide sequence-specifically hybridizes with a portion of a target mRNA and thus targets the mRNA for RNaseH cleavage. In some embodiments, an antisense oligonucleotide can distinguish between wild-type and target mutant alleles. In some embodiments, an antisense oligonucleotide is heavily involved in RNaseH-mediated cleavage of a mutant allele but to a much lesser extent in RNaseH-mediated cleavage of a wild-type allele (e.g., not heavily involved in RNaseH-mediated cleavage of a target wild-type allele).

[0127] Fluctuation Position: As used herein, “fluctuation position” refers to the third position of a codon. In some embodiments, mutations in a DNA molecule within a codon fluctuation position result in silent or conserved mutations at the amino acid level. For example, there are four codons encoding glycine, namely GGU, GGC, GGA, and GGG, and therefore, mutations of any nucleotide at any fluctuation position to any other nucleotide selected from A, U, C, and G result in no change at the amino acid level of the encoded protein and are therefore silent substitutions.

[0128] Silent substitution: A "silent substitution" or "silent mutation" is a mutation in which a nucleotide within a codon is changed, but the amino acid residue encoded by that codon is not altered. An example is a mutation in the third position of a codon, such as the codon "CGG" which encodes Arg, even though the mutation occurs at the first 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 codes for a protein or a portion thereof. The DNA molecule may include an open reading frame that codes for the protein (as an exon sequence) and may further include an intron sequence. In this specification, the term “intron” refers to a DNA sequence present in a given gene that is not translated into a protein, and, in some but not all cases, is found between exons. As is well known in the art, it may be desirable for a gene to be operably bound (or may include) one or more promoters, enhancers, repressors, and / or other regulatory sequences that modulate the activity or expression of the gene.

[0130] Complementary DNA: As used herein, “complementary DNA” or “cDNA” comprises recombinant polynucleotides synthesized by reverse transcription of mRNA, from which intervening sequences (introns) are removed.

[0131] Homology: “Homologie,” “identity,” or “similarity” refers to the sequence similarity between two nucleic acid molecules. Homology and identity can be determined, respectively, by comparing the positions of each sequence that may be placed for comparison. When the same position in the sequences being compared 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 residues (e.g., similar in steric and / or electronic state), then the molecules may be said to be homologous (similar) at that position. The percentage expression of homology / similarity or identity represents a function of the number of identical or similar nucleic acids at the positions shared by the sequences being compared. “Irrelevant” or “non-homologous” sequences share less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with the sequences described herein. When comparing two sequences, the absence or presence of extra residues (amino acids or nucleic acids) also reduces their identity and homology / similarity.

[0132] In some embodiments, the term “homology” describes a mathematically based comparison of sequence similarities used in identical genes with similar functions or motifs. The nucleic acid sequences described herein may 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 may 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, a BLAST nucleotide search may be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. In some embodiments, a gap BLAST may be used to obtain gap alignment for comparison purposes, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and GapBLAST programs, the default parameters for each program (e.g., 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 to the greatest extent possible, 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). The identity determination method is designed to maximize the match between the test sequences. Furthermore, the identity determination method is coded in a publicly available computer program.Computer programming methods for determining the 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, SF 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 can also be used to determine identity.

[0134] Non-homologous: A “non-homologous” region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not found at all in relation to the larger sequence. Therefore, when a non-homologous region codes for a mammalian gene, that gene may typically be on a side of the DNA that is not adjacent to the mammalian genomic DNA in the source genome. Another example of a non-homologous coding sequence is one in which the coding sequence itself is not found at all (e.g., a cDNA in which the genomic coding sequence contains introns or synthetic sequences that have different codons or motifs from the unmodified gene). Allele mutations or spontaneous mutation events do not result in non-homologous regions of DNA as defined herein.

[0135] Transposition mutation: The term "transposition mutation" refers to a change in a DNA sequence where a pyrimidine (cytidine (C) or thymidine (T)) is substituted by another pyrimidine, or a purine (adenosine (A) or guanosine (G)) is substituted by another purine.

[0136] Trans-base mutation: The term "trans-base mutation" refers to a 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 that includes any combination of a nucleic acid base, a modified nucleic acid base, a sugar, a modified sugar, a phosphate bridge, or a modified phosphorus atom bridge (also referred to herein as “nucleotide-nucleotide bond” as further defined herein).

[0138] Oligonucleotides can be single-stranded or double-stranded. In this specification, the term “oligonucleotide chain” encompasses single-stranded oligonucleotides. Single-stranded oligonucleotides may have double-stranded regions, and double-stranded oligonucleotides may have single-stranded regions. Examples of oligonucleotides include, but are not limited to, structural genes, genes containing regulatory and terminal regions, viruses or plasmid DNA, self-renewal systems such as single-stranded and double-stranded siRNAs and other RNA interferants (RNAi agents or iRNA agents), shRNAs, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, ul-adapters, triple-stranded oligonucleotides, guanine quadruple-stranded oligonucleotides, RNA activators, immunostimulant oligonucleotides, and decoy oligonucleotides.

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

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

[0141] Internucleotide linkage: As used herein, the term “internucleotide linkage” generally refers to a phosphorus-containing linkage between nucleotide units of an oligonucleotide and is synonymous with “intersugar linkage” and “phosphorus atom bridge” above and herein. 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 substituted by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are selected from, but are 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. [ka] Alternatively, it is a modified phosphorothioate triester bond. Those skilled in the art will understand that the internucleotide bond may exist as an anion or a cation at a given pH due to the presence of the acidic or base portion of the bond.

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

[0143] Table 1. Examples of modified nucleotide bond formations. [Table 1] TIFF2026086654000003.tif224166TIFF2026086654000004.tif96166

[0144] For example, (Rp,Sp)-ATsCs1GA is 1) a phosphorothioate nucleotide bond between T and C. ( [ka] ) ; and 2) between C and G [ka] It has a phosphorothioate triester nucleotide internucleotide bond having the structure shown. Unless otherwise specified, the Rp / Sp notation preceding an oligonucleotide sequence indicates the stereochemistry of the chiral phosphorus atoms of the nucleotide internucleotide bond sequentially from 5' to 3' in the oligonucleotide sequence. For example, in (Rp,Sp)-ATsCs1GA, the phosphorus in the "s" bond between T and C has the Rp configuration, and the phosphorus in 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 in the oligonucleotide have the same Rp or Sp configuration, respectively. For example, all(Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsCsGsCsAsCsC indicates that all of the chiral phosphorus atoms in the oligonucleotide have an Rp configuration; all(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsGsCsTsCsGsCsAsCsC indicates that all of the chiral phosphorus atoms in the oligonucleotide have an Sp configuration.

[0145] Oligonucleotide type: As used herein, the term “oligonucleotide type” refers to a specific base sequence, skeletal bonding pattern (i.e., internucleotide bonding pattern, e.g., phosphate, phosphorothioate, etc.), skeletal chiral center pattern (i.e., bonding phosphorus stereochemical pattern (Rp / Sp)), and skeletal phosphorus modification pattern (e.g., “-XLR” of formula I). 1 This is used to define oligonucleotides having a "group pattern". Oligonucleotides of a commonly denoted "type" are structurally identical to one another.

[0146] Those skilled in the art will recognize that the synthesis method of the present invention provides some degree of control during the synthesis of oligonucleotide chains, such that each nucleotide unit of an oligonucleotide chain can be designed and / or pre-selected such that it has a specific stereochemistry and / or specific modification and / or specific base and / or specific sugar at the bounding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the bounding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected because it has a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have one or more of the above structural features in a specific combination. The present invention provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chiral-controlled oligonucleotide compositions). In some embodiments, all such molecules are of the same type (i.e., structurally identical to one another). However, in many embodiments, the compositions provided typically contain a plurality of 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 phosphorus within an oligonucleotide chain. The term “chiral controlled oligonucleotide” refers to an oligonucleotide that exists as a single diastereomer with respect to chiral phosphorus. Chiral controlled oligonucleotides are prepared from chiral controlled oligonucleotide synthesis.

[0148] Chiral-controlled oligonucleotide compositions: As used herein, the phrase “chiral-controlled oligonucleotide composition” refers to an oligonucleotide composition comprising a predetermined level of individual oligonucleotide types. For example, in some embodiments, a chiral-controlled oligonucleotide composition comprises one oligonucleotide type. In some embodiments, a chiral-controlled oligonucleotide composition comprises a mixture of multiple oligonucleotide types. Specific chiral-controlled oligonucleotide compositions are further described herein.

[0149] Chiral purity: In this specification, the term “chiral purity” is used to describe a chiral controlled oligonucleotide composition in which all oligonucleotides exist as a single diastereoisomer with respect to the bound phosphorus.

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

[0151] "Predetermined": "Predetermined" means, for example, selected systematically, as the opposite of random occurrence or achievement. Those skilled in the art will read this specification and understand that the present invention provides novel and remarkable techniques that enable the selection of specific oligonucleotide types for the preparation and / or encapsulation of the provided compositions, and further enable controlled preparation of the selected specific types, precisely, in selected specific relative amounts, as the provided compositions are prepared. Such provided compositions are the "predetermined" ones described herein. Compositions that may contain specific individual oligonucleotide types are not "predetermined" compositions because they are created by chance, through processes in which the intentional creation of specific oligonucleotide types is uncontrollable. In some embodiments, the predetermined compositions are those that can be intentionally replicated (for example, through iterations of a controlled process).

