Oligonucleotide compositions and methods thereof

Abstract

To provide chirally controlled oligonucleotides of select designs, to provide chirally controlled oligonucleotide compositions, and to provide methods of making and using the same.SOLUTION: In some embodiments, a provided chirally controlled oligonucleotide composition provides cleavage patterns of a nucleic acid polymer different from a reference oligonucleotide composition. In some embodiments, a provided chirally controlled oligonucleotide composition provides single site cleavage within a complementary sequence of a nucleic acid polymer. In some embodiments, the chiral controlled oligonucleotide compositions have any sequence of bases, and / or a pattern of base modifications, sugar modifications, backbone modifications and / or stereochemistry, or a combination of these elements.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

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

[0003] Among other things, the present disclosure encompasses the recognition that oligonucleotide components, such as base sequence, chemical modifications (e.g., sugar, base, and / or internucleotide linkage modifications and their patterns), and / or stereochemistry (e.g., the stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or their patterns), can significantly affect the properties, e.g., activity, of the oligonucleotide. In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising controlled components, e.g., oligonucleotides with controlled chemical modifications and / or controlled backbone stereochemical patterns, result in unexpected properties, including, but not limited to, those described herein. In some embodiments, the present disclosure demonstrates that the combination of chemical modifications and stereochemistry can result in unexpected and significantly improved properties (e.g., biological activity, selectivity, etc.). In some embodiments, the present disclosure provides oligonucleotide compositions having a particular sequence of bases and / or pattern of sugar modifications (e.g., 2'-OMe, 2'-F, 2'-MOE, etc.) and / or pattern of base modifications (e.g., 5-methylcytosine) and / or pattern of backbone modifications (phosphate or phosphorothioate) and / or pattern of backbone modification stereochemistry (e.g., each phosphorothioate is Sp or Rp). [Means for solving the problem]

[0004] In some embodiments, modification of the internucleotide bond can convert the phosphorus atom in the modified bond into a chiral center. For example, in a phosphorothioate (PS) modification, one of the non-bridging oxygen (O) atoms attached to the phosphorus (P) atom is replaced with a sulfur (S) atom. The result of using a PS modification in oligonucleotide synthesis is that it creates a chiral center at the phosphorus, which can have either an "Sp" or "Rp" configuration. For example, conventional stereorandom PS-modified oligonucleotide compositions with 19 PS bonds (e.g., 20 nucleotides long, each with 19 PS modifications, each with two possible stereochemistries (Sp or Rp)) each have the same nucleotide sequence (e.g., base sequence), but with 500,000 (2 19) stereoisomers; such compositions are "stereorandom" oligonucleotide compositions. In some embodiments, in contrast to stereorandom compositions, chiral-controlled oligonucleotide compositions are substantially pure preparations of single oligonucleotides in that a given level of oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, some oligonucleotide compositions are stereopure (i.e., chiral-controlled oligonucleotide compositions), in which the stereochemistry of each PS is defined as (Sp or Rp). In some embodiments, in a stereorandom composition of oligonucleotides, various oligonucleotides can have the same base sequence, the same pattern of sugar modifications (e.g., 2'-OMe, 2'-F, 2'-OMe, etc.), the same pattern of base modifications (e.g., 5-methylcytosine), and the same pattern of backbone modifications (phosphate or PS), and different patterns of backbone chiral centers, the levels of which are random from non-stereocontrolled synthesis (not predetermined as in stereocontrolled synthesis, such as certain methods exemplified herein using chiral auxiliaries). Chirality-controlled oligonucleotide compositions can be selected to have higher desired biological activity (e.g., higher activity, efficiency, etc. in RNA interference or RNAse H-mediated pathways) and reduced undesired activity (e.g., undesired immunogenicity, toxicity, etc.) than stereorandom preparations of oligonucleotides of the same base sequence. In some embodiments, chirality-controlled oligonucleotide compositions can better distinguish between mutant (mu) and wild-type (wt) HTT sequences (having a single nt difference).

[0005] In particular, the present disclosure encompasses the recognition that stereorandom oligonucleotide preparations contain multiple unique chemical entities that differ from one another, for example, in the stereochemical configuration of individual backbone chiral centers within the oligonucleotide chain. Unless the stereochemistry of the backbone chiral centers is controlled, stereorandom oligonucleotide preparations result in uncontrolled compositions containing undefined levels of oligonucleotide stereoisomers. These stereoisomers may have the same base sequence, but due at least to their different backbone stereochemistry, they are different chemical entities and, as demonstrated herein, may have different properties, e.g., biological activity. Among other things, the present disclosure provides novel compositions that are or contain specific stereoisomers of the subject oligonucleotides. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, its length, its backbone bond pattern, and its backbone chiral center pattern. As understood in the art, in some embodiments, a base sequence may refer to the identity and / or modification state of nucleoside residues (e.g., sugar and / or base moieties relative to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) within an oligonucleotide, and / or the hybridization properties of such residues (i.e., the ability to hybridize with a specific complementary residue).

[0006] The present disclosure demonstrates, inter alia, that individual stereoisomers of a particular oligonucleotide may exhibit different stability and / or activity (e.g., functional and / or toxicological properties) from one another. Furthermore, the present disclosure demonstrates that the improved stability and / or activity achieved by the inclusion and / or location of a particular chiral structure within an oligonucleotide may be comparable to or even better than the improved stability and / or activity achieved by the use of particular backbone linkages, residue modifications, etc. (e.g., by the use of particular types of modified phosphates [e.g., phosphorothioates, substituted phosphorothioates, etc.], sugar modifications [e.g., 2'-modifications, etc.], and / or base modifications [e.g., methylation, etc.]).

[0007] In particular, the present disclosure recognizes that in some embodiments, the properties (e.g., stability and / or activity) of an oligonucleotide may be modulated by optimizing the pattern of its backbone chiral centers, optionally in combination with modulating / optimizing one or more other characteristics of the oligonucleotide (e.g., linkage pattern, nucleoside modification pattern, etc.).

[0008] In some embodiments, the present invention provides oligonucleotide compositions, wherein the oligonucleotides have a common pattern of backbone chiral centers that unexpectedly and significantly improves the stability and / or biological activity of the oligonucleotides. In some embodiments, the pattern of backbone chiral centers results in increased stability. In some embodiments, the pattern of backbone chiral centers surprisingly results in increased activity. In some embodiments, the pattern of backbone chiral centers surprisingly alone alters the cleavage pattern of a target nucleic acid polymer when the oligonucleotide is utilized to cleave a nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers effectively prevents cleavage at a second site. In some embodiments, the pattern of backbone chiral centers creates new cleavage sites. In some embodiments, the pattern of backbone chiral centers minimizes the number of cleavage sites. In some embodiments, the pattern of backbone chiral centers minimizes the number of cleavage sites so that the target nucleic acid polymer is cleaved at only one site within the sequence of the target nucleic acid polymer that is complementary to the oligonucleotide. In some embodiments, the pattern of backbone chiral centers improves cleavage efficiency at the cleavage sites. In some embodiments, the pattern of backbone chiral centers of an oligonucleotide improves cleavage of a target nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers increases selectivity. In some embodiments, the pattern of backbone chiral centers minimizes off-target effects. In some embodiments, the pattern of backbone chiral centers increases selectivity, for example, cleavage selectivity between two target sequences that differ only by a single nucleotide polymorphism (SNP). In some embodiments, the pattern of backbone chiral centers 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 described herein, m is 1 to 50; n is 1 to 10; and t is 1 to 50. In some embodiments, the pattern of backbone chiral centers comprises or is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, the pattern of backbone chiral centers comprises (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) mwhere m>2. In some embodiments, the pattern of backbone chiral centers is a sequence comprising at least 5, 6, 7, 8, 9, or 10 or more consecutive (Sp) positions. In some embodiments, the pattern of backbone chiral centers is a sequence comprising at least 5 consecutive (Sp) positions. In some embodiments, the pattern of backbone chiral centers is a sequence comprising at least 8 consecutive (Sp) positions. In some embodiments, the pattern of backbone chiral centers is a sequence comprising at least 10 consecutive (Sp) positions. In some embodiments, the pattern of backbone chiral centers is a sequence consisting of all (Sp) and a single (Rp). In some embodiments, the pattern of backbone chiral centers is a sequence consisting of all (Sp) and a single (Rp) at or adjacent to an SNP position. In some embodiments, the pattern of backbone chiral centers is a sequence consisting of all (Sp) and a single (Rp), where the molecule has a wing-core-wing format. In some embodiments, the pattern of backbone chiral centers is an all-(Sp) and a single (Rp) sequence, where the molecule has a wing-core-wing format, where the 5'-terminal wings are 1-9 nt long, the core is 1-15 nt long, and the 3'-terminal wings are 1-9 nt long. In some embodiments, the pattern of backbone chiral centers is an all-(Sp) and a single (Rp) sequence, where the molecule has a wing-core-wing format, where the 5'-terminal wings are 5 nt long, the core is 1-15 nt long, and the 3'-terminal wings are 5 nt long. In some embodiments, the pattern of backbone chiral centers is an all-(Sp) and a single (Rp) sequence, where the molecule has a wing-core-wing format, where the 5'-terminal wings are 1-9 nt long, the core is 10 nt long, and the 3'-terminal wings are 1-9 nt long. In some embodiments, the pattern of backbone chiral centers is an all (Sp) and single (Rp) sequence, where the molecule has a wing-core-wing format, where the 5'-terminal wing is 5 nt long, the core is 10 nt long, and the 3'-terminal wing is 5 nt long.In some embodiments, the pattern of backbone chiral centers is an arrangement consisting of all (Sp) and a single (Rp), where the molecule has a wing-core-wing format, where the 5'-terminal wing is 5 nt long, the core is 10 nt long, and the 3'-terminal wing is 5 nt long, and at least one wing comprises a nucleotide with a 2'-OMe modification. In some embodiments, the pattern of backbone chiral centers is an arrangement consisting of all (Sp) and a single (Rp), where the molecule has a wing-core-wing format, where each wing comprises at least one nucleotide with a 2'-OMe modification. In some embodiments, the pattern of backbone chiral centers is an arrangement consisting of all (Sp) and a single (Rp), where the molecule has a wing-core-wing format, where each nucleotide in both wings comprises a 2'-OMe modification. In some embodiments, the pattern of backbone chiral centers is an all (Sp) and single (Rp) sequence, where the molecule has a wing-core-wing format, where the 5'-terminal wing is 5 nt long, the core is 10 nt long, the 3'-terminal wing is 5 nt long, and each nucleotide within each wing has a 2'-OMe modification. In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing format, where the wing at the 5' end of the molecule contains 4 to 8 nt, each of which has a 2'-OMe modification, and where the nt at the 5' end of the molecule has a phosphorothioate in the Sp configuration; the core contains 8 to 12 nt, each of which is DNA(2'-H), and where each has a phosphorothioate in the Sp position except for one nt that has a phosphorothioate in the Rp position; and where the wing at the 3' end of the molecule contains 4 to 8 nt, each of which has a 2'-OMe modification, and where the nt at the 3' end of the molecule has a phosphorothioate in the Sp configuration.In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing format, where the wing at the 5' end of the molecule comprises 6 nt, each of which has a 2'-OMe modification, and where the nt at the 5' end of the molecule has a phosphorothioate in the Sp configuration; the core comprises 10 nt, each of which is DNA(2'-H), and where each has a phosphorothioate in the Sp position except for one nt that has a phosphorothioate in the Rp position; and where the wing at the 3' end of the molecule comprises 6 nt, each of which has a 2'-OMe modification, and where the nt at the 3' end of the molecule has a phosphorothioate in the Sp configuration.

[0009] In some embodiments, the present disclosure recognizes that chemical modifications, such as modifications of nucleosides and internucleotide linkages, can improve properties. In some embodiments, the present disclosure demonstrates that a combination of chemical modifications and stereochemistry can result in unexpected and significantly improved properties (e.g., biological activity, selectivity, etc.). In some embodiments, the combination of chemical modifications, such as modifications of sugars, bases, and / or internucleotide linkages, can result in stereochemical patterns, e.g., (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m In some embodiments, the oligonucleotide compositions provided are chiral and comprise one or more 2'-modifications of the sugar moiety, one or more natural phosphate linkages, one or more phosphorothioate linkages, and (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m (wherein m>2)

[0010] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers wherein the composition is a substantially pure preparation of a single oligonucleotide, wherein a predetermined level of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0011] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; wherein the composition is chirally controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0012] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers The present invention provides a chirality-controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type, characterized by: wherein the composition is a substantially pure preparation of a single oligonucleotide, wherein at least about 10% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0013] Among other things, the present disclosure recognizes that combinations of oligonucleotide building blocks (e.g., patterns of chemical modifications, backbone linkages, backbone chiral centers, and / or backbone phosphorus modifications) can confer properties such as surprisingly improved biological activity. In some embodiments, the present disclosure provides oligonucleotide compositions comprising predetermined levels of oligonucleotides comprising one or more wing regions and a common core region, wherein: each wing region independently has a length of two or more bases and independently optionally contains one or more chiral internucleotide linkages; The core regions independently have a length of two or more bases and independently contain one or more chiral internucleotide linkages, and the common core region comprises: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers It has.