[0152] Binding Phosphorus: When defined herein, the phrase “binding phosphorus” is used to indicate that a particular phosphorus atom represented is present in the internucleotide bond, and that the phosphorus atom corresponds to the phosphorus atom of the phosphodiester in the internucleotide bond occurring in native DNA and RNA. In some embodiments, the binding phosphorus atom is present in the modified internucleotide bond, and each oxygen atom of the phosphodiester bond is optionally and independently substituted by an organic or inorganic moiety. In some embodiments, the binding phosphorus atom is P of formula I. * In some embodiments, the bonded phosphorus atom is chiral. In some embodiments, the chiral bonded phosphorus atom is P of formula I. * That is the case.

[0153] P-modification: As used herein, the term "P-modification" refers to any modification of bound phosphorus other than stereochemical modification. In some embodiments, P-modification includes the addition, substitution, or removal of a covalently bound suspension portion to bound phosphorus. In some embodiments, the "P-modification" is -XLR 1 (wherein X, L and R 1 (These terms are defined and described independently in this specification and below.)

[0154] Blockmer: As used herein, the term “blockmer” refers to an oligonucleotide chain characterized by the presence of at least two consecutive nucleotide units that share a common structural feature in the internucleotide phosphorus bond. The common structural feature means a common stereochemistry or common modification in the bounding phosphorus. In some embodiments, the at least two consecutive nucleotide units that share a common structural feature in the internucleotide phosphorus bond are referred to as a “block.”

[0155] In some embodiments, the blockmer is a “stereoblockmer,” where, for example, at least two consecutive nucleotide units have the same stereochemistry at the bound phosphorus. Such at least two consecutive nucleotide units form a “stereoblockmer.” For example, (Rp,Sp)-ATsCs1GA is a stereoblockmer because at least two consecutive nucleotide units, Ts and Cs1, have the same stereochemistry at the bound phosphorus (both Sp). With the same oligonucleotide, (Rp,Sp)-ATsCs1 forms a block, which is a stereoblock.

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

[0157] In some embodiments, a blockmer is a “binding blockmer,” for example, at least two consecutive nucleotide units having the same stereochemistry and the same modifications at the binding phosphorus. At least two consecutive nucleotide units form a “binding block.” For example, (Rp,Rp)-ATsCsGA is a binding blockmer 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 binding block.

[0158] In some embodiments, the blockmer comprises one or more blocks independently selected from stereo blocks, P-modified blocks, and combined blocks. In some embodiments, the blockmer is a stereo blockmer for one block and / or a P-modified blockmer for another block and / or a combined blockmer for yet another block. For example, (Rp,Rp,Rp,Rp,Rp,Sp,Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG is a stereoblocker with respect to the stereoblock AsTsCsGsAs1 (all Rp in bound phosphorus) or Ts1Cs1Gs1 (all Sp in bound phosphorus), a P-modified blocker with respect to the P-modified block AsTsCsGs (all s bonds) or As1Ts1Cs1Gs1 (all s1 bonds), or a binding blocker with respect to the binding block AsTsCsGs (all Rp and all s bonds in bound phosphorus) or Ts1Cs1Gs1 (all Sp and all s1 bonds in bound phosphorus).

[0159] Altmer: As used herein, the term “altmer” refers to an oligonucleotide chain characterized by the absence of two consecutive nucleotide units in the oligonucleotide chain that share a particular structural feature in the internucleotide phosphorus bond, characterized by the structural feature pattern that characterizes each individual nucleotide unit. In some embodiments, the altmer is designed to include a repeating pattern. In some embodiments, the altmer is designed not to include a repeating pattern.

[0160] In some embodiments, the altomer is a "stereoaltomer," where, for example, there are no two consecutive nucleotide units having the same stereochemistry in the bound phosphorus. For example, (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Rp,Rp,Rp,Rp,Rp,Rp,Rp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

[0161] In some embodiments, the altomer is a "P-modified altomer," where, for example, there are no two consecutive nucleotide units having the same modification in the bound phosphorus. For example, each bound phosphorus has a different P modification from the others, such as a total (Sp)CAs1GsT.

[0162] In some embodiments, the altomer is a "conjugated altomer" in which, for example, there are no two consecutive nucleotide units having the same stereochemistry or the same modifications in the conjugated phosphorus. For example, (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Rp,Rp,Rp,Rp,Rp,Rp,Rp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC.

[0163] Unimer: As used herein, the term "unimer" refers to an oligonucleotide chain in which the structural feature pattern characterizing each individual nucleotide unit is such that all nucleotide units in the chain share at least one common structural feature in the internucleotide phosphorus bond. The common structural feature means a common stereochemistry or common modification in the phosphorus bond.

[0164] In some embodiments, the unimer is a "stereounimer," where, for example, all nucleotide units have the same stereochemistry at the linked phosphorus. For example, all (Sp)-CsAs1GsT, where all the bonds have Sp phosphorus.

[0165] In some embodiments, the unimer is a "P-modified unimer," where, for example, all nucleotide units have the same modification in the linked phosphorus. For example, (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Rp,Rp,Rp,Rp,Rp,Rp,Rp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

[0166] In some embodiments, the unimer is a "binding unimer," where, for example, all nucleotide units have the same stereochemistry and the same modifications in the binding phosphorus. For example, all (Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

[0167] Gapmer: As used herein, the term “gapmer” refers to an oligonucleotide chain characterized by at least one internucleotide phosphorus bond being a phosphate diester bond, such as those found in native DNA or RNA. In some embodiments, one or more internucleotide phosphorus bonds of the oligonucleotide chain are phosphate diester bonds, such as those found in native DNA or RNA. For example, a total (Sp)-CAs1GsT chain where the internucleotide bond between C and A is a phosphate diester bond.

[0168] Skimmer: As used herein, the term “skimmer” refers to a type of gapmer in which the alternating internucleotide phosphorus bonds of the oligonucleotide chain are phosphate diester bonds, such as those found in natural DNA or RNA, and the alternating internucleotide phosphorus bonds of the oligonucleotide chain are modified internucleotide bonds, for example, all (Sp)-AsTCs1GAs2TCs3G.

[0169] For the purposes of this invention, the chemical elements are identified according to the periodic table of elements on the inside cover of the CAS ed., Handbook of Chemistry and Physics, 67th edition, 1986-87.

[0170] The methods and structures described herein relating to the compounds and compositions of the present invention also apply to pharmaceutically acceptable acids or base-adding acids, as well as to all stereoisomers of these compounds and compositions. [Brief explanation of the drawing]

[0171] [Figure 1] An example of the dose-response of C3a complement activation by a human SOD1-targeting oligonucleotide in pooled serum (from three individual cynomolgus monkeys), measured by C3a. 40-minute incubation at 37°C.

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

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

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

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

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

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

[0178] [Figure 8] An example of albumin binding by an oligonucleotide targeting human SOD1. [Modes for carrying out the invention]

[0179] Synthetic oligonucleotides provide useful molecular tools for a variety of applications. For example, oligonucleotides are useful for 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, various synthetic counterparts have been developed to circumvent these drawbacks. These synthetic counterparts include synthetic oligonucleotides that undergo chemical modifications, such as base modifications, sugar modifications, and skeleton modifications, and in particular, chemical modifications that make these molecules less susceptible to denaturation and improve other properties of the oligonucleotide. Chemical modifications can sometimes lead to certain undesirable effects, such as increased toxicity. From a structural standpoint, modifications to phosphate crosslinks between nucleotides introduce chirality, and certain properties of oligonucleotides can be influenced by the conformation of the phosphorus atoms that form the oligonucleotide skeleton. For example, in vitro studies have shown that the properties of antisense nucleotides, such as binding affinity, sequences that specifically bind to complementary RNA, and nuclease stability, are particularly influenced by the chirality of the cytoskeleton (e.g., the conformation of the phosphorus atom).

[0180] In particular, this disclosure includes the recognition that structural elements of oligonucleotides, such as the base sequence, chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide crosslinks, and their patterns), and / or stereochemistry (e.g., stereochemistry of the chiral centers in the skeleton (chiral internucleotide crosslinks), and / or their patterns), can significantly affect properties such as the activity and toxicity of oligonucleotides, and that these can be regulated to modify the properties of oligonucleotides. In some embodiments, oligonucleotide properties can be regulated by optimizing chemical modifications (modifications of bases, sugars, and / or internucleotide crosslinks) and / or stereochemistry (pattern of chiral centers in the skeleton).

[0181] In some embodiments, the disclosure shows that oligonucleotide compositions comprising oligonucleotides having controlled structural elements (e.g., controlled chemical modifications and / or controlled skeletal stereochemical patterns) provide unexpected properties, including, but not limited to, those described herein. In some embodiments, provided compositions comprising oligonucleotides having chemical modifications (e.g., base modifications, sugar modifications, internucleotide crosslink modifications, etc.) have improved properties, such as lower toxicity, an improved protein binding profile, or improved delivery. In some embodiments, the provided oligonucleotides in the provided composition (e.g., a first plurality of oligonucleotides) include base modifications, sugar modifications, and / or internucleotide crosslink modifications. In some embodiments, the provided oligonucleotides include base modifications and sugar modifications. In some embodiments, the provided oligonucleotides include base modifications and internucleotide crosslink modifications. In some embodiments, the provided oligonucleotides include sugar modifications and internucleotide modifications. In some embodiments, the provided compositions include base modifications, sugar modifications, and internucleotide crosslink modifications. Examples of chemical modifications (e.g., base modifications, sugar modifications, nucleotide crosslink modifications, etc.) are widely known in the art, including but not limited to those described herein. 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 modification. 1 In some embodiments, the 2'-modification is 2'-OR 1 And here, R 1is an alkyl which may be optionally substituted. 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 crosslinked bicyclic or polycyclic ring. In some embodiments, the modified sugar moiety is a crosslinked bicyclic or polycyclic ring containing 5 to 20 ring atoms, one or more of which may independently be heteroatoms. Examples of ring structures are widely known in the art and are found, for example, in BNA, LNA, etc. In some embodiments, the oligonucleotides provided contain both one or more modified internucleotide crosslinks and one or more natural phosphate crosslinks. In some embodiments, oligonucleotides and compositions containing both modified internucleotide crosslinks and natural phosphate crosslinks provide improved properties, such as activity and toxicity. In some embodiments, the modified internucleotide crosslinks are chiral internucleotide crosslinks. In some embodiments, the modified internucleotide crosslinks are phosphorothioate crosslinks. In some embodiments, the modified internucleotide crosslinks are substituted phosphorothioate crosslinks. In particular, this disclosure includes the recognition that sterically random oligonucleotide preparations contain multiple distinct chemical moies that differ from one another, for example, in the stereochemical structure of the individual chiral centers in the oligonucleotide chain. Without controlling the stereochemistry of the chiral centers in the chiral centers, sterically random oligonucleotide preparations give an uncontrolled composition containing an undetermined amount of oligonucleotide stereoisomers. These stereoisomers may have the same base sequence, but they are at least different chemical moies due to their different skeletal stereochemistry and may have different properties (e.g., activity, toxicity, etc.) as shown herein. In particular, this disclosure provides novel compositions that are specific stereoisomers of an oligonucleotide of interest, or that contain such stereoisomers. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, its length, the crosslinking pattern of its skeleton, and the pattern of chiral centers in its skeleton. As understood in the art, in some embodiments, the nucleotide sequence may refer to the identity and / or modification state of the nucleoside residue in the oligonucleotide (e.g., the identity and / or modification state of the sugar and / or base elements 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 specific complementary residues). In some embodiments, the oligonucleotides in the provided composition include sugar modifications, e.g., 2'-modifications, in the wing regions. In some embodiments, the oligonucleotides in the provided composition include a region in the center (e.g., the core region) that does not have sugar modifications. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a predetermined level of individual types of oligonucleotides, where the individual types of oligonucleotides are chemically identical, e.g., they have the same nucleotide sequence, the same pattern of nucleoside modifications (if any, modifications to the sugar and base portions), the same pattern of chiral centers in the skeleton, and the same pattern of phosphorus modifications in the skeleton.This disclosure shows, in particular, that individual stereoisomers of a particular oligonucleotide may exhibit different stabilities and / or activities (e.g., functional properties and / or toxic properties) from one another. In some embodiments, the disclosure shows that suitable improvements achieved by including and / or positioning a particular chiral structure in an oligonucleotide may be comparable to, or better than, those achieved by using (e.g., by using a particular type of modified phosphate [e.g., phosphorothioates, substituted phosphorothioates, etc.], sugar modifications [e.g., 2'-modifications, etc.], and / or base modifications [e.g., methylation, etc.]) the crosslinking of a particular skeleton, residue modifications, etc. In particular, this disclosure recognizes that in some embodiments, the properties of an oligonucleotide (e.g., activity, toxicity, etc.) can be modulated by optimizing the pattern of chiral centers in the skeleton, sometimes in combination with the modulation / optimization of one or more other features of the oligonucleotide (e.g., crosslinking pattern, nucleoside modification pattern, etc.). As illustrated by various examples of this disclosure, the chirality-controlled oligonucleotide compositions provided may exhibit improved properties, such as lower toxicity, improved protein binding profiles, and improved delivery.

[0182] In some embodiments, oligonucleotide properties can be tuned by optimizing stereochemistry (the pattern of chiral centers in the backbone) and chemical modifications (modification of bases, sugars, and / or internucleotide crosslinks). In particular, this disclosure shows that stereochemistry can further improve the properties of oligonucleotides including chemical modifications. In some embodiments, this disclosure provides oligonucleotide compositions in which the oligonucleotide comprises nucleoside modifications, chiral internucleotide crosslinks, and native phosphate crosslinks. For example, WV-1092 comprises 2'-OMe modifications, phosphate and phosphorothioate crosslinks in its 5'-wing and 3'-wing regions, and a phosphorothioate crosslink in its core region.

[0183] In some embodiments, this disclosure provides oligonucleotide compositions that unexpectedly greatly improve the properties of oligonucleotides. In some embodiments, the oligonucleotide compositions provided offer surprisingly low toxicity. In some embodiments, the oligonucleotide compositions provided offer a surprisingly improved protein binding profile. In some embodiments, the oligonucleotide compositions provided offer surprisingly enhanced delivery. In some embodiments, improvements in specific properties, such as low toxicity, an improved protein binding profile, and / or enhanced delivery, are achieved without sacrificing other properties, such as activity, specificity, etc. In some embodiments, the compositions provided offer low toxicity, an improved protein binding profile, 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, increased target specificity, increased cleavage site specificity, etc.) is described in, but is not limited to, WO / 2014 / 012081 and WO / 2015 / 107425.

[0184] In some embodiments, the chiral center pattern of the skeleton provides increased stability. In some embodiments, the chiral center pattern of the skeleton provides remarkably increased activity. In some embodiments, the chiral center pattern of the skeleton provides increased stability and activity. In some embodiments, the chiral center pattern of the skeleton provides remarkably low toxicity. In some embodiments, the chiral center pattern of the skeleton provides remarkably low immune response. In some embodiments, the chiral center pattern of the skeleton provides remarkably low complement activation. In some embodiments, the chiral center pattern of the skeleton provides remarkably low complement activation via an alternative pathway. In some embodiments, the chiral center pattern of the skeleton provides a remarkably improved protein binding profile. In some embodiments, the chiral center pattern of the skeleton provides remarkably increased binding to specific proteins. In some embodiments, the chiral center pattern of the skeleton provides remarkably enhanced delivery. In some embodiments, the chiral center pattern of the skeleton 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 chiral center pattern of the skeleton includes 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 chiral center pattern of the skeleton includes 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 chiral center pattern of the skeleton includes one or more achiral native phosphate crosslinks. In some embodiments, the chiral center pattern of the skeleton is (Sp) m (Rp) n , (Rp) n (Sp) m , (Np) t(Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m This includes, or includes one or more repetitions of these. In some embodiments described herein, m is 1 to 50; n is 1 to 10; t is 1 to 50. In some embodiments, the chiral center pattern of the skeleton includes, or includes (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 chiral center pattern of the skeleton is (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) mThe chiral center pattern of the skeleton is an array containing at least 5, 6, 7, 8, 9 or 10 or more consecutive (Sp) positions. In some embodiments, the chiral center pattern of the skeleton is an array containing at least 5 consecutive (Sp) positions. In some embodiments, the chiral center pattern of the skeleton is an array containing at least 8 consecutive (Sp) positions. In some embodiments, the chiral center pattern of the skeleton is an array containing at least 10 consecutive (Sp) positions. In some embodiments, the chiral center pattern of the skeleton is an array containing one (Rp) and all consisting of (Sp). In some embodiments, the chiral center pattern of the skeleton is an array containing one (Rp) at or adjacent to the SNP position and all consisting of (Sp). In some embodiments, the chiral center pattern of the skeleton is an array containing one (Rp) and all consisting of (Sp), where the molecule has a wing-core-wing morphology. In some embodiments, the chiral center pattern of the skeleton is an arrangement containing one (Rp) and all consisting of (Sp), where the molecule has a wing-core-wing morphology, with the wing at the 5' end being 1-9nt long, the core being 1-15nt long, and the wing at the 3' end being 1-9nt long. In some embodiments, the chiral center pattern of the skeleton is an arrangement containing one (Rp) and all consisting of (Sp), where the molecule has a wing-core-wing morphology, with the wing at the 5' end being 5nt long, the core being 1-15nt long, and the wing at the 3' end being 5nt long. In some embodiments, the chiral center pattern of the skeleton is an arrangement containing one (Rp) and all consisting of (Sp), where the molecule has a wing-core-wing morphology, with the wing at the 5' end being 1-9nt long, the core being 10nt long, and the wing at the 3' end being 1-9nt long. In some embodiments, the chiral center pattern of the skeleton is an array containing one (Rp) and all consisting of (Sp), where the molecule has a wing-core-wing morphology, with the wing at the 5' end being 5nt long, the core being 10nt long, and the wing at the 3' end being 5nt long.In some embodiments, the chiral center pattern of the skeleton is a sequence consisting of one (Rp) and all (Sp), where the molecule has a wing-core-wing morphology, with the wing at the 5' end being 5nt long, the core being 10nt long, and the wing at the 3' end being 5nt long, and at least one wing containing a nucleotide having a 2'-OMe modification. In some embodiments, the chiral center pattern of the skeleton is a sequence consisting of one (Rp) and all (Sp), where the molecule has a wing-core-wing morphology, with each wing containing at least one nucleotide having a 2'-OMe modification. In some embodiments, the chiral center pattern of the skeleton is a sequence consisting of one (Rp) and all (Sp), where the molecule has a wing-core-wing morphology, with each wing in both wings containing a 2'-OMe modification. In some embodiments, the chiral center pattern of the skeleton is a sequence containing one (Rp) and all consisting of (Sp), where the molecule has a wing-core-wing morphology, with the wing at the 5' end being 5nt long, the core being 10nt long, and the wing at the 3' end being 5nt long, and each nucleotide in each wing having a 2'-OMe modification. In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing configuration, the 5' terminal wing of the molecule comprises 4-8 nts, each having a 2'-OMe modification, and the 5' terminal nts of the molecule have a phosphorothioate in the Sp conformation; the core comprises 8-12 nts, each being DNA(2'-H), each having a phosphorothioate at the Sp position except for one nt having a phosphorothioate at the Rp position; the 3' terminal wing of the molecule comprises 4-8 nts, each having a 2'-OMe modification, and the 3' terminal nts of the molecule have a phosphorothioate in the Sp conformation.In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing configuration, the 5' terminal wing of the molecule contains 6 nts, each having a 2'-OMe modification, and the 5' terminal nts of the molecule have a phosphorothioate in the Sp conformation; the core contains 10 nts, each being DNA(2'-H), each having a phosphorothioate at the Sp position except for one nt having a phosphorothioate at the Rp position; the 3' terminal wing of the molecule contains 6 nts, each having a 2'-OMe modification, and the 3' terminal nts of the molecule have a phosphorothioate in the Sp conformation.