[0014] In some embodiments, within an oligonucleotide comprising a wing-core-wing format, the "wings" are portions of the oligonucleotide at the 5' or 3' end of the core, with the "core" (alternatively referred to as a "gap") between the two wings. In some embodiments, an oligonucleotide can have a single wing and a single core; in such cases, the wings are at the 5' or 3' end of the oligonucleotide. The wings and core can be defined by any of several components (e.g., modifications or patterns of modifications to the sugar, base, backbone, or backbone stereochemistry). In some embodiments, the wings and core are defined by nucleoside modifications, where the wings include nucleoside modifications that the core region does not have. In some embodiments, the oligonucleotides in the provided compositions have a wing-core structure of nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a core-wing structure of nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a wing-core-wing structure of nucleoside modifications. In some embodiments, the wings and core are defined by modifications to the sugar moiety. In some embodiments, the wings and core are defined by modifications to 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 any sugar modification in the core region. In some embodiments, each sugar moiety in the wing region has the same 2'-modification, and the core region has no 2'-modification. In some embodiments, when more than one wing is present, each sugar moiety in the wing region has the same 2'-modification, but the common 2'-modification in a first wing region can be the same as or different from the common 2'-modification in a second wing region.

[0015] In some embodiments, each wing comprises at least one chiral internucleotide linkage and at least one natural phosphate linkage. In some embodiments, each wing comprises at least one modified sugar moiety. In some embodiments, each wing sugar moiety is modified. In some embodiments, the wing sugar moiety is modified with a modification that is not present in the core region. In some embodiments, only the wing region has a modified internucleotide linkage at one or both of its termini. In some embodiments, only the wing region has a modified internucleotide linkage at its 5'-terminus. In some embodiments, only the wing region has a modified internucleotide linkage at its 3'-terminus. In some embodiments, only the wing region has modified internucleotide linkages at its 5'-terminus and 3'-terminus. In some embodiments, the wing is to the 5'-terminus of the core and only the wing has a modified internucleotide linkage at its 5'-terminus. In some embodiments, the wing is to the 5'-terminus of the core and only the wing has a modified internucleotide linkage at its 3'-terminus. In some embodiments, the wings are to the 5'-end of the core and only the wings have modified internucleotide linkages at both their 5'-end and 3'-end. In some embodiments, the wings are to the 3'-end of the core and only the wings have modified internucleotide linkages at their 5'-end. In some embodiments, the wings are to the 3'-end of the core and only the wings have modified internucleotide linkages at their 3'-end. In some embodiments, the wings are to the 3'-end of the core and only the wings have modified internucleotide linkages at both their 5'-end and 3'-end. In some embodiments, the modification(s) to the sugar moiety or internucleotide linkage or other modification in one wing can differ from the modifications in another wing.

[0016] In some embodiments, each internucleotide linkage of the core region is modified. In some embodiments, each internucleotide linkage of the core region is chiral. In some embodiments, the core region comprises (Sp) m (Rp) n , (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m In some embodiments, the core region has a pattern of backbone chiral centers of (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m where m>2. In particular, the present disclosure shows that in some embodiments, such patterns can result in or enhance controlled cleavage of target sequences, such as RNA sequences.

[0017] In some embodiments, the oligonucleotides in the provided compositions have a common pattern of backbone phosphorus modifications. In some embodiments, the provided compositions are chiral controlled oligonucleotide compositions in that they comprise predetermined levels of oligonucleotides of individual oligonucleotide types, where the oligonucleotide types are: 1) Nucleotide sequence; 2) pattern of skeletal bonding; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification is defined as:

[0018] As noted above and understood in the art, in some embodiments, the base sequence of an oligonucleotide may refer to the identity and / or modification state of the nucleoside residues (e.g., sugar and / or base moieties, relative to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) within the oligonucleotide, and / or the hybridization properties of such residues (i.e., the ability to hybridize with specific complementary residues).

[0019] In some embodiments, a particular oligonucleotide type is 1A) base identity; 1B) base modification patterns; 1C) Glycosylation patterns; 2) pattern of skeletal bonding; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification It may be defined by: Thus, in some embodiments, oligonucleotides of a particular type may share the same bases but differ in their patterns of base and / or sugar modifications, hi some embodiments, oligonucleotides of a particular type may share the same bases and patterns of base modifications (including, for example, the absence of base modifications), but differ in their patterns of sugar modifications.

[0020] In some embodiments, oligonucleotides of a particular type have the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same pattern of backbone linkages (e.g., natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., the pattern of chiral internucleotide linkage stereochemistry (Rp / Sp)), and the same pattern of backbone phosphorus modifications (e.g., -S - and -LR of formula I 1They are chemically identical in that they have the same pattern of modifications to the phosphorus atom of an internucleotide such as ribonucleotides.

[0021] In some embodiments, the sequence of the oligonucleotide comprises or consists of the sequence of any oligonucleotide disclosed herein. In some embodiments, the sequence of the oligonucleotide comprises or consists of the sequence of any oligonucleotide selected from Table N1, Table N2, Table N3, Table N4, and Table 8. In some embodiments, the sequence of the oligonucleotide comprises or consists of the sequence of any oligonucleotide selected from Table N1A, Table N2A, Table N3A, Table N4A, and Table 8. In some embodiments, the sequence of the oligonucleotide in the stereopure (chiral controlled) oligonucleotide composition comprises or consists of the sequence of WV-1092, WVE120101, WV-2603, or WV-2595. In some embodiments, the sequence of an oligonucleotide may be determined by a number of factors, including base sequence (including length); the pattern of chemical modifications to the sugar and base moieties; the pattern of backbone linkages; the pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof; the pattern of backbone chiral centers; the pattern of chiral internucleotide linkage stereochemistry (Rp / Sp); the pattern of backbone phosphorus modifications; -S - and -LR of formula I 1 The internucleotide may include any one or more of the patterns of modification to the phosphorus atom such as:

[0022] In particular, the present disclosure recognizes the problem of stereoselective (as opposed to stereorandom or racemic) preparation of oligonucleotides. In particular, the present disclosure provides methods and reagents for the stereoselective preparation of oligonucleotides containing multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotide linkages, particularly for oligonucleotides containing multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotide linkages. In some embodiments, in stereorandom or racemic preparations of oligonucleotides, at least one chiral internucleotide linkage is formed with a diastereoselectivity of less than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral-controlled preparations of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 95:5. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 96:4. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 97:3. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 98:2. In some embodiments, for stereoselective or chiral-controlled preparation of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 99:1.In some embodiments, the diastereoselectivity of a chiral internucleotide linkage within an oligonucleotide may be measured by a model reaction, e.g., the formation of a dimer under substantially the same or equivalent conditions, in which the dimer has the same internucleotide linkage as the chiral internucleotide linkage, the 5'-nucleoside of the dimer is the same as the nucleoside to the 5'-terminus of the chiral internucleotide linkage, and the 3'-nucleoside of the dimer is the same as the nucleoside to the 3'-terminus of the chiral internucleotide linkage.

[0023] In particular, it has been surprisingly found that certain provided oligonucleotide compositions provide unprecedented control over the cleavage of target sequences, e.g., cleavage of target RNA by RNase H. In some embodiments, the present disclosure demonstrates that precise control of the chemical and stereochemical properties of oligonucleotides provides improved activity of oligonucleotide preparations compared to comparable preparations where the stereochemical properties are not otherwise controlled. In particular, the present disclosure demonstrates improved cleavage rates, degrees of cleavage, and / or cleavage specificity of nucleic acid targets to which the provided oligonucleotides hybridize.

[0024] In some embodiments, the present disclosure provides various uses for oligonucleotide compositions. In particular, the present disclosure demonstrates that the properties of oligonucleotides can be significantly improved by controlling the components of the oligonucleotide, such as, for example, base sequence, chemical modification, and stereochemistry. For example, in some embodiments, the present disclosure provides methods for highly selective suppression of transcription of a target nucleic acid sequence. In some embodiments, the present disclosure provides methods for treating a subject by suppressing transcription from a disease-causing copy (e.g., a disease-causing allele). In some embodiments, the present disclosure provides methods for designing and preparing oligonucleotide compositions with surprisingly improved activity and / or selectivity when suppressing transcription of a target sequence. In some embodiments, the present disclosure provides methods for designing and / or preparing oligonucleotide compositions that result in allele-specific suppression of transcription from a target nucleic acid sequence.

[0025] In some embodiments, the disclosure provides a method for controlled cleavage of a nucleic acid polymer, the method comprising: 1) a consensus sequence and length that is complementary to or includes a target sequence found in a nucleic acid polymer; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; contacting a nucleic acid polymer, the nucleotide sequence of which comprises a target sequence, with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having a particular base sequence and length.

[0026] In some embodiments, the disclosure provides methods for altering the cleavage pattern observed when a nucleic acid polymer, the nucleotide sequence of which comprises a target sequence, is contacted with a reference oligonucleotide composition comprising oligonucleotides having a particular base sequence and length, the particular base sequence being or including a sequence that is complementary to the target sequence, the method comprising: contacting a nucleic acid polymer with a chiral controlled oligonucleotide composition of oligonucleotides having a specific base sequence and length, the composition having, relative to a substantially racemic preparation of oligonucleotides having the specific base sequence and length: 1) specific base sequence and length; 2) a specific pattern of skeletal bonds; and 3) Specific patterns of backbone chiral centers The chirality is controlled in that it is enriched for oligonucleotides of a single oligonucleotide type characterized by:

[0027] In some embodiments, the disclosure provides a method for the suppression of transcription from a target nucleic acid sequence for which one or more similar nucleic acid sequences are present within a population, wherein each of the target sequence and the similar sequences comprises a sequence element characteristic of a specific nucleotide that defines the target sequence relative to the similar sequence, the method comprising: 1) consensus sequence and length; and 2) common patterns of skeletal bonding; contacting a sample containing a transcript of a target nucleic acid sequence with an oligonucleotide composition comprising an oligonucleotide having: Here, the common base sequence is or includes a sequence that is complementary to a characteristic sequence element that defines the target nucleic acid sequence, and the composition is characterized in that when the composition is contacted with a system containing transcripts of both the target nucleic acid sequence and the similar nucleic acid sequence, transcription of the target nucleic acid sequence is suppressed at a level higher than the level of suppression observed in the similar nucleic acid sequence.

[0028] In some embodiments, the disclosure provides a method for the suppression of transcription from a target nucleic acid sequence for which one or more similar nucleic acid sequences are present within a population, wherein each of the target sequence and the similar sequences comprises a sequence element characteristic of a specific nucleotide that defines the target sequence relative to the similar sequence, the method comprising: 1) consensus sequence and length; and 2) common patterns of skeletal bonding; 3) common patterns of backbone chiral centers; contacting a sample containing a transcript of a target nucleic acid sequence with an oligonucleotide composition comprising an oligonucleotide having: Here, the common base sequence is or includes a sequence that is complementary to a characteristic sequence element that defines the target nucleic acid sequence, and the composition is characterized in that when the composition is contacted with a system containing transcripts of both the target nucleic acid sequence and the similar nucleic acid sequence, transcription of the target nucleic acid sequence is suppressed at a level higher than the level of suppression observed in the similar nucleic acid sequence.

[0029] In some embodiments, transcription of the target nucleic acid sequence is repressed at a level greater than the level of repression observed for any one of the similar nucleic acid sequences.

[0030] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target nucleic acid sequence for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising: 1) consensus sequence and length; and 2) common patterns of skeletal bonding; contacting a sample containing a transcript of a target nucleic acid sequence with an oligonucleotide composition comprising an oligonucleotide having: wherein the consensus base sequence is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition is characterized in that when the composition is contacted with a system containing transcripts of both the target allele and another allele of the same nucleic acid sequence, the transcript of the particular allele is suppressed at a level greater than the level of suppression observed in another allele of the same nucleic acid sequence.

[0031] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target nucleic acid sequence for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; 3) common patterns of backbone chiral centers; contacting a sample containing a transcript of a target nucleic acid sequence with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length; wherein the common base sequence of the oligonucleotides of a particular oligonucleotide type is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition is characterized in that when the composition is contacted with a system containing transcripts of both the target allele and another allele of the same nucleic acid sequence, the transcript of the particular allele is suppressed at a level greater than the level of suppression observed in another allele of the same nucleic acid sequence.

[0032] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target gene, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; contacting a sample containing a transcript of a target gene with an oligonucleotide composition comprising an oligonucleotide having: wherein the consensus base sequence is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and wherein the composition is characterized in that, when the composition is contacted with a system containing transcripts of both the target allele and another allele of the same gene, the transcript of the particular allele is suppressed at a level that is at least two-fold higher than the level of suppression observed in another allele of the same gene.

[0033] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target gene, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; 3) common patterns of backbone chiral centers; contacting a sample containing a transcript of a target gene with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length; wherein the common base sequence of the oligonucleotides of a particular oligonucleotide type is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition is characterized in that when the composition is contacted with a system containing transcripts of both the target allele and another allele of the same gene, the transcript of the particular allele is suppressed at a level that is at least two-fold higher than the level of suppression observed in another allele of the same gene.

[0034] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target gene, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; 3) common patterns of backbone chiral centers; contacting a sample containing a transcript of a target gene with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length; wherein the common base sequence of the oligonucleotides of a particular oligonucleotide type is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition is characterized in that when the composition is contacted with a system that expresses transcripts of both the target allele and another allele of the same gene, the transcript of the particular allele is suppressed at a level that is at least two-fold higher than the level of suppression observed in another allele of the same gene.