[0185] In some embodiments, the disclosure acknowledges that chemical modifications, such as modifications of nucleosides and internucleotide bonds, can improve properties. In some embodiments, the disclosure shows that combinations of chemical modifications and stereochemistry can result in unexpectedly significant improvements in properties (e.g., bioactivity, selectivity, etc.). In some embodiments, combinations such as modifications of sugars, bases, and / or internucleotide bonds can result in stereochemical patterns, e.g., (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m The present invention provides oligonucleotides and compositions thereof that, when combined with other elements, exhibit remarkably improved properties. In some embodiments, the provided oligonucleotide compositions are chiralally controlled and include 2'-modification of one or more sugar moieties, one or more native phosphate bonds, one or more phosphorothioate bonds, and (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) mThis includes combinations of stereochemical patterns of the form (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 Disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, The first group of oligonucleotides have the same base sequence; The first plurality of oligonucleotides each contain one or more modified sugar moieties, or one or more native phosphate crosslinks and one or more modified internucleotide crosslinks.

[0187] In some embodiments, the first plurality of oligonucleotides include one or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes one or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes two or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes three or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes four or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes five or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes six or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes seven or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes eight or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes nine or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes ten or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes fifteen or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes twenty or more modified sugar moieties. In some embodiments, the provided oligonucleotide includes twenty-five or more modified sugar moieties. In some embodiments, the provided oligonucleotide comprises 30 or more modified sugar moieties.

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

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

[0190] The oligonucleotides provided may contain varying numbers of natural phosphate crosslinks. In some embodiments, the oligonucleotides provided do not contain any natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain one natural phosphate crosslink. In some embodiments, the oligonucleotides provided contain two or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain three or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain four or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain five or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain six or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain seven or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain eight or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain nine or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain ten or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain fifteen or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain twenty or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain twenty-five or more natural phosphate crosslinks. In some embodiments, the oligonucleotides provided contain 30 or more natural phosphate crosslinks.

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

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

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

[0194] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently contains one or more modified internucleotide crosslinks and optionally one or more native phosphate crosslinks, and the core region independently contains one or more modified internucleotide crosslinks; or The present invention provides an oligonucleotide composition in which each wing region independently contains one or more modified sugar moieties, and the core region contains one or more unmodified sugar moieties.

[0195] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; and The present invention provides an oligonucleotide composition in which each wing region independently comprises one or more modified internucleotide crosslinks and optionally one or more native phosphate crosslinks, and the core region independently comprises one or more modified internucleotide crosslinks.

[0196] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; and The present invention provides an oligonucleotide composition in which each wing region independently contains one or more modified sugar moieties, and the core region contains one or more unmodified sugar moieties.

[0197] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently comprises one or more modified internucleotide crosslinks and optionally one or more native phosphate crosslinks, and the core region independently comprises one or more modified internucleotide crosslinks; and The present invention provides an oligonucleotide composition in which each wing region independently contains one or more modified sugar moieties, and the core region contains one or more unmodified sugar moieties.

[0198] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks, and the core region independently contains one or more modified internucleotide crosslinks; and The present invention provides an oligonucleotide composition in which each wing region independently contains one or more modified sugar moieties, and the core region contains one or more unmodified sugar moieties.

[0199] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and optionally one or more native phosphate crosslinks; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0200] In some embodiments, the present disclosure relates to oligonucleotide compositions, A first plurality of oligonucleotides comprising one or more wing regions and one core region, The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0201] In some embodiments, the present disclosure relates to oligonucleotide compositions, It comprises a first plurality of oligonucleotides, each containing two wing regions and one core region. The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0202] In some embodiments, the present disclosure relates to oligonucleotide compositions, It comprises a first plurality of oligonucleotides, each containing two wing regions and one core region. The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; The wing region relative to the 5'-terminus of the core region contains, within the wing, at least one modified internucleotide crosslink followed by a native phosphate crosslink; and The wing region relative to the 3'-terminus of the core region contains, within the wing, at least one modified internucleotide crosslink followed by a native phosphate crosslink; The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0203] In some embodiments, the present disclosure relates to oligonucleotide compositions, It comprises a first plurality of oligonucleotides, each containing one wing region and one core region. The first group of oligonucleotides have the same base sequence; The wing region has a length of 2 nucleotides or more and contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; The wing region is relative to the 5'-terminus of the core region and contains a modified internucleotide bridge between two nucleosides at its 3'-terminus, or the wing region is relative to the 3'-terminus of the core region and contains a modified internucleotide bridge between two nucleosides at its 5'-terminus; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

[0204] In some embodiments, the present disclosure relates to oligonucleotide compositions, It comprises a first plurality of oligonucleotides, each containing two wing regions and one core region. The first group of oligonucleotides have the same base sequence; Each wing region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks and one or more native phosphate crosslinks; The wing region relative to the 5' end of the core region contains a modified internucleotide bridge between two nucleosides at its 3' end; The wing region relative to the 3' end of the core region contains a modified internucleotide bridge between two nucleosides at its 5' end; and The present invention provides an oligonucleotide composition in which the core region independently has a length of 2 nucleotides or more and independently contains one or more modified internucleotide crosslinks.

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

[0206] In some embodiments, the first plurality of oligonucleotides include two wings 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 includes two or more modified internucleotide crosslinks. In some embodiments, the wing region includes three or more modified internucleotide crosslinks. In some embodiments, the wing region includes four or more modified internucleotide crosslinks. In some embodiments, the wing region includes five or more modified internucleotide crosslinks. In some embodiments, the wing region includes six or more modified internucleotide crosslinks. In some embodiments, the wing region includes seven or more modified internucleotide crosslinks. In some embodiments, the wing region includes eight or more modified internucleotide crosslinks. In some embodiments, the wing region includes nine or more modified internucleotide crosslinks. In some embodiments, the wing region includes ten or more modified internucleotide crosslinks. In some embodiments, the wing region includes eleven or more modified internucleotide crosslinks. In some embodiments, the wing region includes twelve or more modified internucleotide crosslinks. In some embodiments, the wing region includes thirteen or more modified internucleotide crosslinks. In some embodiments, the wing region includes fourteen or more modified internucleotide crosslinks. In some embodiments, the wing region includes fifteen or more modified internucleotide crosslinks. In some embodiments, the wing region includes two or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes three or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes four or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes five or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes six or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes seven or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes eight or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes nine or consecutive modified internucleotide crosslinks.In some embodiments, the wing region includes 10 or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes 11 or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes 12 or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes 13 or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes 14 or consecutive modified internucleotide crosslinks. In some embodiments, the wing region includes 15 or consecutive modified internucleotide crosslinks. In some embodiments, each internucleotide crosslink in the wing region is independently a modified internucleotide crosslink.