[0035] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target nucleic acid sequence for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; contacting a sample containing a transcript of a target nucleic acid sequence with an oligonucleotide composition comprising an oligonucleotide having: wherein the consensus base sequence is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and wherein the composition, when contacting the composition with a system containing a transcript of the same target nucleic acid sequence: a) higher than when the composition is absent; b) greater than the level of suppression observed in another allele of the same nucleic acid sequence; or c) greater than in the absence of the composition and greater than the level of suppression observed in another allele of the same nucleic acid sequence These are characterized by showing suppression of the transcript of a particular allele at the level of the gene.

[0036] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target nucleic acid sequence for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; 3) common patterns of backbone chiral centers; contacting a sample containing a transcript of a target nucleic acid sequence with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length; wherein the common base sequence of the oligonucleotides of a particular oligonucleotide type is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition, when contacted with a system containing a transcript of the same target nucleic acid sequence: a) higher than when the composition is absent; b) greater than the level of suppression observed in another allele of the same nucleic acid sequence; or c) greater than in the absence of the composition and greater than the level of suppression observed in another allele of the same nucleic acid sequence These are characterized by showing suppression of the transcript of a particular allele at the level of the gene.

[0037] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target gene, the method comprising: 1) consensus sequence and length; and 2) common patterns of skeletal bonding; contacting a sample containing a transcript of a target gene with an oligonucleotide composition comprising an oligonucleotide having: wherein the consensus sequence is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition is characterized in that, when the composition is contacted with a system that expresses a transcript of the target gene, a) at least two-fold, in that transcripts from a particular allele are detected at half the amount when the composition is present compared to when the composition is absent; b) at least two-fold higher than the level of suppression observed in another allele of the same gene; or c) at least two-fold, in that the transcript from a particular allele is detected at half the amount in the presence of the composition compared to the absence of the composition, and at least two-fold greater than the level of repression observed for another allele of the same gene; These are characterized by exhibiting suppression of expression of the transcript of a particular allele at the level of the gene.

[0038] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target gene, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; 3) common patterns of backbone chiral centers; contacting a sample containing a transcript of a target gene with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length; wherein the common base sequence of the oligonucleotides of a particular oligonucleotide type is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition, when contacted with a system that expresses a transcript of the target gene, a) at least two-fold, in that transcripts from a particular allele are detected at half the amount when the composition is present compared to when the composition is absent; b) at least two-fold higher than the level of suppression observed in another allele of the same gene; or c) at least two-fold, in that the transcript from a particular allele is detected at half the amount in the presence of the composition compared to the absence of the composition, and at least two-fold greater than the level of repression observed for another allele of the same gene; These are characterized by exhibiting suppression of expression of the transcript of a particular allele at the level of the gene.

[0039] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target gene, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; contacting a sample containing a transcript of a target gene with an oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: wherein the consensus sequence is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition is characterized in that, when the composition is contacted with a system that expresses a transcript of the target gene, a) at least two-fold, in that transcripts from a particular allele are detected at half the amount when the composition is present compared to when the composition is absent; b) at least two-fold higher than the level of suppression observed in another allele of the same gene; or c) at least two-fold, in that the transcript from a particular allele is detected at half the amount in the presence of the composition compared to the absence of the composition, and at least two-fold greater than the level of repression observed for another allele of the same gene; These are characterized by exhibiting suppression of expression of the transcript of a particular allele at the level of the gene.

[0040] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which multiple alleles are present in a population, each of which contains a sequence element characteristic of a specific nucleotide that defines the allele relative to other alleles of the same target gene, the method comprising: 1) consensus sequence and length; 2) common patterns of skeletal bonding; 3) common patterns of backbone chiral centers; contacting a sample containing a transcript of a target gene with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length; wherein the common base sequence of the oligonucleotides of a particular oligonucleotide type is or includes a sequence that is complementary to a characteristic sequence element that defines a particular allele, and the composition, when contacted with a system that expresses a transcript of the target gene, a) at least two-fold, in that transcripts from a particular allele are detected at half the amount when the composition is present compared to when the composition is absent; b) at least two-fold higher than the level of suppression observed in another allele of the same gene; or c) at least two-fold, in that the transcript from a particular allele is detected at half the amount in the presence of the composition compared to the absence of the composition, and at least two-fold greater than the level of repression observed for another allele of the same gene; These are characterized by exhibiting suppression of expression of the transcript of a particular allele at the level of the gene.

[0041] In some embodiments, the nucleotide signature sequence comprises a mutation that defines the target sequence relative to other similar sequences. In some embodiments, the nucleotide signature sequence comprises a point mutation that defines the target sequence relative to other similar sequences. In some embodiments, the nucleotide signature sequence comprises a SNP that defines the target sequence relative to other similar sequences.

[0042] In some embodiments, the present disclosure provides: 1) A consensus sequence that is identical to a specific sequence; 2) common patterns of skeletal bonding; and 3) Common pattern of skeletal chiral centers (This pattern is (Sp) m (Rp) n , (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m (In the formula: m is 1 to 50; n is 1 to 10; t is between 1 and 50; and Each Np is independently Rp or Sp. The present invention provides a method for preparing an oligonucleotide composition comprising an oligonucleotide of a specific sequence that results in selective inhibition of transcription of a target sequence, the method comprising the step of providing a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by:

[0043] Generally, the activity of the oligonucleotide compositions described herein can be assessed using any suitable assay. The relative activities of different compositions (e.g., stereoregulated versus non-stereoregulated compositions, and / or different stereoregulated compositions) are typically measured, desirably in the same assay, in some embodiments substantially simultaneously, and in some embodiments with reference to historical results.

[0044] Those skilled in the art will recognize and / or be able to readily develop assays appropriate for particular oligonucleotide compositions. The present disclosure describes certain assays that may be useful, for example, in assessing one or more characteristics of the behavior of an oligonucleotide composition with respect to RNAse H cleavage of a target sequence.

[0045] For example, particular assays that may be useful in assessing one or more characteristics of RNase H cleavage (e.g., rate, extent and / or selectivity of cleavage) may include any of the assays described and / or exemplified herein (e.g., in one or more of Examples 4, 9-10, 12, 14, 17-20, etc.).

[0046] In some embodiments, the present disclosure recognizes that the base sequence can affect the properties of an oligonucleotide. The present disclosure demonstrates that chemical and steric modifications combined with a designed base sequence can provide oligonucleotide compositions with unexpectedly improved properties (e.g., surprisingly higher activity and / or selectivity). In some embodiments, oligonucleotides having a common base sequence complementary to a distinctive sequence element of a target nucleic acid sequence provide better activity than another common base sequence complementary to a distinctive sequence element of a target nucleic acid sequence. In some embodiments, oligonucleotides having a common base sequence complementary to a distinctive sequence element of a target nucleic acid sequence provide better selectivity than another common base sequence complementary to a distinctive sequence element of a target nucleic acid sequence.

[0047] In some embodiments, a composition of oligonucleotides having a common base sequence complementary to a distinctive sequence element of a target nucleic acid sequence results in a higher cleavage rate of transcripts from the target nucleic acid sequence and / or a cleavage pattern with only one major cleavage site, the major cleavage site being within or close to the sequence characteristic of the nucleotide, compared to another composition of oligonucleotides having another common base sequence complementary to the sequence characteristic of the target nucleic acid sequence. In some embodiments, a composition of oligonucleotides having a complementary common base sequence results in a higher cleavage rate of transcripts from the target nucleic acid sequence and a cleavage pattern with only one major cleavage site, the major cleavage site being within or close to the sequence characteristic of the nucleotide, compared to another composition of oligonucleotides having another complementary common base sequence. In some embodiments, more than 50%, 60%, 70%, 80%, or 90% of cleavage occurs at one major cleavage site, for example, as measured by a suitable method, such as an RNase H assay. In some embodiments, a composition of oligonucleotides having a complementary common base sequence results in a higher cleavage rate of transcripts from a target nucleic acid sequence and a cleavage pattern with only one primary cleavage site when compared to another composition of oligonucleotides having another complementary common base sequence, and the primary cleavage site is within or close to a mutation or SNP that defines the target sequence relative to other similar sequences. In some embodiments, the mutation is a point mutation. In some embodiments, the primary cleavage site is adjacent to a mutation or SNP that defines the target sequence relative to other similar sequences. In some embodiments, each common base sequence is 100% complementary to a distinctive sequence element of the target nucleic acid sequence. In some embodiments, the primary cleavage site is within 5, 4, 3, or less than 1 internucleotide bond from a mutation or SNP that defines the target sequence relative to other similar sequences.In some embodiments, the primary cleavage site is within less than 5, 4, 3, or 1 internucleotide bond from the mutation or SNP that defines the target sequence relative to other similar sequences, and within less than 5, 4, 3, or 1 internucleotide bond from the cleavage site when a stereorandom composition of oligonucleotides having the same consensus sequence and / or a composition of DNA oligonucleotides having the same consensus sequence is used. In some embodiments, the primary cleavage site is the cleavage site when a stereorandom composition of oligonucleotides having the same consensus sequence is used. In some embodiments, the primary cleavage site is the cleavage site when a stereorandom composition of oligonucleotides having the same consensus sequence is used. In some embodiments, the primary cleavage site is the cleavage site when a composition of DNA oligonucleotides having the same consensus sequence is used. In some embodiments, the primary cleavage site is the cleavage site when a composition of DNA oligonucleotides having the same consensus sequence is used.

[0048] In some embodiments, when comparing the effects of a first and second common base sequence, a stereorandom composition of oligonucleotides having a first common base sequence may be compared to a stereorandom composition of oligonucleotides having a second common base sequence. In some embodiments, the stereorandom composition is a composition of oligonucleotides having a common base sequence, a common pattern of nucleoside modifications, and a common pattern of backbone linkages. In some embodiments, the stereorandom composition is a composition of oligonucleotides having a common base sequence, a common pattern of nucleoside modifications, and a common pattern of backbone linkages, where each internucleotide linkage is phosphorothioate. In some embodiments, when comparing the effects of a first and second common base sequence, a chirality-controlled oligonucleotide composition of oligonucleotides having a first common base sequence may be compared to a chirality-controlled oligonucleotide composition of oligonucleotides having a second common base sequence. In some embodiments, the oligonucleotides in the chirality-controlled oligonucleotide composition have a common base sequence, a common pattern of nucleoside modifications, a common pattern of backbone linkages, a common pattern of backbone chiral centers, and a common pattern of backbone phosphorus modifications. In some embodiments, each internucleotide linkage is phosphorothioate.

[0049] In some embodiments, the oligonucleotide compositions and techniques described herein are particularly useful in the treatment of Huntington's disease. For example, in some embodiments, the present disclosure defines stereochemically controlled oligonucleotide compositions that direct cleavage (e.g., RNase H-mediated cleavage) of nucleic acids associated with Huntington's disease. In some embodiments, such compositions direct preferential cleavage of Huntington's disease-associated alleles of a particular target sequence compared to one or more (e.g., all non-Huntington's disease-associated) other alleles of the sequence.

[0050] Huntington's disease is a genetic disorder that causes progressive degeneration of brain neurons, potentially affecting a subject's motor and cognitive abilities. In some embodiments, Huntington's disease is an autosomal dominant disorder. In some embodiments, Huntington's disease is caused by mutations in the huntingtin gene. A normal HTT gene contains 10 to 35 CAG trinucleotide repeats. People with 40 or more repeats often develop the disorder. In some embodiments, an expanded CAG segment in the first exon of the HTT gene leads to the production of an abnormally long version of the huntingtin protein (an expanded polyglutamine tract) that binds and accumulates in neurons, disrupting the normal function of these cells, and is cleaved into smaller, toxic fragments. Warby et al. (Am J Hum Genet. 2009, 84(3), 351-366) reported a number of SNPs associated with the disease chromosome that are more strongly associated with CAG expansions than previously reported. Many SNPs strongly associated with CAG expansions do not segregate independently and are in linkage disequilibrium with each other. In particular, the present disclosure recognizes that the strong association between specific SNP and CAG expansion chromosome provides attractive therapeutic opportunities for, for example, the treatment of Huntington's disease by antisense therapy.Furthermore, the association of specific SNP combined with the high frequency of heterozygosity in HD patients provides a suitable target for the allele-specific knockdown of mutant gene products.For example, see Liu et al., Journal of Huntington's Disease 2,2013,491-500; Aronin, Neil and Pfister, Edith, International Publication No. 2010 / 118263(A1); Pfister et al., Current Biology 2009,19,774-778.

[0051] In some embodiments, the target SNPs of the present disclosure have a high frequency of heterozygosity in HD and have specific variants associated with mutant HTT alleles. In some embodiments, the SNP is rs362307. In some embodiments, the SNP is rs7685686. In some embodiments, the SNPs may be unlinked but have a high frequency of heterozygosity. In some embodiments, the SNP is rs362268 (3'-UTR region). In some embodiments, the SNP is rs362306 (3'-UTR region). In some embodiments, the SNP is rs2530595. In some embodiments, the SNP is rs362331.

[0052] In some embodiments, provided are methods for treating or preventing Huntington's disease in a subject, comprising administering to the subject a provided oligonucleotide composition. 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; 10. A method for treating or preventing Huntington's disease in a subject, comprising administering to the subject a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The method is chiral controlled in that the composition is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0053] In some embodiments, provided methods ameliorate symptoms of Huntington's disease. In some embodiments, provided methods delay the onset of Huntington's disease. In some embodiments, provided methods slow the progression of Huntington's disease.