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

[0209] In some embodiments, the wing region contains two or more natural phosphate crosslinks. In some embodiments, the wing region contains three or more natural phosphate crosslinks. In some embodiments, the wing region contains four or more natural phosphate crosslinks. In some embodiments, the wing region contains five or more natural phosphate crosslinks. In some embodiments, the wing region contains six or more natural phosphate crosslinks. In some embodiments, the wing region contains seven or more natural phosphate crosslinks. In some embodiments, the wing region contains eight or more natural phosphate crosslinks. In some embodiments, the wing region contains nine or more natural phosphate crosslinks. In some embodiments, the wing region contains ten or more natural phosphate crosslinks. In some embodiments, the wing region contains eleven or more natural phosphate crosslinks. In some embodiments, the wing region contains twelve or more natural phosphate crosslinks. In some embodiments, the wing region contains thirteen or more natural phosphate crosslinks. In some embodiments, the wing region contains fourteen or more natural phosphate crosslinks. In some embodiments, the wing region contains fifteen or more natural phosphate crosslinks. In some embodiments, the wing region includes two or consecutive natural phosphate crosslinks. In some embodiments, the wing region includes three or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains four or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains five or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains six or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains seven or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains eight or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains nine or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains ten or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains eleven or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains twelve or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains thirteen or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains fourteen or consecutive natural phosphate crosslinks. In some embodiments, the wing region contains fifteen or consecutive natural phosphate crosslinks. In some embodiments, each internucleotide crosslink in the wing region is independently a natural phosphate crosslink.

[0210] In some embodiments, 5% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 10% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 15% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 20% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 25% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 30% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 35% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 40% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 45% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 50% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, 55% or more of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, more than 60% of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, more than 65% of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, more than 70% of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, more than 75% of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, more than 80% of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, more than 85% of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks.In some embodiments, more than 90% of the internucleotide crosslinks of the provided oligonucleotide are natural phosphate crosslinks. In some embodiments, more than 95% of the internucleotide crosslinks of the wing regions are natural phosphate crosslinks. In some embodiments, each internucleotide crosslink of the wing region is a natural phosphate crosslink.

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

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

[0213] In some embodiments, the present disclosure relates to an oligonucleotide composition with controlled chirality, (1) Common base sequence and length; (2) Common skeletal cross-linking patterns; and (3) Pattern of chiral centers of the common skeleton A plurality of first oligonucleotides defined by having The composition provides a chirality-controlled oligonucleotide composition in which a predetermined level of oligonucleotides in the composition is substantially a pure preparation of a single oligonucleotide, having a common base sequence and length, a common backbone crosslinking pattern, and a common backbone chiral center pattern.

[0214] In some embodiments, a common base sequence and length may be referred to as the common base sequence. In some embodiments, oligonucleotides having a common base sequence may have the same pattern of nucleoside modifications, e.g., sugar modifications, base modifications, etc. In some embodiments, the pattern of nucleoside modifications may be represented by a combination of position and modification. For example, for WV-1092, the nucleoside crosslink pattern is 5×2'-OMe(2'-OMe modification on the sugar portion)-DNA(no 2'-modification on the sugar portion)-5×2'-OMe, from the 5'-terminus to the 3'-terminus. In some embodiments, the crosslink pattern of the backbone includes the position and type of each internucleotide crosslink (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.). For example, for WV-1092, the crosslink pattern of the backbone is 1×PS(phosphorothioate)-3×PO(phosphate)-11×PS-3×PO-1×PS. The chiral center pattern of the oligonucleotide skeleton can be indicated by the combination of the stereochemistry (Rp / Sp) of the crosslinking 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 crosslinks (e.g., PO) may be omitted. As illustrated above, the positions of non-chiral crosslinks may be obtained, for example, from the crosslinking pattern of the skeleton.

[0215] In some embodiments, the present disclosure relates to an oligonucleotide composition with controlled chirality, (1) Common base sequence and length; (2) Common skeletal cross-linking patterns; and (3) Pattern of chiral centers of the common skeleton It comprises a first plurality of oligonucleotides of a specific oligonucleotide type characterized by, The composition provides an oligonucleotide composition in which chirality is controlled, in that for a particular oligonucleotide type, it is concentrated compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0216] As those skilled in the art will understand, 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 auxiliaries, chiral modifying reagents, and / or chiral catalysts. In some embodiments, in substantially racemic (or chiral-uncontrolled) preparations of oligonucleotides, all or most of the coupling steps are chiral-uncontrolled, in that the coupling steps are not specifically performed to improve stereoselectivity. An example of a substantially racemic preparation of oligonucleotides is a preparation of phosphorothioate oligonucleotides by a commonly used phosphoramidite oligonucleotide synthesis process known in the art, which sulfides phosphite triesters using either tetraethylthiuram disulfide, i.e., (TETD), or 3H-1,2-benzodithiol (bensodithiol)-3-one 1,1-dioxide (BDTD). In some embodiments, a substantially racemic preparation of oligonucleotides results in a substantially racemic oligonucleotide composition (or chiral-uncontrolled oligonucleotide composition). In some embodiments, the coupling of at least one nucleotide monomer has diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, the coupling of at least two nucleotide monomers has diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, the coupling of at least three nucleotide monomers has diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1.In some embodiments, the coupling of at least four nucleotide monomers has diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, the coupling of at least five nucleotide monomers has diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, in a stereorandom preparation or a racemic preparation, at least one internucleotide bond has diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, in stereorandom or racemic preparations, at least two internucleotide links have diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, in stereorandom or racemic preparations, at least three internucleotide links have diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, in stereorandom or racemic preparations, at least four internucleotide links have diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, in stereorandom or racemic preparations, at least five internucleotide links have diastereoselectivity of about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or less than 99:1. In some embodiments, at least two internucleotide links have diastereoselectivity of about 60:40.In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 70:30. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 80:20. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 90:10. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 91:9. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 92:8. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 93:7. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 94:6. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 95:5. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 96:4. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 97:3. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 98:2. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 99:1. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 91:9. In some embodiments, at least one coupling has diastereoselectivity less than about 90:10. In some embodiments, at least two couplings have diastereoselectivity less than about 90:10. In some embodiments, at least three couplings have diastereoselectivity less than about 90:10. In some embodiments, at least four couplings have diastereoselectivity less than about 90:10.In some embodiments, at least five couplings have diastereoselectivity less than about 90:10. In some embodiments, at least one internucleotide bond has diastereoselectivity less than about 90:10. In some embodiments, at least two internucleotide bonds have diastereoselectivity less than about 90:10. In some embodiments, at least three internucleotide bonds have diastereoselectivity less than about 90:10. In some embodiments, at least four internucleotide bonds have diastereoselectivity less than about 90:10. In some embodiments, at least five internucleotide bonds have diastereoselectivity less than about 90:10.

[0217] As will be understood by those skilled in the art, in some embodiments, the diastereoselectivity of coupling or bonding can be evaluated by the diastereoselectivity of dimer formation under the same or equivalent conditions, where the dimers have the same 5'-nucleoside and 3'-nucleoside as well as internucleotide bonds. 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 evaluated by the coupling of the two G moieties under the same or equivalent conditions (e.g., monomer, chiral auxiliaries, solvent, activator, temperature, etc.).

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

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

[0220] In some embodiments, the present disclosure relates to an oligonucleotide composition with controlled chirality, (1) Common base sequence and length; (2) Common skeletal cross-linking patterns; and (3) Pattern of chiral centers of the common skeleton It comprises a first plurality of oligonucleotides of a specific oligonucleotide type characterized by, The composition provides an oligonucleotide composition in which chirality is controlled, in that for a particular oligonucleotide type, it is concentrated 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 skeletal bonding, and a common pattern of skeletal chiral centers have a common pattern of skeletal phosphorus modification and a common pattern of base modification. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers have a common pattern of skeletal phosphorus modification and a common pattern of nucleoside modification. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers have the same structure.

[0222] In some embodiments, oligonucleotides of the oligonucleotide type have a common pattern of phosphate modification of the skeleton and a common pattern of sugar modification. In some embodiments, oligonucleotides of the oligonucleotide type have a common pattern of phosphate modification of the skeleton and a common pattern of base modification. In some embodiments, oligonucleotides of the oligonucleotide type have a common pattern of phosphate modification of the skeleton and a common pattern of nucleoside modification. In some embodiments, oligonucleotides of the oligonucleotide type are identical.

[0223] In some embodiments, the chiral-controlled oligonucleotide composition is a substantially pure preparation of the oligonucleotide type, where the oligonucleotides in the composition that are not of the oligonucleotide type are (form) impurities from the preparation process of the oligonucleotide type (and possibly after certain purification steps).

[0224] In some embodiments, at least about 20% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 25% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 30% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 35% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 40% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 45% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 50% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 55% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 60% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 65% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 70% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers.In some embodiments, at least about 75% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 80% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 85% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 90% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 92% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, at least about 94% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal 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 skeletal bonding, and a common pattern of skeletal 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 skeletal bonding, and a common pattern of skeletal 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 skeletal bonding, and a common pattern of skeletal chiral centers. In some embodiments, the purity of a chiral-controlled oligonucleotide composition can be expressed as the percentage of oligonucleotides in the composition having a common base sequence and length, a common pattern of skeletal bonding, and a common pattern of skeletal chiral centers.

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

[0226] In some embodiments, the oligonucleotides in the provided composition have a common phosphorus modification pattern of the backbone. In some embodiments, the common base sequence is the base sequence of a certain type of oligonucleotide. In some embodiments, the provided composition is a chirality-controlled oligonucleotide composition in which the composition contains a predetermined amount of a first plurality of oligonucleotides of each individual type, and the types of oligonucleotides are (1) Base sequence; (2) Cross-linking pattern of the skeleton; (3) Pattern of chiral centers in the skeleton; and (4) Defined by the pattern of phosphorus modification of the skeleton.

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

[0228] In some embodiments, a particular type of oligonucleotide is 1A) Base identity; 1B) Patterns of base modification; 1C) Patterns of sugar modification; 2) Patterns of skeletal connections; 3) Patterns of skeletal chiral centers; and 4) Patterns of skeletal phosphorus modification It may also be defined by: Therefore, in some embodiments, certain types of oligonucleotides may share the same base, but their patterns of base modification and / or sugar modification may differ.

[0229] In some embodiments, certain types of oligonucleotides have the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same pattern of skeletal bonding (e.g., patterns of native phosphate bonds, phosphorothioate bonds, phosphorothioate triester bonds, and combinations thereof), the same pattern of skeletal chiral centers (e.g., patterns of the stereochemistry (Rp / Sp) of chiral internucleotide bonds), and the same pattern of skeletal phosphorus modifications (e.g., -S - , and -LR of formula I 1They are identical in that they have patterns of modification to the internucleotide phosphate atom, such as the above.

[0230] In some embodiments, the purity of an oligonucleotide-type chiral controlled oligonucleotide composition is expressed as the percentage of oligonucleotides in the composition that are of the oligonucleotide type. In some embodiments, at least about 10% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 20% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 30% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 40% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 50% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 60% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 70% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 80% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 90% of the oligonucleotides in the chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 92% of the oligonucleotides in the chiral-controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 94% of the oligonucleotides in the chiral-controlled oligonucleotide composition are of the same oligonucleotide type.In some embodiments, at least about 95% of the oligonucleotides in the chiral-controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 96% of the oligonucleotides in the chiral-controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 97% of the oligonucleotides in the chiral-controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 98% of the oligonucleotides in the chiral-controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 99% of the oligonucleotides in the 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 binding step in its preparation process. In some embodiments, the binding step has 60% stereoselectivity (e.g., diastereoselectivity) (60% of the new internucleotide bonds formed by the binding step have the desired stereochemistry). The new internucleotide bonds formed after such a binding step are sometimes said to have 60% purity. In some embodiments, each binding step has at least 60% stereoselectivity. In some embodiments, each binding step has at least 70% stereoselectivity. In some embodiments, each binding step has at least 80% stereoselectivity. In some embodiments, each binding step has at least 85% stereoselectivity. In some embodiments, each binding step has at least 90% stereoselectivity. In some embodiments, each binding step has at least 91% stereoselectivity. In some embodiments, each binding step has at least 92% stereoselectivity. In some embodiments, each binding step has at least 93% stereoselectivity. In some embodiments, each binding step has at least 94% stereoselectivity. In some embodiments, each binding step has at least 95% stereoselectivity. In some embodiments, each binding step has at least 96% stereoselectivity. In some embodiments, each binding step has at least 97% stereoselectivity. In some embodiments, each binding step has at least 98% stereoselectivity. In some embodiments, each binding step has at least 99% stereoselectivity. In some embodiments, each binding step has substantially 100% stereoselectivity. In some embodiments, the binding step has substantially 100% stereoselectivity, where all products from the binding step, detectable by analytical methods (e.g., NMR, HPLC, etc.), have the desired stereoselectivity.

[0232] In particular, this disclosure acknowledges that combinations of oligonucleotide components (e.g., patterns of chemical modifications, skeletal bonding, skeletal chiral centers and / or skeletal phosphorus modifications) can confer properties such as remarkably improved biological activity.

[0233] In some embodiments, the Disclosure relates to an oligonucleotide composition comprising a predetermined amount of a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides comprises one or more wing regions and one common core region, Each wing region independently has a length of 2 nucleotides or more and independently contains, optionally, one or more chiral internucleotide bridges; The core region independently has a length of 2 nucleotides or more, independently contains one or more chiral internucleotide crosslinks, and the common core region is (1) Common base sequence and length; (2) Common pattern skeletal crosslinking; and (3) Provide an oligonucleotide composition containing a common pattern of skeletal chiral centers.

[0234] In some embodiments, the wing region includes structural features not present in the core region. In some embodiments, the wing and core can be defined by any constituent elements, such as base modifications (e.g., methylation / demethylation, methylation at position 1 / methylation at position 2, etc.), sugar modifications (e.g., modified / unmodified, 2'-modification / another type of modification, one type of 2'-modification / another type of 2'-modification, etc.), skeletal bonding type (e.g., phosphate / phosphorothioate, phosphorothioate / substituted phosphorothioate, etc.), skeletal chiral center stereochemistry (e.g., total Sp / total Rp, (SpRp) repeat / total Rp, etc.), skeletal phosphorus modification type (e.g., s1 / s2, s1 / s3, etc.), etc.

[0235] In some embodiments, the wings and core are defined by nucleoside modifications, and the wings include nucleoside modifications not present in the core region. In some embodiments, the wings and core are defined by sugar modifications, and the wings include sugar modifications not present 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 examples of sugar modifications are described in this disclosure. In some embodiments, the wings and core are defined by internucleotide crosslinks, and the wings include a type of internucleotide crosslink that the core region does not have (e.g., natural phosphate crosslinks, certain modified internucleotide crosslinks, etc.). In some embodiments, the wings and core are defined by internucleotide crosslinks, and the wings have a different skeletal crosslink pattern than the core's skeletal crosslinks.

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

[0237] In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an antisense oligonucleotide (e.g., chiromersen). In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an siRNA oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition provided is an oligonucleotide that may be an antisense oligonucleotide, an antagonist mir, a microRNA, a premicroRNs, an anti-mir, a super-mir, a ribozyme, an Ul adapter, an RNA activator, an RNAi agent, a decoy oligonucleotide, a triple-stranding oligonucleotide, an aptamer, or an adjuvant. In some embodiments, the chiral-controlled oligonucleotide composition is an antisense oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is an antagonist mir oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is a microRNA oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is a premicroRNA oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is an anti-mir oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is a super-mir oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is a ribozyme oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is an UL adapter oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is an RNA activator oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is an RNAi agent oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is a decoy oligonucleotide.In some embodiments, the chiral-controlled oligonucleotide composition is a triple-chain-forming oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is an aptamer oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide composition is an adjuvant oligonucleotide.

[0238] In some embodiments, the chiral-controlled (and / or stereochemically pure) preparations provided are oligonucleotides comprising one or more modified skeletal bonds, bases, and / or sugars.

[0239] In some embodiments, the oligonucleotide provided contains one or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide provided contains two or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide provided contains three or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide provided contains four or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide provided contains five or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide provided contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or twenty-five chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains five or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains six or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 7 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 8 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 9 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 10 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 11 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 12 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 13 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 14 or more chiral modified phosphate bonds.In some embodiments, the oligonucleotide type provided contains 15 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 16 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 17 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 18 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 19 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 20 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 21 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 22 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 23 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 24 or more chiral modified phosphate bonds. In some embodiments, the oligonucleotide type provided contains 25 or more chiral modified phosphate bonds.

[0240] In some embodiments, the oligonucleotides provided 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 groups. Exemplary such chiral modified phosphate groups are described above and herein. In some embodiments, the oligonucleotides provided 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 groups in the Sp configuration.

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

[0242] In some embodiments, the chiral modified phosphate bonds are chiral phosphorothioate bonds, i.e., phosphorothioate internucleotide bonds. In some embodiments, the 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 phosphorothioate internucleotide bonds. In some embodiments, all chiral modified phosphate bonds are chiral phosphorothioate internucleotide bonds. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide bonds in the provided oligonucleotides are of the Sp structure. In some embodiments, at least about 10% of the chiral phosphorothioate nucleotide interbondings in the provided oligonucleotide are of the Sp structure. In some embodiments, at least about 20% of the chiral phosphorothioate nucleotide interbondings in the provided oligonucleotide are of the Sp structure. In some embodiments, at least about 30% of the chiral phosphorothioate nucleotide interbondings in the provided oligonucleotide are of the Sp structure. In some embodiments, at least about 40% of the chiral phosphorothioate nucleotide interbondings in the provided oligonucleotide are of the Sp structure. In some embodiments, at least about 50% of the chiral phosphorothioate nucleotide interbondings in the provided oligonucleotide are of the Sp structure. In some embodiments, at least about 60% of the chiral phosphorothioate nucleotide interbondings in the provided oligonucleotide are of the Sp structure. In some embodiments, at least about 70% of the chiral phosphorothioate nucleotide interbondings in the provided oligonucleotide are of the Sp structure. In some embodiments, at least about 80% of the chiral phosphorothioate nucleotide interbonds of the provided oligonucleotide are of the Sp structure.In some embodiments, at least about 90% of the chiral phosphorothioate nucleotide interlinks of the provided oligonucleotide are in an Sp structure. In some embodiments, at least about 95% of the chiral phosphorothioate nucleotide interlinks of the provided oligonucleotide are in an Sp structure.