[0054] In some embodiments, the present disclosure provides a method for identifying patients for a given oligonucleotide composition.In some embodiments, the present disclosure provides a method for patient stratification.In some embodiments, the provided method comprises identifying mutations and / or SNPs associated with disease-causing alleles.For example, in some embodiments, the provided method comprises identifying SNPs associated with Huntington's disease or associated with the expanded CAG repeat that causes Huntington's disease in a subject.

[0055] In some embodiments, a subject has a SNP in the subject's huntingtin gene. In some embodiments, the subject has a SNP, where one allele is a mutant huntingtin associated with an expanded CAG repeat. In some embodiments, the subject has a SNP selected from rs362307, rs7685686, rs362268, rs2530595, rs362331, or rs362306. In some embodiments, the oligonucleotides of the provided compositions have a sequence complementary to a sequence containing the SNP from a disease-causing allele (mutation), and the composition selectively suppresses expression from the disease-causing allele.

[0056] In some embodiments, the sequence of an oligonucleotide in the provided technology (compounds, compositions, methods, etc.) comprises, consists of, or is the sequence of any of the oligonucleotides described herein. In some embodiments, the sequence is selected from Table N1A, Table N2A, Table N3A, Table N4A, or Table 8; or WV-1092, WVE120101, WV-2603, or WV-2595. In some embodiments, the sequence is selected from the sequences of WV-1092, WVE120101, WV-2603, or WV-2595. In some embodiments, the provided oligonucleotide is an oligonucleotide of the type defined by WV-1092, WVE120101, WV-2603, or WV-2595. In some embodiments, the provided oligonucleotide is an oligonucleotide of the type defined by WV-1092. In some embodiments, the provided oligonucleotide is an oligonucleotide of the type defined by WVE120101. In some embodiments, the provided oligonucleotide is an oligonucleotide of the type defined by WV-2603. In some embodiments, the provided oligonucleotide is an oligonucleotide of the type defined by WV-2595.

[0057] In some embodiments, provided oligonucleotide compositions comprise a lipid and an oligonucleotide. In some embodiments, the lipid is associated with the oligonucleotide.

[0058] In some embodiments, a composition comprises an oligonucleotide and a lipid selected from the list of: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid, and dilinoleyl. In some embodiments, a composition comprises an oligonucleotide and a lipid selected from the list of: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid, and dilinoleyl.

[0059] In some embodiments, the composition comprises an oligonucleotide and a lipid selected from: [ka] TIFF2025038091000002.tif87166

[0060] In some embodiments, the composition comprises an oligonucleotide and a lipid, wherein the lipid comprises one or more C 1-4 Saturated or partially unsaturated straight-chain C optionally substituted with aliphatic groups 10 -C 40 Contains aliphatic chains.

[0061] In some embodiments, the oligonucleotide composition comprises a plurality of oligonucleotides, which are: 1) consensus sequence; 2) common patterns of skeletal bonding; and 3) common patterns of backbone phosphorus modifications; wherein one or more of the plurality of oligonucleotides are individually bound to a lipid.

[0062] In some embodiments, the chiral controlled oligonucleotide composition comprises a plurality of oligonucleotides, which are: 1) consensus sequence; 2) common patterns of skeletal bonding; and 3) common patterns of backbone phosphorus modifications; Share it here: The composition is chiral controlled in that the oligonucleotides share the same stereochemistry at one or more chiral internucleotide linkages; one or more of the plurality of oligonucleotides are individually bound to a lipid; and One or more of the plurality of oligonucleotides are optionally individually bound to a target compound or moiety.

[0063] In some embodiments, a method for delivering an oligonucleotide to a cell or tissue of a human subject comprises: (a) providing a composition according to any one of the embodiments described herein; and (b) administering the composition to a human subject such that the oligonucleotide is delivered to a cell or tissue of the subject. Includes.

[0064] In some embodiments, a method for delivering an oligonucleotide to a cell or tissue includes preparing a composition according to any one of the embodiments described herein and contacting the cell or tissue with the composition.

[0065] In some embodiments, a method for modulating the level of a gene transcript or gene product in a cell, comprising contacting the cell with a composition according to any one of the embodiments described herein, wherein the oligonucleotide is capable of modulating the level of the transcript or gene product.

[0066] In some embodiments, a method for inhibiting expression of a gene in a cell or tissue includes preparing a composition according to any one of the embodiments described herein and treating the cell or tissue with the composition.

[0067] In some embodiments, a method for inhibiting expression of a gene in a cell or tissue of a mammal comprises preparing a composition according to any one of the embodiments described herein and administering the composition to a mammal.

[0068] In some embodiments, a method for treating a disease caused by overexpression of one or several proteins in cells or tissues of a subject, the method comprising administering to the subject a composition according to any one of the embodiments described herein.

[0069] In some embodiments, a method for treating a disease caused by reduced, suppressed, or missing expression of one or several proteins in a subject, the method comprising administering to the subject a composition according to any one of the embodiments described herein.

[0070] In some embodiments, a method for eliciting an immune response in a subject, comprising administering to the subject a composition according to any one of the embodiments described herein, wherein the biologically active compound is an immune modulatory nucleic acid.

[0071] In some embodiments, a method for treating signs and / or symptoms of Huntington's disease by providing a composition according to any one of the embodiments described herein and administering the composition to a subject.

[0072] In some embodiments, a method for modulating the amount of RNaseH-mediated cleavage in a cell, comprising contacting the cell with a composition according to any one of the embodiments described herein, wherein the oligonucleotide is capable of modulating the amount of RNaseH-mediated cleavage.

[0073] In some embodiments, a method of administering an oligonucleotide to a subject in need thereof comprises providing a composition comprising a drug lipid and administering the composition to the subject, wherein the drug is any drug disclosed herein and the lipid is any lipid disclosed herein.

[0074] In some embodiments, a method of treating a disease in a subject, comprising providing a composition comprising a drug lipid, and administering a therapeutically effective amount of the composition to the subject, wherein the drug is any drug disclosed herein, the lipid is any lipid disclosed herein, and the disease is any disease disclosed herein.

[0075] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated C 10 -C 40 Contains aliphatic chains.

[0076] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated linear C 10 -C 40 Contains aliphatic chains.

[0077] In some embodiments, the lipid comprises one or more C 1-4 Saturated or partially unsaturated straight-chain C optionally substituted with aliphatic groups 10 -C 40 Contains aliphatic chains.

[0078] In some embodiments, the lipid is an unsubstituted saturated or partially unsaturated linear C 10 -C 40 Contains aliphatic chains.

[0079] In some embodiments, the lipid comprises no more than one optionally substituted saturated or partially unsaturated linear C 10 -C 40 Contains aliphatic chains.

[0080] In some embodiments, the lipid comprises two or more optionally substituted saturated or partially unsaturated linear C 10 -C 40 Contains aliphatic chains.

[0081] In some embodiments, the lipid does not include a tricyclic or polycyclic moiety.

[0082] In some embodiments, the lipid is R 1 -COOH(wherein, R 1 is an optionally substituted saturated or partially unsaturated C 10 -C 40 It has the structure of an aliphatic chain.

[0083] 17. The composition or method of claim 16, wherein the lipid is attached through its carboxyl group.

[0084] Lipids: [ka] The composition or method according to any one of the embodiments described herein, selected from:

[0085] In some embodiments, the lipid is conjugated to the oligonucleotide.

[0086] In some embodiments, the lipid is directly attached to the oligonucleotide.

[0087] In some embodiments, the lipid is attached to the oligonucleotide via a linker.

[0088] In some embodiments, the linker is selected from: an uncharged linker; a charged linker; an alkyl-containing linker; a phosphate-containing linker; a branched linker; an unbranched linker; a linker comprising at least one cleavage group; a linker comprising at least one redox cleavage group; a linker comprising at least one phosphate-based cleavage group; a linker comprising at least one acid cleavage group; a linker comprising at least one ester-based cleavage group; and a linker comprising at least one peptide-based cleavage group.

[0089] In some embodiments, each oligonucleotide in the plurality is individually bound to the same lipid at the same location.

[0090] In some embodiments, the lipid is attached to the oligonucleotide by a linker.

[0091] In some embodiments, one or more of the plurality of oligonucleotides is independently bound to a target compound or moiety.

[0092] In some embodiments, one or more of the plurality of oligonucleotides are independently bound to a lipid and a targeting compound or moiety.

[0093] In some embodiments, one or more of the plurality of oligonucleotides are independently attached to a lipid at one end and to a targeting compound or moiety at the other end.

[0094] In some embodiments, the oligonucleotides in the plurality share the same chemical modification pattern.

[0095] In some embodiments, the oligonucleotides in the plurality share the same chemical modification pattern, including one or more base modifications.

[0096] In some embodiments, the oligonucleotides in the plurality share the same chemical modification pattern, including one or more sugar modifications.

[0097] In some embodiments, the consensus sequence is capable of hybridizing to a transcript in a cell that contains a mutation associated with Huntington's disease or whose level, activity and / or distribution is associated with Huntington's disease.

[0098] In some embodiments, the oligonucleotide is a nucleic acid.

[0099] In some embodiments, the oligonucleotide is an oligonucleotide.

[0100] In some embodiments, the oligonucleotide is an oligonucleotide that participates in RNase H-mediated cleavage of mutant huntingtin gene mRNA.

[0101] In some embodiments, the disease or disorder is Huntington's disease.

[0102] In some embodiments, the lipid is a C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, C1-C6 heteroaliphatic moiety, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O )N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O Optionally substituted saturated or partially unsaturated C alkyl groups, in which one or more methylene units are optionally independently replaced with an optionally substituted group selected from —N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, where each variable is independently as defined and described herein. 10 -C 80 Contains aliphatic groups.

[0103] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated C 10 -C 80 Contains aliphatic chains.

[0104] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated linear C 10 -C 80 Contains aliphatic chains.

[0105] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated C 10 -C 60 Contains aliphatic chains.

[0106] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated linear C 10 -C 60 Contains aliphatic chains.

[0107] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated C 10 -C 40 Contains aliphatic chains.

[0108] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated linear C 10 -C 40 Contains aliphatic chains.

[0109] In some embodiments, the lipid is a C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, C1-C6 heteroaliphatic moiety, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O )N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O Optionally substituted saturated or partially unsaturated C alkyl groups, in which one or more methylene units are optionally independently replaced with an optionally substituted group selected from —N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, where each variable is independently as defined and described herein. 10 -C 60 Contains aliphatic groups.

[0110] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated C 10 -C 80 Contains aliphatic chains.

[0111] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated linear C 10 -C 60 Contains aliphatic chains.

[0112] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated linear C 10 -C 40 Contains aliphatic chains.

[0113] In some embodiments, the lipid is selected from the group consisting of C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, C1-C6 heteroaliphatic moiety, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R') an optionally substituted saturated or partially unsaturated C alkyl group, in which one or more methylene units are optionally independently replaced with an optionally substituted group selected from -, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, and -C(O)O-, where each variable is independently as defined and described herein; 10 -C 40 Contains aliphatic groups.

[0114] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated C 10 -C 40 Contains aliphatic chains.

[0115] In some embodiments, the lipid is an optionally substituted saturated or partially unsaturated linear C 10 -C 40 Contains aliphatic chains.

[0116] In some embodiments, the composition further comprises one or more additional components selected from the following: a polynucleotide, a carbonic anhydrase inhibitor, a dye, an intercalating agent, an acridine, a crosslinker, a psoralen, a mitomycin C, a porphyrin, a TPPC4, a texaphyrin, a sapphyrin, a polycyclic aromatic hydrocarbon phenazine, a dihydrophenazine, an artificial endonuclease, a chelating agent, an EDTA, an alkylating agent, a phosphate, an amino, a mercapto, a PEG, a PEG-40K, a MPEG, a [MPEG]2, a polyamino, an alkyl, a substituted alkyl, a radiolabeled marker, an enzyme, a hapten biotin, a transport / absorption enhancer, aspirin, vitamin E, folic acid, a synthetic ribonuclease, a protein, a glycoprotein, a peptide, a molecule with specific affinity for a co-ligand, an antibody, a hormone, a hormone receptor, a non-peptide species, a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, a selectivity agent, or a drug. In some embodiments, the composition further comprises one or more additional components selected from the following: a polynucleotide, a carbonic anhydrase inhibitor, a dye, an intercalating agent, an acridine, a crosslinker, a psoralen, a mitomycin C, a porphyrin, a TPPC4, a texaphyrin, a sapphyrin, a polycyclic aromatic hydrocarbon phenazine, a dihydrophenazine, an artificial endonuclease, a chelating agent, an EDTA, an alkylating agent, a phosphate, an amino, a mercapto, a PEG, a PEG-40K, a MPEG, a [MPEG]2, a polyamino, an alkyl, a substituted alkyl, a radiolabeled marker, an enzyme, a hapten biotin, a transport / absorption enhancer, aspirin, vitamin E, folic acid, a synthetic ribonuclease, a protein, a glycoprotein, a peptide, a molecule with specific affinity for a co-ligand, an antibody, a hormone, a hormone receptor, a non-peptide species, a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, or a drug.

[0117] In some embodiments, the present disclosure provides an oligonucleotide conjugated to a selectivity agent. In some embodiments, the present disclosure provides a composition comprising an oligonucleotide or oligonucleotide type comprising a selectivity agent. In some embodiments, the selectivity agent specifically binds to one or more neurotransmitter transporters selected from the group consisting of dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). In some embodiments, the selectivity agent is selected from the group consisting of dopamine reuptake inhibitors (DRIs), selective serotonin reuptake inhibitors (SSRIs), noradrenaline reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs). In some embodiments, the selectivity agent is selected from the group consisting of triple reuptake inhibitors, noradrenaline-dopamine double reuptake inhibitors, serotonin single reuptake inhibitors, noradrenaline single reuptake inhibitors, and dopamine single reuptake inhibitors. In some embodiments, the selectivity agent is selected from the group consisting of dopamine reuptake inhibitors (DRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs). In some embodiments, the selectivity agent is selected from the selectivity agents described in U.S. Patent Nos. 9,084,825 and 9,193,969, and WO 2011131693 and WO 2014064258.

[0118] In some embodiments, the lipid is a saturated or partially unsaturated linear C 10 -C 80 Contains aliphatic chains.

[0119] In some embodiments, the composition further comprises a linker connecting the oligonucleotide and the lipid, wherein the linker is selected from the following: an uncharged linker; a charged linker; an alkyl-containing linker; a phosphate-containing linker; a branched linker; an unbranched linker; a linker comprising at least one cleavage group; a linker comprising at least one redox cleavage group; a linker comprising at least one phosphate-based cleavage group; a linker comprising at least one acid cleavage group; a linker comprising at least one ester-based cleavage group; or a linker comprising at least one peptide-based cleavage group.

[0120] In some embodiments, the oligonucleotide comprises, consists of, or is an oligonucleotide or oligonucleotide composition or chiral controlled oligonucleotide composition.

[0121] In some embodiments, the oligonucleotide comprises, consists of, or is an oligonucleotide composition or a chiral controlled oligonucleotide composition, wherein the sequence of the oligonucleotide comprises or consists of the sequence of any of the oligonucleotides described herein.

[0122] In some embodiments, the oligonucleotide comprises, consists of, or is an oligonucleotide composition or a chiral controlled oligonucleotide composition, wherein the sequence of the oligonucleotide comprises or consists of the sequence of any of the oligonucleotides listed in Table 4.

[0123] In some embodiments, the oligonucleotide comprises, consists of, or is an oligonucleotide composition or a chiral controlled oligonucleotide composition, wherein the sequence of the oligonucleotide comprises or consists of the sequence of a splice switch oligonucleotide.

[0124] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is a chiral controlled oligonucleotide composition.

[0125] The composition or method of any of the embodiments described herein, wherein the disease or disorder is Huntington's disease.

[0126] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is capable of participating in RNase H-mediated cleavage of mutant huntingtin gene mRNA.

[0127] The composition or method of any of the embodiments described herein, wherein the oligonucleotide comprises, consists of, or is the sequence of any of the oligonucleotides disclosed herein.

[0128] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is capable of distinguishing between wild-type and mutant huntingtin alleles.

[0129] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is capable of participating in RNase H-mediated cleavage of mutant huntingtin gene mRNA.

[0130] The composition or method of any of the embodiments described herein, wherein the oligonucleotide comprises, consists of, or is the sequence of any of the oligonucleotides disclosed in Table 4.

[0131] In some embodiments, the oligonucleotide comprises, consists of, or is an oligonucleotide or oligonucleotide composition or chiral controlled oligonucleotide composition, wherein the sequence of the oligonucleotide comprises or consists of the sequence of any of WV-1092, WV-2595, or WV-2603.

[0132] In some embodiments, the sequence of an oligonucleotide may be determined by a number of factors, including base sequence (including length); the pattern of chemical modifications to the sugar and base moieties; the pattern of backbone linkages; the pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof; the pattern of backbone chiral centers; the pattern of chiral internucleotide linkage stereochemistry (Rp / Sp); the pattern of backbone phosphorus modifications; -S - and -LR of formula I 1 The internucleotide may include any one or more of the patterns of modification to the phosphorus atom such as:

[0133] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; The present invention provides a chirality-controlled oligonucleotide composition comprising an oligonucleotide of a particular oligonucleotide type, characterized by: wherein the composition is chirality-controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, the oligonucleotide targets a mutant huntingtin gene and is about 10 to about 50 nucleotides in length, the backbone linkage comprises at least one phosphorothioate, and the pattern of backbone chiral centers comprises at least one chiral center in an Rp configuration and at least one chiral center in an Sp configuration.

[0134] In some embodiments, the present disclosure provides a method for cleaving a nucleic acid having a base sequence that includes a target sequence, the method comprising: (a) 1) a common base sequence and length that is or contains a sequence that is complementary to a target sequence within a nucleic acid; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; contacting a nucleic acid having a base sequence including a target sequence with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral-controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having a particular base sequence and length, the oligonucleotides targeting a mutant huntingtin gene and being about 10 to about 50 nucleotides in length, the backbone linkages comprising at least one phosphorothioate, and the pattern of backbone chiral centers comprising at least one chiral center in an Rp configuration and at least one chiral center in an Sp configuration.

[0135] In some embodiments, the present disclosure provides a method for cleaving a nucleic acid having a base sequence that includes a target sequence, the method comprising: (a) 1) a common base sequence and length that is or contains a sequence that is complementary to a target sequence within a nucleic acid; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; contacting a nucleic acid having a base sequence including a target sequence with a chiral-controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type, characterized by: The composition is chiral-controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having a particular base sequence and length, the oligonucleotides targeting a mutant huntingtin gene and being about 10 to about 50 nucleotides in length, the backbone linkages comprising at least one phosphorothioate, and the pattern of backbone chiral centers comprising at least one chiral center in an Rp configuration and at least one chiral center in an Sp configuration; and (b) cleaving the nucleic acid, mediated by RNAse H or RNA interference machinery; Includes.

[0136] In some embodiments, the compositions provided are selected from the group of compounds that specifically bind to one or more neurotransmitter transporters selected from the group consisting of dopamine transporters (DAT), serotonin transporters (SERT), and norepinephrine transporters (NET); dopamine reuptake inhibitors (DRIs), selective serotonin reuptake inhibitors (SSRIs), noradrenaline reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine transporters (NETs). the group consisting of triple reuptake inhibitors, noradrenaline-dopamine double reuptake inhibitors, serotonin single reuptake inhibitors, noradrenaline single reuptake inhibitors, and dopamine single reuptake inhibitors; and the group consisting of dopamine reuptake inhibitors (DRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs).

[0137] In some embodiments, provided compositions comprise oligonucleotides, wherein the base sequence, backbone bond pattern and / or backbone chiral center pattern of the oligonucleotide comprises or consists of any of the base sequence, backbone bond pattern and / or backbone chiral center pattern of any oligonucleotide selected from Table N1A, Table N2A, Table N3A, Table N4A and Table 8; and WV-1092, WV-2595 and WV-2603.

[0138] In some embodiments, provided compositions comprise oligonucleotides, wherein the base sequence, backbone bond pattern and / or backbone chiral center pattern of the oligonucleotide comprises or consists of any of the base sequence and backbone bond pattern and / or backbone chiral center pattern of any oligonucleotide selected from Table N1A, Table N2A, Table N3A, Table N4A and Table 8; and WV-1092, WV-2595 and WV-2603.

[0139] In some embodiments, provided compositions comprise oligonucleotides, wherein the base sequence, backbone bond pattern and / or backbone chiral center pattern of the oligonucleotide comprises or consists of any of the base sequence and backbone bond pattern and backbone chiral center pattern of any oligonucleotide selected from Table N1A, Table N2A, Table N3A, Table N4A and Table 8; and WV-1092, WV-2595 and WV-2603.

[0140] definition Aliphatic: As used herein, the terms "aliphatic" or "aliphatic group" refer to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or polycyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbocycle," "alicyclic," or "cycloalkyl"). In some embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. Unless otherwise specified, an aliphatic group contains 1-10 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-4 aliphatic carbon atoms. In still other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in yet other embodiments, an aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic or bicyclic C3-C6 ring that is fully saturated or contains one or more unsaturated units, but is not aromatic, with one point of attachment to another molecule. 10 refers to a hydrocarbon. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, with one point of attachment to another molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, and composites thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0141] Alkylene: The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n-, where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0142] Alkenylene: The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene group is a polymethylene group containing at least one double bond in which one or more hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0143] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human, at any stage of development. In some embodiments, "animal" refers to a non-human animal, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0144] Approximately: As used herein, the word "approximately" or "about" when describing a number is generally intended to include numbers within a range of 5%, 10%, 15%, or 20% in either direction (greater or less) of that number, unless otherwise stated or otherwise apparent from the context (unless such number is less than 0% or more than 100% of its possible values). In some embodiments, the use of the word "about" when describing dosage means ±5 mg / kg / day.

[0145] Aryl: The term "aryl," used alone or as part of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring structures in which at least one ring of the structure is aromatic and each ring of the structure contains 3 to 7 ring members, with a total of 5 to 14 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present invention, "aryl" refers to aromatic ring structures, including but not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, are also included within the scope of the term "aryl" herein.

[0146] Characteristic portion: As used herein, the phrase "characteristic portion" of a protein or polypeptide includes a stretch of amino acids, or a set of stretches of amino acids, that, taken together, are characteristic of the protein or polypeptide. Each such stretch will generally contain at least two amino acids. Moreover, one of skill in the art will recognize that at least 5, 10, 15, 20, or more amino acids are usually required to be characteristic of a protein. Generally, a characteristic portion shares at least one functional characteristic with the related intact protein, in addition to the specific sequence homology described above.

[0147] Signature Sequence: A "signature sequence" is a sequence that is found in all members of a family of polypeptides or nucleic acids and therefore can be used by those of skill in the art to define the members of that family.

[0148] Characteristic structural element: The term "characteristic structural element" refers to a distinct structural element (e.g., backbone structure, collection of pendant moieties, sequence element, etc.) found in all members of a family of polypeptides, small molecules, or nucleic acids, and thus can be used by those skilled in the art to define the members of that family.

[0149] Equivalent: The term "equivalent" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit a comparison of the results obtained or the phenomena observed. In some embodiments, equivalent sets of conditions or circumstances are characterized by a plurality of substantially the same characteristics and one or a few altered characteristics. Those skilled in the art will recognize that sets of conditions are equivalent to one another when they are characterized by a sufficient number and type of substantially the same characteristics such that the differences in the results obtained or the phenomena observed under the different sets of conditions or circumstances can justify a reasonable conclusion that the sets of conditions are caused by or exhibit differences in those altered characteristics.

[0150] Dosage regimen: As used herein, a "dosage regimen" or "treatment regimen" refers to a set of unit doses (usually one or more) administered individually to a subject, usually separated by a period of time. In some embodiments, a given therapeutic agent has a required dosing regimen that may include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated from the other by a period of equal length; in some embodiments, a dosing regimen includes multiple doses and at least two different periods that separate the separate doses. In some embodiments, all doses within a dosing regimen are the same unit dosage. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is different from the first dosage amount. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is the same as the first dosage amount.

[0151] Equivalent Agents: Upon reading this disclosure, those skilled in the art will recognize that the scope of agents useful in the context of the present invention is not limited to those specifically mentioned or exemplified herein. Specifically, those skilled in the art will recognize that active agents typically have a structure consisting of a backbone and attached pendant moieties, and will therefore understand that simple modifications of such backbone and / or pendant moieties do not significantly alter the activity of the agent. For example, in some embodiments, replacement of one or more pendant moieties with groups of equivalent three-dimensional structure and / or chemical reactivity properties can produce substituted compounds or moieties equivalent to the parent reference compound or moiety. In some embodiments, addition or removal of one or more pendant moieties can produce substituted compounds equivalent to the parent reference compound. In some embodiments, for example, alteration of the backbone structure by addition or removal of a small number of bonds (usually no more than 5, 4, 3, 2, or 1 bond, and often only a single bond) can produce substituted compounds equivalent to the parent reference compound. In many embodiments, equivalent compounds can be synthesized, for example, by the methods shown in the following general reaction schemes, or modifications thereof, using readily available materials, reagents, and conventional or provided synthetic procedures. Modifications to these reactions that are known per se but not mentioned here can also be employed.

[0152] Equivalent Dosage: The term "equivalent dosage" is used herein to compare dosages of different pharmaceutically active agents that produce the same biological result. Doses of two different agents are considered "equivalent" to one another according to the present invention if they achieve the same level or degree of biological result. In some embodiments, equivalent dosages of different pharmaceuticals used according to the present invention are determined using the in vitro and / or in vivo assays described herein. In some embodiments, one or more lysosomotropic agents used according to the present invention are utilized at a dose equivalent to that of a reference lysosomotropic agent; in some embodiments, such a reference lysosomotropic agent is selected from the group consisting of small molecule allosteric activators (e.g., pyrazolpyrimidines), iminosugars (e.g., isofagomine), antioxidants (e.g., n-acetylcysteine), and regulators of cellular trafficking (e.g., Rab1a polypeptides).

[0153] Heteroaliphatic: The term "heteroaliphatic" refers to an aliphatic group in which one or more units selected from C, CH, CH, or CH are independently replaced by a heteroatom. In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0154] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl" or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 pi electrons shared in the cyclic arrangement; and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and the quaternized form of any basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the aromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "aromatic heterocycle," any of which includes optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, where the alkyl and heteroaryl portions independently may be optionally substituted.