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

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

[0245] In some embodiments, the chiral phosphorothioate nucleotide bonds are chiral phosphorothioate diester bonds. In some embodiments, each chiral phosphorothioate nucleotide bond is independently a chiral phosphorothioate diester bond. In some embodiments, each nucleotide bond is independently a chiral phosphorothioate diester bond. In some embodiments, each nucleotide bond is independently a chiral phosphorothioate diester bond, with only one bond being Rp.

[0246] In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide containing one or more modified bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide without modified bases. Exemplary such modified bases are described above and herein.

[0247] In some embodiments, the oligonucleotide of the provided composition contains at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 1 natural phosphate bond. In some embodiments, the oligonucleotide of the provided composition contains at least 2 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 3 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 4 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 5 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 6 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 7 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 8 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 9 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 10 native phosphate bonds.

[0248] In some embodiments, the oligonucleotide of the provided composition contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains one natural phosphate bond. In some embodiments, the oligonucleotide of the provided composition contains two natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains three natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains four natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains five natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains six natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains seven natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains eight natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains nine natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains ten natural phosphate bonds.

[0249] In some embodiments, the oligonucleotide of the provided composition contains at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 2 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 3 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 4 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 5 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 6 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 7 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 8 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains at least 9 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition comprises at least 10 consecutive native phosphate bonds.

[0250] In some embodiments, the oligonucleotide of the provided composition contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 2 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 3 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 4 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 5 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 6 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 7 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 8 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 9 consecutive natural phosphate bonds. In some embodiments, the oligonucleotide of the provided composition contains 10 consecutive natural phosphate bonds.

[0251] In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 8 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 9 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 10 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 11 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 12 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 13 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 14 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 15 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 16 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 17 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 18 bases.In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 19 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 20 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 21 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 22 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 23 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 24 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparation provided is an oligonucleotide having a common base sequence of at least 25 bases. In some embodiments, the chiral-controlled (and / or stereochemically pure) preparations provided are 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, the provided composition comprises an oligonucleotide containing one or more residues modified at the sugar moiety. In some embodiments, the provided composition comprises an oligonucleotide containing one or more residues modified at the 2' position of the sugar moiety (hereinafter referred to herein as "2'-modification"). Examples of such modifications are, but are not limited to, those described above and herein, 2'-OMe, 2'-MOE, 2'-LNA, 2'-F, FRNA, FANA, S-cEt, etc. In some embodiments, the provided composition comprises an oligonucleotide containing one or more residues that are 2'-modified. For example, in some embodiments, the provided oligonucleotide contains one or more residues that are 2'-O-methoxyethyl (2'-MOE)-modified residues. In some embodiments, the provided composition comprises an oligonucleotide that does not contain 2'-modification. In some embodiments, the provided composition is an oligonucleotide that does not contain a 2'-MOE residue. That is, in some embodiments, the provided oligonucleotide is not MOE-modified. Further examples of sugar modifications are described herein.

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

[0254] In some embodiments, the oligonucleotides provided have a wing-core motif represented as XY, or a core-wing motif represented as YX, where the residues in the "X" portion are of a particular type of glycosylation, and the residues in the core "Y" portion are not of the same particular type of glycosylation. In some embodiments, the oligonucleotides provided have a wing-core-wing motif represented as XYX, where the residues in each "X" portion are of a particular type of glycosylation, and the residues in the core "Y" portion are not of the same particular type of glycosylation. In some embodiments, the oligonucleotides provided have a wing-core motif represented as XY, or a core-wing motif represented as YX, where the residues in the "X" portion are of a particular type of 2'-modification, and the residues in the core "Y" portion are not of the same particular type of 2'-modification. In some embodiments, the oligonucleotides provided have a wing-core motif represented as XY, where the residues in the "X" portion are of a particular type of 2'-modification, and the residues in the core "Y" portion are not of the same particular type of 2'-modification. In some embodiments, the oligonucleotide provided has a core-wing motif represented as YX, where the residues in the "X" portion are of a particular type of 2'-modified residue, and the residues in the core "Y" portion are not of the same particular type of 2'-modified residue. In some embodiments, the oligonucleotide provided has a wing-core-wing motif represented as XYX, where the residues in each "X" portion are of a particular type of 2'-modified residue, and the residues in the core "Y" portion are not of the same particular type of 2'-modified residue. In some embodiments, the oligonucleotide provided has a wing-core motif represented as XY, where the residues in the "X" portion are of a particular type of 2'-modified residue, and the residues in the core "Y" portion are 2'-deoxyribonucleosides. In some embodiments, the oligonucleotide provided has a core-wing motif represented as YX, where the residues in the "X" portion are of a particular type of 2'-modified residue, and the residues in the core "Y" portion are 2'-deoxyribonucleosides.In some embodiments, the oligonucleotide provided has a wing-core-wing motif represented as XYX, where each "X" residue is a specific type of 2'-modified residue and the core "Y" residue is a 2'-deoxyribonucleoside. In some embodiments, the oligonucleotide provided has a wing-core-wing motif represented as XYX, where each "X" residue is a specific type of 2'-modified residue and the core "Y" residue is a 2'-deoxyribonucleoside. For example, in some embodiments, the oligonucleotide provided has a wing-core-wing motif represented as XYX, where each "X" residue is a 2'-MOE-modified residue and the core "Y" residue is not a 2'-MOE-modified residue. In some embodiments, the oligonucleotides provided have a wing-core-wing motif represented as XYX, where each "X" portion is a 2'-MOE-modified residue and the core "Y" portion is a 2'-deoxyribonucleoside. Those skilled 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 intended.

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

[0256] In some embodiments, the wing has a length of 1 base. In some embodiments, the wing has a length of 2 bases. In some embodiments, the wing has a length of 3 bases. In some embodiments, the wing has a length of 4 bases. In some embodiments, the wing has a length of 5 bases. In some embodiments, the wing has a length of 6 bases. In some embodiments, the wing has a length of 7 bases. In some embodiments, the wing has a length of 8 bases. In some embodiments, the wing has a length of 9 bases. In some embodiments, the wing has a length of 10 bases. In some embodiments, the wing has a length of 11 bases. In some embodiments, the wing has a length of 12 bases. In some embodiments, the wing has a length of 13 bases. In some embodiments, the wing has a length of 14 bases. In some embodiments, the wing has a length of 15 bases. In some embodiments, the wing has a length of 16 bases. In some embodiments, the wing has a length of 17 bases. In some embodiments, the wing has a length of 18 bases. In some embodiments, the wing has a length of 19 bases. In some embodiments, the wing has a length of 10 bases.

[0257] In some embodiments, the wing comprises one or more chiral internucleotide bonds. In some embodiments, the wing comprises one or more native phosphate bonds. In some embodiments, the wing comprises one or more chiral internucleotide bonds and one or more native phosphate bonds. In some embodiments, the wing comprises one or more chiral internucleotide bonds and two or more native phosphate bonds. In some embodiments, the wing comprises one or more chiral internucleotide bonds and two or more native phosphate bonds, where the two or more native phosphate bonds are contiguous. In some embodiments, the wing does not contain chiral internucleotide bonds. In some embodiments, each wing bond is a native phosphate bond. In some embodiments, the wing does not contain phosphate bonds. In some embodiments, each wing is independently a chiral internucleotide bond.

[0258] In some embodiments, each wing region independently comprises one or more chiral internucleotide crosslinks. In some embodiments, each wing region independently comprises one or more native phosphate crosslinks. In some embodiments, each wing region independently comprises one or more chiral internucleotide crosslinks and one or more native phosphate crosslinks. In some embodiments, each wing region independently comprises one or more chiral internucleotide crosslinks and two or more native phosphate crosslinks. In some embodiments, each wing region independently comprises one or more chiral internucleotide crosslinks and two or more native phosphate crosslinks, where the two or more native phosphate crosslinks are continuous.

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

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

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

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

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

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

[0265] In some embodiments, each wing region independently contains at least one continuous natural phosphate crosslink. In some embodiments, each wing region independently contains at least two continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least three continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least four continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least five continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least six continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least seven continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least eight continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least nine continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least ten continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least eleven continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 12 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 13 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 14 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 15 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 16 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 17 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 18 consecutive natural phosphate crosslinks.In some embodiments, each wing region independently contains at least 19 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains at least 20 consecutive natural phosphate crosslinks.