[0155] Heteroatom: The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, boron, selenium, or silicon (any oxidized form of nitrogen, boron, selenium, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or a heterocycle, such as N (in 3,4-dihydro-2H-pyrrolyl), NH (in pyrrolidinyl), or NR + (including the substitutable nitrogen of) in N-substituted pyrrolidinyl).

[0156] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used to refer to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, wherein the nitrogen is N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + It can be NR (as in N-substituted pyrrolidinyl).

[0157] A heterocycle can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl groups, heteroaryl groups, or aliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. The heterocyclyl ring can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions are independently optionally substituted.

[0158] Intraperitoneal: As used herein, the phrases "intraperitoneal administration" and "administered intraperitoneally" have their art-recognized meaning to refer to administration of a compound or composition into the peritoneal membrane of a subject.

[0159] In vitro: As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal, plant, and / or microorganism) but in an artificial environment, e.g., in a test tube or reactor, in cell culture, etc.

[0160] In vivo: As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, a plant, and / or a microorganism).

[0161] Lower alkyl: The term "lower alkyl" refers to a C 1~4It represents a straight or branched chain alkyl group. Illustrative lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0162] Lower haloalkyl: The term "lower haloalkyl" refers to a C alkyl group substituted with one or more halogen atoms. 1~4 It represents a straight or branched chain alkyl group.

[0163] Optionally substituted: As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted," with or without the word "may," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, where one or more positions in any given structure may be the same or different at each position. Combinations of substituents contemplated by the present invention are preferably those that result in the production of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are substantially unchanged when in a condition that allows for their preparation, detection, and, in certain embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0164] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently: halogen; -(CH) 0~4 R ○ ;-(CH2) 0~4 OR ○ ;-O(CH2) 0~4 R ○ , -O-(CH2) 0~4 C(O)OR ○ ;-(CH2) 0~4 CH(OR ○ )2;-(CH2) 0~4 SR ○ ;R ○ may be substituted with (CH2) 0~4 Ph;R ○ may be substituted with (CH2) 0~4O(CH2) 0~1 Ph;R ○ CH=CHPh optionally substituted with R ○ may be substituted with (CH2) 0~4 O(CH2) 0~1 -pyridyl; -NO2; -CN; -N3; ​​(CH2) 0~4 N(R ○ )2;-(CH2) 0~4 N(R ○ )C(O)R ○ ;-N(R ○ )C(S)R ○ ;-(CH2) 0~4 N(R ○ )C(O)NR ○ 2;N(R ○ )C(S)NR ○ 2;-(CH2) 0~4 N(R ○ )C(O)OR ○ ;-N(R ○ )N(R ○ )C(O)R ○ ;N(R ○ )N(R ○ )C(O)NR ○ 2;N(R ○ )N(R ○ )C(O)OR ○ ;-(CH2) 0~4 C(O)R ○ ;-C(S)R ○ ;-(CH2) 0~4 C(O)OR ○ ;-(CH2) 0~4 C(O)SR ○ ;(CH2) 0~4 C(O)OSiR ○ 3;-(CH2) 0~4 OC(O)R ○ ;-OC(O)(CH2) 0~4 SR-, SC(S)SR ○ ;-(CH2) 0~4 SC(O)R ○ ;-(CH2) 0~4 C(O)NR ○ 2;-C(S)NR ○ 2;-C(S)SR ○ ;-SC(S)SR ○ , (CH2)0~4 OC(O)NR ○ 2;C(O)N(OR ○ )R ○ ;-C(O)C(O)R○;-C(O)CH2C(O)R○;-C(NOR○)R ○ ;(CH2) 0~4 SSR ○ ;-(CH2) 0~4 S(O)2R ○ ;-(CH2) 0~4 S(O)2OR ○ ;-(CH2) 0~4 OS(O)2R ○ ;-S(O)2NR ○ 2;(CH2) 0~4 S(O)R ○ ;N(R ○ )S(O)NR ○ 2;-N(R ○ )S(O)2R ○ ;-N(OR ○ )R ○ ;-C(NH)NR ○ 2;-P(O)2R ○ ;P(O)R ○ 2;OP(O)R ○ 2;-OP(O)(OR ○ )2;-SiR○3;-(C 1~4 Linear or branched alkylene)ON(R ○ )2; or -(C 1~4 Linear or branched alkylene)C(O)ON(R ○ )2(in the formula, each R ○ are optionally substituted as described below and independently represent hydrogen, C 1~6 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, -CH2- (5- to 6-membered heteroaryl ring) or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two independently occurring R ○taken together with their intervening atoms form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as described below.

[0165] R ○ The appropriate monovalent substituent (or two independently occurring R ○ together with the intervening atoms to form a ring) are independently halogen, -(CH2) 0~2 R ● ,-(Halo R ● ), -(CH2) 0~2 OH, -(CH2) 0~2 OR ● , -(CH2) 0~2 CH(OR ● )2;O(Halo R ● ), -CN, -N3, -(CH2) 0~2 C(O)R ● , -(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR ● , -(CH2) 0~2 SR ● , -(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR ● , -(CH2) 0~2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. C(O)SR ● , -(C 1~4 Straight or branched chain alkylene)C(O)OR ● , or -SSR ● (In the formula, each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and, independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. ○ Suitable divalent substituents on a saturated carbon atom of include ═O and ═S.

[0166] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2~3 O-, or -S(C(R * 2)) 2~3 S-(wherein each R * can be substituted with hydrogen, 1~6 aliphatic, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to adjacent substitutable carbon atoms of an "optionally substituted" group include: -O(CR * 2) 2~3 O-(wherein each R * can be substituted with hydrogen, 1~6 aliphatic, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0167] Suitable substituents on the aliphatic group of R* include halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ●2, or -NO2 (wherein each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and, independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0168] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † ;(In the formula, each R † are independently selected from halogen, and the following may be substituted C 1~6 an aliphatic, unsubstituted -OPh, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, notwithstanding the above definition, two independently occurring R † together with their intervening atoms form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0169] Suitable substituents on an aliphatic group of R† are independently halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2 (wherein each R● is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and is independently a C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0170] Oral: As used herein, the phrases "oral administration" and "orally administered" refer to administration of a compound or composition by mouth and have their art-recognized meaning.

[0171] Parenteral: As used herein, the phrases "parenteral administration" and "parenterally administered" have their art-recognized meaning to refer to modes of administration that are not enteral or topical, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0172] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0173] As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., for buccal, sublingual, and systemic absorption, boluses, powders, granules, and pastes to be applied to the tongue; parenteral administration, e.g., as a sterile solution or suspension, or sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray to be applied to the skin, lungs, or oral cavity; intravaginally or rectally, e.g., as a pessary, cream, or foam; sublingually; ophthalmically; transdermally; or intranasally to the lungs and other mucosal surfaces.

[0174] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that, within sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0175] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the transport or transfer of a substance from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0176] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to a salt of such a compound that is suitable for use in a pharmaceutical context, i.e., a salt that is suitable for use in contact with the tissues of humans and lower animals, within the scope of sound medical judgment, at a reasonable benefit / risk ratio, and without undue harmful effects, irritation, allergic response, etc. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxypropyl methylcellulose ... Examples of suitable salts include, but are not limited to, sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates and arylsulfonates having 1 to 6 carbon atoms, as appropriate.

[0177] Prodrug: Generally, a "prodrug," as the term is used herein, and as understood in the art, is an entity that, when administered to an organism, is metabolized in the body to deliver an active (e.g., therapeutic or diagnostic) agent of interest. Typically, such metabolism results in the removal of at least one "prodrug moiety," so that the active agent is produced. Various forms of "prodrugs" are well known in the art. Examples of such prodrug moieties include: a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, 42:309-396, edited by K. Widder, et al. (Academic Press, 1985); b)Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley, 2011); c)Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley, 2011); d)A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen; e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H. Bundgaard, p. 113-191 (1991); f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992); g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988); and h)Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984) reference.

[0178] Like other compounds described herein, prodrugs can be provided in any of a variety of forms, e.g., crystalline forms, salt forms, etc. In some embodiments, the prodrug is provided as a pharmaceutically acceptable salt thereof.

[0179] Protecting group: As used herein, the term "protecting group" is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd edition, John Wiley & Sons, 1999 (the entire text of which is incorporated herein by reference). It also includes protecting groups specifically adapted to nucleotides and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012 (the entire text of Chapter 2 is incorporated herein by reference). Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenanthyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2- Phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitro p-Toluenesulfonylmethylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethylcarbamate, diphenylmethylcarbamate, 2-methylthioethylcarbamate, 2-methylsulfonylethylcarbamate, 2-(p-toluenesulfonyl)ethylcarbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthio Phenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acryloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-Dimethoxycarbonylvinylcarbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methyl 1-methyl-1-cyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamates, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenoxy)acetamide o-(methyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-Tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, Quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np- Methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylideneamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenyl Carbolic acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridine sulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4- Methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0180] Suitable protected carboxylic acids include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

[0181] Suitable hydroxyl protecting groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (ME), and the like. M), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl] -4-Methoxypiperazin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trimethyl- methylethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazolinone) (4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthiazolyl Silyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate ester, benzoylformate ester, acetate ester, chloroacetate ester, dichloroacetate ester, trichloroacetate ester, trifluoroacetate ester, methoxyacetate ester, triphenylmethoxyacetate ester, phenoxyacetate ester, p-chlorophenoxyacetate Acid esters, 3-phenylpropionate esters, 4-oxopentanoate esters (levulinate esters), 4,4-(ethylenedithio)pentanoate esters (levulinoyl dithioacetal), pivalate esters, adamantate, crotonate esters, 4-methoxycrotonate esters, benzoate esters, p-phenylbenzoate esters, 2,4,6-trimethylbenzoate esters (mesitoate), alkyl methyl carbonates, 9-fluorenylmethyl carbonates (Fmoc), alkyl ethyl carbonates, alkyl 2,2,2-Trichloroethyl (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoic acid ester, 4-azidobutyric acid ester, 4-nitro-4-methylpentanoic acid ester, o-(dibromomethyl)benzoic acid ester, 2-formylbenzenesulfonic acid ester, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyric acid Acid esters, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoic acid, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothionyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,Examples of suitable boronic acid derivatives include 4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl borate, and phenyl borate.

[0182] In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4′-dimethoxytrityl, ( DMTr) and 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting groups are selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.

[0183] In some embodiments, the phosphite protecting group is a group attached to an internucleotide phosphite bond throughout oligonucleotide synthesis. In some embodiments, the phosphite protecting group is attached to the sulfur atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is attached to the oxygen atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is attached to the oxygen atom of an internucleotide phosphate bond. In some embodiments, the phosphite protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0184] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked to one another by peptide bonds). In some embodiments, a protein contains only naturally occurring amino acids. In some embodiments, a protein contains one or more non-naturally occurring amino acids (e.g., a moiety that forms one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues of a protein chain contain a non-amino acid moiety (e.g., a glycan, etc.). In some embodiments, a protein contains more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means. In some embodiments, a protein contains L-amino acids, D-amino acids, or both; in some embodiments, a protein contains one or more amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 20 amino acids, or less than about 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0185] Sample: As used herein, a "sample" refers to a specific organism or material obtained from. In some embodiments, a sample is a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, a biological sample includes biological tissue or biological fluid. In some embodiments, a biological sample is, or includes any one or more of, bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; suspended nucleic acid; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural fluid; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washings or lavage fluids, such as ductal lavage or bronchoalveolar lavage; aspirates; scrapings; bone marrow specimen; tissue biopsy specimen; surgical specimen; feces, other body fluids, secretions, and / or cells therefrom, etc. In some embodiments, a biological sample is, or includes, cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), etc. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), for example, by filtration through a semipermeable membrane. Such a "processed sample" can include, for example, nucleic acids or proteins obtained by extraction from a sample or processing a primary sample by techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components, etc. In some embodiments, the sample is an organism. In some embodiments, the sample is a plant. In some embodiments, the sample is an animal. In some embodiments, the sample is a human. In some embodiments, the sample is a non-human organism.

[0186] Stereochemical isomers: As used herein, the phrase "stereochemical isomers" refers to different compounds constructed of the same atoms connected by the same series of bonds but having different, incompatible three-dimensional structures. In some embodiments of the present invention, the provided chemical compositions may be or contain a pure synthesis of an individual stereochemical isomer of a compound; in some embodiments, the provided chemical compositions may be or contain a mixture of two or more stereochemical isomers of the compound. In certain embodiments, such mixtures contain equal amounts of different stereochemical isomers; in certain embodiments, such mixtures contain unequal amounts of at least two different stereochemical isomers. In some embodiments, the chemical composition may contain all diastereomers and / or enantiomers of the compound. In some embodiments, the chemical composition may contain less than all diastereomers and / or enantiomers of the compound. In some embodiments, if a specific enantiomer of a compound of the present invention is desired, it may be synthesized, for example, by asymmetric synthesis or derivatization with a chiral auxiliary, and the resulting diastereomeric mixture separated and the auxiliary cleaved to yield the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as amino, diastereomeric salts can be formed with a suitable optically active acid and resolved, for example, by fractional crystallization.

[0187] Subject: As used herein, the term "subject" or "subject" refers to any organism to which provided compounds or compositions are administered in accordance with the present invention, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject is suffering from and / or susceptible to a disease, disorder, and / or condition.

[0188] Substantial: As used herein, the term "substantial" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property under consideration. Those skilled in the biological arts will understand that biological and chemical phenomena rarely eschew completeness and / or completion or accomplishment or absolute results. Thus, the term "substantial" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0189] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0190] Susceptible (to a disease): An individual who is "susceptible to" a disease, disorder, and / or condition is one who is at a higher risk of developing the disease, disorder, and / or condition than members of the general community. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not be diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0191] Systemic: As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" have their art-recognized meaning of referring to administering a compound or composition so that it enters the recipient's entire body.

[0192] Tautomers: As used herein, the phrase "tautomers" is used to describe different isomers of an organic compound that are readily interchangeable. Tautomers can be characterized by the formal migration of a hydrogen atom or a proton accompanied by the shifting of a single bond and an adjacent double bond. In some embodiments, tautomers can result from proton tautomerism (i.e., relocation of a proton). In some embodiments, tautomers can result from valence tautomerism (i.e., rapid relocation of bonding electrons). All such tautomers are intended to be included within the scope of the present invention. In some embodiments, tautomers of a compound exist in mobile equilibrium with one another such that attempts to synthesize the separate substances would result in mixtures. In some embodiments, tautomers of a compound are separable and isolatable compounds. In some embodiments of the present invention, chemical compositions can be provided that are or contain a pure composition of a single tautomer of a compound. In some embodiments of the present invention, chemical compositions can be provided as mixtures of two or more tautomers of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomers; in certain embodiments, such mixtures contain different amounts of at least two tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain all tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain fewer than all tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain one or more tautomers of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the present invention, the tautomer is keto-enol tautomerism. One skilled in the chemical arts can "capture" (i.e., chemically modify to retain the "enol" form) keto-enol tautomerism using any suitable reagent known in the chemical arts to obtain an enol derivative that can be subsequently isolated using one or more suitable techniques known in the art. Unless otherwise indicated, the present invention encompasses all tautomers of the relevant compounds, whether in pure form or in mixtures with each other.

[0193] Therapeutic Agent: As used herein, the term "therapeutic agent" refers to any agent that, when administered to a subject, induces a therapeutic effect and / or a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0194] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be recognized by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and others. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, suppresses, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0195] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, suppress, prevent, delay onset, reduce severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows only early signs of the disease, disorder, and / or condition, for example, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0196] Unsaturated: As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0197] Unit dose: As used herein, the phrase "unit dose" refers to an amount administered as a single dose of a pharmaceutical composition and / or in a physically discrete unit. In many embodiments, a unit dose contains a predetermined amount of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, one or more unit doses are administered to achieve the entire single dose. In some embodiments, administration of multiple unit doses is necessary or expected to be necessary to achieve the intended effect. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of a solid form, sustained-release formulation, or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose can be in a formulation containing any variety of ingredients in addition to the therapeutic agent. For example, an acceptable carrier (e.g., a pharmaceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, etc., as described below, can be included. It will be understood by those skilled in the art that in many embodiments, an appropriate total daily dosage of a particular therapeutic agent may comprise a fraction or multiple unit doses, and may be determined, for example, by an attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dosage level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active compound used; the specific composition used; the age, weight, health, sex, and diet of the subject; the number of administrations and the excretion rate of the specific compound used; the duration of treatment; drugs and / or additional therapies used in combination or simultaneously with the specific compound used, and similar factors well known in the medical arts.

[0198] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning of referring to an entity having a structure and / or activity actually found in a "normal" (as opposed to mutant, diseased, altered, etc.) state or context. Those of skill in the art will recognize that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0199] Nucleic Acid: The term "nucleic acid" includes any nucleotide, modified variants thereof, analogs thereof, and polymers thereof. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), or modified variants or analogs thereof. These terms refer to the primary structure of the molecule and thus include double- and single-stranded DNA and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. These terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from nucleobases and / or modified nucleobase N- or C-glycosides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages"). The terms encompass nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. The prefix poly- refers to nucleic acids containing from 2 to about 10,000 nucleotide monomer units, while the prefix oligo- refers to nucleic acids containing from 2 to about 200 nucleotide monomer units.

[0200] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more phosphate groups or phosphorus-containing internucleotide linkages. Naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but natural and unnatural base analogs are also understood to be included. Natural sugars are pentoses (five-carbon sugars), deoxyribose (forming DNA), or ribose (forming RNA), but natural and unnatural sugar analogs are also understood to be included. Nucleotides are linked via internucleotide linkages to form nucleic acids or polynucleotides. Many internucleotide linkages are known in the art (e.g., but not limited to, phosphate, phosphorothioate, boranophosphate, etc.). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothioates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. Other analogs (e.g., artificial nucleic acids, or components that can be incorporated into nucleic acids or artificial nucleic acids) include boranophosphate RNA, FANA, locked nucleic acids (LNA), morpholinos, peptide nucleic acids (PNAs), threose nucleic acids (TNAs), and glycol nucleic acids (GNAs). Those skilled in the art will recognize a variety of modified nucleotides or nucleotide analogs, including, for example, those described in any of the following: Gryaznov, S; Chen, J.-KJ Am. Chem. Soc. 1994, 116, 3143; Hendrix et al., 1997 Chem. Eur. J. 3:110; Hyrup et al., 1996 Bioorg. Med. Chem. 4:5; Jepsen et al., 2004 Oligo. 14:130-146; Jones et al., J. Org. Chem. 1993, 58, 2983; Koizumi et al., 2003 Nuc. Acids Res.12:3267-3273; Koshkin et al., 1998 Tetrahedron 54:3607-3630; Kumar et al., 1998 Bioo.Med.Chem.Let. 8:2219-2222; Lauritsen et al., 2002 Chem.Comm. 5:530-531; Lauritsen et al., 2003 Bioo.Med.Chem.Lett. 13:253-256; Mesmaeker et al., Angew.Chem., Int. Ed. Engl. 1994, 33, 226; Morita et al., 2001 Nucl.Acids Res.Supp. 1:241-242; Morita et al., 2002 Bioo.Med.Chem.Lett. 12:73-76; Morita et al., 2003 Bioo.Med.Chem.Lett.2211-2226;Nielsen et al., 1997 Chem.Soc.Rev.73;Nielsen et al., 1997 J.Chem.Soc.PerkinsTransl.1:3423-3433;Obika et al., 1997 Tetrahedron Lett.38(50):8735-8;Obika et al., 1998 Tetrahedron Lett.39:5401-5404;Pallan et al., 2012 Chem.Comm.48:8195-8197;Petersen et al., 2003 TRENDS Biotech.21:74-81;Rajwanshi et al., 1999 Chem.Commun.1395-1396;Schultz et al., 1996 Nucleic Acids Res.24:2966;Seth et al., 2009 J. Med.Chem.52:10-13;Seth et al., 2010 J.Med.Chem.53:8309-8318;Seth et al., 2010 J.Org.Chem.75:1569-1581;Seth et al., 2012 Bioo.Med.Chem.Lett.22:296-299;Seth et al., 2012 Mol.Ther-Nuc.Acids.1,e47;Seth,Punit P;Siwkowski,Andrew;Allerson,Charles R;Vasquez,Guillermo;Lee,Sam;Prakash,Thazha P;Kinberger,Garth;Migawa,Michael T;Gaus,Hans;Bhat,Balkrishen;et al. From Nucleic Acids Symposium Series(2008),52(1),553-554;Singh et al., 1998 Chem.Comm.1247-1248;Singh et al., 1998 J.Org.Chem.63:10035-39;Singh et al., 1998 J.Org.Chem.63:6078-6079;Sorensen 2003 Chem.Comm. 2130-2131; Ts'o et al., Ann.NY Acad.Sci. 1988, 507, 220; Van Aerschot et al., 1995 Angew.Chem.Int.Ed.Engl. 34:1338; Vasseur et al., J.Am.Chem.Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; or WO 2016 / 079181.

[0201] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar.

[0202] Sugar: The term "sugar" refers to a monosaccharide in closed and / or open form. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term also encompasses structural analogs used in place of normal sugar molecules, such as glycols, whose polymers form the backbone of nucleic acid analogs, glycol nucleic acids ("GNAs").

[0203] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar, such that the modified sugar mimics the spatial arrangement, electronic state, or some other physicochemical property of a sugar.

[0204] Nucleobase: The term "nucleobase" refers to a nucleic acid moiety that participates in hydrogen bonds to bind one nucleic acid strand to another complementary strand in a sequence-specific manner. Most naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase," e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobases mimic the spatial arrangement, electronic state, or some other physicochemical property of nucleobases and retain the hydrogen-bonding properties that allow one nucleic acid strand to bind to another complementary strand in a sequence-specific manner. In some embodiments, the modified nucleobases can pair with all five natural bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex.

[0205] Chiral Ligand: The term "chiral ligand" or "chiral auxiliary" refers to a moiety that is chiral and can be incorporated into a reactant so that a reaction can be carried out with a particular stereoselectivity.

[0206] Condensing Reagent: In a condensation reaction, the term "condensing reagent" refers to a reagent that activates a less reactive site, making it more susceptible to action with another reagent. In some embodiments, such another reagent is a nucleophile.

[0207] Blocking group: The term "blocking group" refers to a group that masks the reactivity of a functional group that can be subsequently unmasked by removal of the blocking group. In some embodiments, a blocking group is a protecting group.

[0208] Moiety: The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often recognized as a chemical entity incorporated into or added to a molecule.

[0209] Solid support: The term "solid support" refers to any support that allows for the synthesis of nucleic acids. In some embodiments, the term refers to glass or polymer that is insoluble in the medium used in the reaction step to carry out nucleic acid synthesis and derivatize to introduce reactive groups. In some embodiments, the solid support is highly cross-linked polystyrene (HCP) or controlled pore glass (CPG). In some embodiments, the solid support is controlled pore glass (CPG). In some embodiments, the solid support is a composite support of controlled pore glass (CPG) and highly cross-linked polystyrene (HCP).

[0210] Linking moiety: The term "linking moiety" refers to any moiety that may be located between the terminal nucleotide and the solid support or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0211] DNA molecule: The term "DNA molecule" refers to either its single-stranded form or the polymeric form of double-helical deoxyribonucleotides (adenine, guanine, thymine, or cytosine). The term refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found in, inter alia, linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing specific double-stranded DNA molecule structures, the sequence may be described herein according to the convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA).

[0212] Coding sequence: A DNA "coding sequence" or "coding region" is a double-stranded DNA that is transcribed and translated into an in vivo polypeptide when placed under the control of appropriate expression control sequences. The boundaries of the coding sequence ("open reading frame" or "ORF") are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxylic) terminus. Coding sequences include, but are not limited to, prokaryotic sequences, cDNA from prokaryotic mRNA, genomic DNA sequences from prokaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence. The terms "non-coding sequence" or "non-coding region" refer to regions of a polynucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0213] Reading Frame: The term "reading frame" refers to one of six possible reading frames, three in each direction of a double-stranded DNA molecule. The reading frame used determines which codons are used to code for amino acids within the coding sequence of the DNA molecule.

[0214] Antisense: As used herein, an "antisense" nucleic acid molecule comprises a nucleotide sequence that is complementary to a protein-encoding "sense" nucleic acid, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence, or complementary to the coding strand of a gene. Thus, an antisense nucleic acid molecule can associate with a sense nucleic acid molecule through hydrogen bonds. In some embodiments, the antisense oligonucleotide is an oligonucleotide involved in RNase H-mediated cleavage; for example, the antisense oligonucleotide sequence-specifically hybridizes to a portion of the target mRNA, thus targeting the mRNA for cleavage by RNase H. In some embodiments, the antisense oligonucleotide can distinguish between wild-type and targeted mutant alleles. In some embodiments, the antisense oligonucleotide significantly participates in RNase H-mediated cleavage of the mutant allele, but to a much lesser extent in RNase H-mediated cleavage of the wild-type allele (e.g., does not significantly participate in RNase H-mediated cleavage of the targeted wild-type allele).

[0215] Wobble position: As used herein, "wobble position" refers to the third position of a codon. In some embodiments, a mutation in a DNA molecule within the wobble position of a codon results in a silent or conservative mutation at the amino acid level. For example, there are four codons that code for glycine, namely, GGU, GGC, GGA, and GGG; therefore, mutation of any nucleotide at any wobble position to another nucleotide selected from A, U, C, and G will not result in a change at the amino acid level of the encoded protein, and is therefore a silent substitution.

[0216] Silent Substitution: A "silent substitution" or "silent mutation" is one in which a nucleotide in a codon is changed but does not result in a change in the amino acid residue encoded by the codon. Examples include mutations in the first position of a particular codon, such as the codon "CGG," which when mutated to AGG still encodes Arg, as well as mutations in the third position of the codon.

[0217] Gene: As used herein, the terms "gene," "recombinant gene," and "gene construct" refer to a DNA molecule, or portion of a DNA molecule, that encodes a protein or portion thereof. The DNA molecule may include an open reading frame that encodes the protein (as an exon sequence) and may further include intron sequences. As used herein, the term "intron" refers to a DNA sequence present in a given gene that is not translated into protein, and in some, but not all, cases, found between exons. As is well known in the art, it may be desirable for a gene to be operably associated with (or may include) one or more promoters, enhancers, repressors, and / or other control sequences that regulate the activity or expression of the gene.

[0218] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes recombinant polynucleotides synthesized by reverse transcription of mRNA, from which intervening sequences (introns) have been removed.