[0266] In some embodiments, each wing region independently contains one continuous natural phosphate crosslink. In some embodiments, each wing region independently contains two continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains three continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains four continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains five continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains six continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains seven continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains eight continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains nine continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains ten continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains eleven continuous natural phosphate crosslinks. In some embodiments, each wing region independently contains 12 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 13 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 14 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 15 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 16 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 17 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 18 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 19 consecutive natural phosphate crosslinks. In some embodiments, each wing region independently contains 20 consecutive natural phosphate crosslinks.

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

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

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

[0270] In some embodiments, the 3'-terminal wing includes a modified internucleotide bridge having one or more native phosphate bridges connecting two or more nucleosides before the modified internucleotide bridge in the 3'-terminal wing (relative to the 5'-terminus). For example, the 3'-terminal wing mAmCmUmU *SmC has three native phosphate bridges connecting four nucleosides (mAmCmUmU) before a modified internucleotide bridge in the 3'-terminal wing, with a modified internucleotide bridge (mU * S Includes mC). In some embodiments, the 3'-terminal wing includes one or more native phosphate crosslinks and / or one or more modified internucleotide crosslinks prior to the modified internucleotide crosslinks, and the 3'-terminal wing includes one or more native phosphate crosslinks prior to them (e.g., mAmCmU * SmU * mU during SmC * S mU and mU * S mC). In some embodiments, the 3'-terminal wing contains one or more native phosphate bridges in the 3'-terminal wing prior to the modified internucleotide bridge. In some embodiments, the 3'-terminal wing contains one or more consecutive native phosphate bridges in the 3'-terminal wing prior to the modified internucleotide bridge. In some embodiments, the 3'-terminal wing contains a native phosphate bridge between two nucleosides at its 5'-terminus. For example, mAmCmUmU * The 3'-terminal wing, which has an SmC structure, contains a natural phosphate crosslink between two nucleosides at its 5'-terminus. 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, the wing contains only one chiral internucleotide bond. In some embodiments, the 5'-terminal wing contains only one chiral internucleotide bond. In some embodiments, the 5'-terminal wing contains only one chiral internucleotide bond at its 5'-end. In some embodiments, the 5'-terminal wing contains only one chiral internucleotide bond at its 5'-end, and the chiral internucleotide bond is Rp. In some embodiments, the 5'-terminal wing contains only one chiral internucleotide bond at its 5'-end, and the chiral internucleotide bond is Sp. In some embodiments, the 3'-terminal wing contains only one chiral internucleotide bond at its 3'-end. In some embodiments, the 3'-terminal wing contains only one chiral internucleotide bond at its 3'-end, and the chiral internucleotide bond is Rp. In some embodiments, the 3'-terminal wing contains only one chiral internucleotide bond at its 3'-end, and the chiral internucleotide bond is Sp.

[0273] In some embodiments, the wing contains two or more natural phosphate bonds. In some embodiments, all phosphate bonds within the wing are contiguous, and no non-phosphate bonds exist between any two phosphate bonds within the wing.

[0274] In some embodiments, when describing bonds, such as bond chemistry or bond stereochemistry, the bonds connecting the wing and the core are considered 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 bond may be considered part of the core (bold), its 5'-wing (having 2'-OMe in the sugar portion) having a single Sp phosphorothioate bond at its 5'-terminus, its 3'-wing (having 2'-OMe in the sugar portion) having a single Sp phosphorothioate bond at its 3'-terminus, and its core having no 2'-modification of the sugar).

[0275] In some embodiments, the 5'-internucleotide bond attached to the unmodified sugar moiety is a modified bond. In some embodiments, the 5'-internucleotide bond attached to the unmodified sugar moiety is a bond having the structure of formula I. In some embodiments, the 5'-internucleotide bond attached to the unmodified sugar moiety is a phosphorothioate bond. In some embodiments, the 5'-internucleotide bond attached to the unmodified sugar moiety is a substituted phosphorothioate bond. In some embodiments, the 5'-internucleotide bond attached to the unmodified sugar moiety is a phosphorothioate triester bond. In some embodiments, each 5'-internucleotide bond attached to the unmodified sugar moiety is a modified bond. In some embodiments, each 5'-internucleotide bond attached to the unmodified sugar moiety is a bond having the structure of formula I. In some embodiments, each 5'-internucleotide bond attached to the unmodified sugar moiety is a phosphorothioate bond. In some embodiments, each 5'-internucleotide bond attached to the unmodified sugar moiety is a substituted phosphorothioate bond. In some embodiments, each 5'-internucleotide bond attached to the 2'-unmodified sugar moiety is a phosphorothioate triester bond.

[0276] In some embodiments, the 3'-internucleotide bond attached to the unmodified sugar moiety is a modified bond. In some embodiments, the 3'-internucleotide bond attached to the unmodified sugar moiety is a bond having the structure of formula I. In some embodiments, the 3'-internucleotide bond attached to the unmodified sugar moiety is a phosphorothioate bond. In some embodiments, the 3'-internucleotide bond attached to the unmodified sugar moiety is a substituted phosphorothioate bond. In some embodiments, the 3'-internucleotide bond attached to the unmodified sugar moiety is a phosphorothioate triester bond. In some embodiments, each 3'-internucleotide bond attached to the unmodified sugar moiety is a modified bond. In some embodiments, each 3'-internucleotide bond attached to the unmodified sugar moiety is a bond having the structure of formula I. In some embodiments, each 3'-internucleotide bond attached to the unmodified sugar moiety is a phosphorothioate bond. In some embodiments, each 3'-internucleotide bond attached to the unmodified sugar moiety is a substituted phosphorothioate bond. In some embodiments, each 3'-internucleotide bond attached to the 2'-unmodified sugar moiety is a phosphorothioate triester bond.

[0277] In some embodiments, both internucleotide bonds attached to the unmodified sugar moiety are modified bonds. In some embodiments, both internucleotide bonds attached to the unmodified sugar moiety are bonds having the structure of formula I. In some embodiments, both internucleotide bonds attached to the unmodified sugar moiety are phosphorothioate bonds. In some embodiments, both internucleotide bonds attached to the unmodified sugar moiety are substituted phosphorothioate bonds. In some embodiments, both internucleotide bonds attached to the unmodified sugar moiety are phosphorothioate triester bonds. In some embodiments, each internucleotide bond attached to the unmodified sugar moiety is a modified bond. In some embodiments, each internucleotide bond attached to the unmodified sugar moiety is a bond having the structure of formula I. In some embodiments, each internucleotide bond attached to the unmodified sugar moiety is a phosphorothioate bond. In some embodiments, each internucleotide bond attached to the unmodified sugar moiety is a substituted phosphorothioate bond. In some embodiments, each internucleotide bond attached to the 2'-unmodified sugar moiety is a phosphorothioate triester bond.

[0278] In some embodiments, the unmodified sugar moiety is a sugar moiety found in natural DNA nucleosides.

[0279] In some embodiments, in a wing-core-wing structure, the 5'-terminal wing contains only one chiral internucleotide bond. In some embodiments, in a wing-core-wing structure, the 5'-terminal wing contains only one chiral internucleotide bond at its 5'-end. In some embodiments, in a wing-core-wing structure, the 3'-terminal wing contains only one chiral internucleotide bond. In some embodiments, in a wing-core-wing structure, the 3'-terminal wing contains only one chiral internucleotide bond at its 3'-end. In some embodiments, in a wing-core-wing structure, each wing contains only one chiral internucleotide bond. In some embodiments, in a wing-core-wing structure, each wing contains only one chiral internucleotide bond, where the 5'-terminal wing contains only one chiral internucleotide bond at its 5'-end; and the 3'-terminal wing contains only one chiral internucleotide bond at its 3'-end...

Claims

1. A chiral-controlled oligonucleotide composition, (1) Base sequence; (2) Cross-linking pattern of the skeleton; (3) Pattern of chiral centers of the skeleton; and (4) Patterns of phosphorus modification of the skeleton It includes multiple oligonucleotides of a specific oligonucleotide type as defined by The above composition is concentrated in relation to a racemic preparation of oligonucleotides having the same base sequence for a specific oligonucleotide type. The plurality of oligonucleotides have a wing-core-wing structure and have the same base sequence. Each wing region independently has a length of 2 or more base pairs. The bonds connecting the sugars in the wing region include one or more phosphate crosslinks, each independently being either a phosphate bond or a phosphorothioate bond. Each sugar in the wing region is independently a modified sugar. Each of the bonds connecting the sugar in the wing region and the sugar in the core region is independently a phosphorothioate bond. Each sugar in the core region is independently a sugar found in natural DNA. Each of the bonds connecting the sugars in the core region is independently a phosphorothioate bond. More than 60% of the inter-oligonucleotide crosslinks of the aforementioned plurality of oligonucleotides are phosphorothioate nucleotide bonds. Oligonucleotide composition.

2. A chiral-controlled oligonucleotide composition according to claim 1, A composition wherein the chiral center pattern of the aforementioned skeleton includes at least one Rp nucleotide bond and at least one Sp nucleotide bond.

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