[0219] Homology: "Homology" or "identity" or "similarity" refers to the sequence similarity between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence that can be positioned for comparison purposes. When the same position in the compared sequences is occupied by the same base, then the molecules are identical at that position; when the same site is occupied by the same or similar nucleic acid residue (e.g., similar in steric and / or electronic state), then the molecules can be said to be homologous (similar) at that position. Expression of percentage homology / similarity or identity represents a function of the number of identical or similar nucleic acids at a position shared by the compared sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with a sequence described herein. When comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of extra residues also reduces the identity and homology / similarity.

[0220] In some embodiments, the term "homology" describes a mathematically based comparison of sequence similarity used for identical genes with similar functions or motifs. The nucleic acid sequences described herein can be used as "query sequences" to perform searches against public databases, for example, to identify other family members, related sequences, or homologs. In some embodiments, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments, BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. In some embodiments, gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, to obtain gapped alignments for comparison purposes. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).

[0221] Identity: As used herein, "identity" means the percentage of identical nucleotide residues at corresponding positions in two or more sequences when the sequences are aligned for maximum sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to maximize the match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The well known Smith-Waterman algorithm may also be used to determine identity.

[0222] Heterologous: A "heterologous" region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not found in relation to the larger sequence. Thus, when a heterologous region encodes a mammalian gene, the gene may be located on a side of DNA that is not normally adjacent to the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a sequence in which the coding sequence itself is not found at all (e.g., a cDNA containing introns or synthetic sequences in which the genomic coding sequence has codons or motifs that differ from the native gene). Allelic variation or natural mutational events do not give rise to a non-homologous region of DNA as defined herein.

[0223] Transition Mutation: The term "transition mutation" refers to a base change in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T)) is replaced by another pyrimidine, or a purine (adenosine (A) or guanosine (G)) is replaced by another purine.

[0224] Transversion Mutation: The term "transversion mutation" refers to a base change in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T)) is replaced by a purine, or a purine (adenosine (A) or guanosine (G)) is replaced by a pyrimidine.

[0225] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotide monomers containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages," as further defined herein).

[0226] Oligonucleotides can be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" encompasses single-stranded oligonucleotides. Single-stranded oligonucleotides can have double-stranded regions, and double-stranded oligonucleotides can have single-stranded regions. Illustrative oligonucleotides include, but are not limited to, structural genes, genes including regulatory and terminal regions, viral or plasmid DNA, self-replicating systems such as single-stranded and double-stranded siRNAs and other RNA interference agents (RNAi agents or iRNA agents), shRNAs, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adapters, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0227] The double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference are also referred to herein as siRNA, RNAi agent or iRNA agent.In some embodiments, these RNA interference-inducing oligonucleotides are associated with the cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC).In many embodiments, single-stranded and double-stranded RNAi agents are long enough that they can be cut by endogenous molecules, such as Dicer, to produce smaller oligonucleotides that can enter RISC mechanism and participate in the RISC-mediated cleavage of target sequence, such as target mRNA.

[0228] Oligonucleotides of the present invention can vary in length. In certain embodiments, oligonucleotides can range from about 2 to about 200 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides can range in length from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides. In some embodiments, the oligonucleotides are about 9 to about 39 nucleotides in length. In some embodiments, the oligonucleotides are at least 4 nucleotides in length. In some embodiments, the oligonucleotides are at least 5 nucleotides in length. In some embodiments, the oligonucleotides are at least 6 nucleotides in length. In some embodiments, the oligonucleotides are at least 7 nucleotides in length. In some embodiments, the oligonucleotides are at least 8 nucleotides in length. In some embodiments, the oligonucleotides are at least 9 nucleotides in length. In some embodiments, the oligonucleotides are at least 10 nucleotides in length. In some embodiments, the oligonucleotides are at least 11 nucleotides in length. In some embodiments, the oligonucleotides are at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a double-stranded complementary strand at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a double-stranded complementary strand at least 21 nucleotides in length.

[0229] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to a phosphorus-containing bond between nucleotide units of an oligonucleotide, and is synonymous above and herein with "intersugar linkage" and "phosphorus atom bridge." In some embodiments, the internucleotide linkage is a phosphodiester linkage found in natural DNA and RNA molecules. In some embodiments, the internucleotide linkage is a "modified internucleotide linkage" in which each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are selected from, but not limited to, =S, =Se, =NR', -SR', -SeR', -N(R')2, B(R')3, -S-, -Se-, and -N(R')- (wherein each R' is independently as defined and described below). In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a phosphodiester diester linkage. [ka] or a modified phosphorothioate triester bond. Those skilled in the art will appreciate that the internucleotide linkage may exist as an anion or cation at a given pH depending on the presence of an acid or base moiety in the linkage.

[0230] Unless otherwise specified, when used with an oligonucleotide sequence, each s, s1, s2, s3, s4, s5, s6 and s7 independently represents the following modified internucleotide linkages shown in Table 1 below:

[0231] Table 1. Illustrative modified internucleotide linkages [Table 1] TIFF2025038091000006.tif224166TIFF2025038091000007.tif96166

[0232] For example, (Rp,Sp)-ATsCs1GA has 1) a phosphorothioate internucleotide bond between T and C; ( [ka] ) and 2) between C and G [ka] Unless otherwise specified, the Rp / Sp designation preceding an oligonucleotide sequence refers to the configuration of the chiral phosphorus atoms of the internucleotide linkages sequentially from 5' to 3' of the oligonucleotide sequence. For example, in (Rp,Sp)-ATsCs1GA, the phosphorus of the "s" bond between T and C has the Rp configuration, and the phosphorus of the "s1" bond between C and G has the Sp configuration. In some embodiments, "all (Rp)" or "all (Sp)" is used to indicate that all chiral phosphorus atoms of the oligonucleotide have the same Rp or Sp configuration, respectively. For example, all (Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linking phosphorus atoms of the oligonucleotide have the Rp configuration; all (Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linking phosphorus atoms of the oligonucleotide have the Sp configuration.

[0233] Oligonucleotide Type: As used herein, the phrase "oligonucleotide type" refers to an oligonucleotide having a particular base sequence, backbone linkage pattern (i.e., internucleotide linkage pattern, e.g., phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., linked phosphorus stereochemistry pattern (Rp / Sp)), and backbone phosphorus modification pattern (e.g., "-XLR" in Formula I). 1 "Type" is used to define oligonucleotides having a pattern of "groups." Oligonucleotides of a commonly designated "type" are structurally identical to each other.

[0234] Those skilled in the art will recognize that the synthesis methods of the present invention provide a degree of control during the synthesis of an oligonucleotide chain, such that each nucleotide unit of the oligonucleotide chain can be designed and / or preselected to have a specific stereochemistry at the linking phosphorus and / or a specific modification at the linking phosphorus and / or a specific base and / or a specific sugar. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the linking phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the above structural features. The present invention provides compositions (e.g., chiral-controlled oligonucleotide compositions) comprising or consisting of a plurality of oligonucleotide molecules. In some embodiments, all such molecules are of the same type (i.e., structurally identical to each other). However, in many embodiments, the provided compositions typically contain multiple oligonucleotides of different types in predetermined relative amounts.

[0235] Chiral control: As used herein, "chiral control" refers to the ability to control the stereochemical representation of any chiral linking phosphorus within an oligonucleotide chain. The phrase "chiral controlled oligonucleotide" refers to an oligonucleotide that exists in a single diastereomeric form with respect to the chiral linking phosphorus. Chiral controlled oligonucleotides are prepared by chiral controlled oligonucleotide synthesis.

[0236] Chiral control oligonucleotide composition: As used herein, the phrase "chiral control oligonucleotide composition" refers to an oligonucleotide composition that contains a predetermined level of an individual oligonucleotide type. For example, in some embodiments, the chiral control oligonucleotide composition contains one oligonucleotide type. In some embodiments, the chiral control oligonucleotide composition contains a mixture of multiple oligonucleotide types. Illustrative chiral control oligonucleotide compositions are further described herein.

[0237] Chiral pure: The phrase "chiral pure" is used herein to describe chiral controlled oligonucleotide compositions in which the entire oligonucleotide exists in a single diastereoisomer with respect to the attached phosphorus.

[0238] Chiral homogeneous: As used herein, the phrase "chiral homogeneous" is used to describe an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the junction phosphorus. For example, an oligonucleotide in which all nucleotide units have Rp stereochemistry at the junction phosphorus is chiral homogeneous. Similarly, an oligonucleotide in which all nucleotide units have Sp stereochemistry at the junction phosphorus is chiral homogeneous.

[0239] Predetermined: Predetermined means deliberately selected, e.g., as opposed to randomly occurring or achieved. Those skilled in the art will understand upon reading this specification that the present invention provides new and surprising technology that allows for the selection of specific oligonucleotide types for formulation and / or inclusion in provided compositions, and further allows for the controlled formulation of the selected specific types, optionally in selected specific relative amounts, precisely so that provided compositions are formulated. Such provided compositions are "predetermined" as described herein. Compositions that may contain specific individual oligonucleotide types are not "predetermined" compositions because they were created through a process that, by chance, does not control the intentional creation of specific oligonucleotide types. In some embodiments, a predetermined composition is one that can be intentionally replicated (e.g., through the repetition of a controlled process).

[0240] Bound phosphorus: As defined herein, the phrase "bound phosphorus" is used to indicate that the particular phosphorus atom depicted is present in an internucleotide linkage, and that the phosphorus atom corresponds to the phosphorus atom of the phosphodiester internucleotide linkage that occurs in natural DNA and RNA. In some embodiments, the bound phosphorus atom is in a modified internucleotide linkage, where each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the bound phosphorus atom is represented by P * In some embodiments, the linking phosphorus atom is chiral. In some embodiments, the chiral linking phosphorus atom is P of Formula I * is.

[0241] P modification: As used herein, the term "P modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In some embodiments, a P modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus. In some embodiments, the "P modification" ... 1 (Wherein X, L and R 1 are independently as defined and described herein and below).

[0242] Blockmir: As used herein, the term "blockmir" refers to an oligonucleotide chain in which the pattern of structural features that characterize each individual nucleotide unit is characterized by the presence of at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus linkage. A common structural feature refers to a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus. In some embodiments, the at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus linkage are referred to as a "block."

[0243] In some embodiments, the blockmirror is a "stereoblockmirror," e.g., at least two consecutive nucleotide units have the same stereochemistry at the linked phosphorus. Such at least two consecutive nucleotide units form a "stereoblockmirror." For example, (Rp,Sp)-ATsCs1GA is a stereoblockmirror because at least two consecutive nucleotide units, Ts and Cs1, have the same stereochemistry at the linked phosphorus (both Sp). In the same oligonucleotide, (Rp,Sp)-ATsCs1 forms a block, which is a stereoblock.

[0244] In some embodiments, a blockmir is a "P-modified blockmir," e.g., at least two consecutive nucleotide units have the same modification at the linked phosphorus. Such at least two consecutive nucleotide units form a "P-modified block." For example, (Rp,Sp)-ATsCsGA is a P-modified blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same P modification (i.e., both are phosphorothioate diester). In the same oligonucleotide of (Rp,Sp)-ATsCsGA, Ts and Cs form a block, which is a P-modified block.

[0245] In some embodiments, a blockmir is a "linked blockmir," e.g., at least two consecutive nucleotide units have the same stereochemistry and the same modification at the linking phosphorus. At least two consecutive nucleotide units form a "linked block." For example, (Rp,Rp)-ATsCsGA is a linked blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same stereochemistry (both Rp) and P modification (both phosphorothioate). In the same oligonucleotide of (Rp,Rp)-ATsCsGA, TsCs forms a block and is a linked block.

[0246] In some embodiments, the blockmir comprises one or more blocks independently selected from a stereoblock, a P-modified block, and a linking block, hi some embodiments, the blockmir is a stereoblockmir to one block, and / or a P-modified blockmir to another block, and / or a linking blockmir to yet another block. For example, (Rp,Rp,Rp,Rp,Rp,Sp,Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG is a stereoblockmir with respect to the stereoblock AsTsCsGsAs1 (all Rp's at the bound phosphorus) or Ts1Cs1Gs1 (all Sp's at the bound phosphorus), a P-modified blockmir with respect to the P-modified block AsTsCsGs (all s-bonds) or As1Ts1Cs1Gs1 (all s1-bonds), or a bonded blockmir with respect to the bonded block AsTsCsGs (all Rp's and all s-bonds at the bound phosphorus) or Ts1Cs1Gs1 (all Sp's and all s1-bonds at the bound phosphorus).

[0247] Altmer: As used herein, the term "altmer" refers to an oligonucleotide chain whose structural feature pattern characterizing each individual nucleotide unit is characterized by the absence of two consecutive nucleotide units in the oligonucleotide chain that share a particular structural feature in the internucleotide phosphorus bond. In some embodiments, an altmer is designed so that it contains a repeating pattern. In some embodiments, an altmer is designed so that it does not contain a repeating pattern.

[0248] In some embodiments, the altomer is a "stereoaltomer," e.g., no two consecutive nucleotide units have the same stereochemistry at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

[0249] In some embodiments, the altmer is a "P-modified altmer," e.g., no two consecutive nucleotide units have the same modification at the linked phosphorus, e.g., all (Sp)CAs1GsT, where each linked phosphorus has a different P modification than the others.

[0250] In some embodiments, the altmer is a "linked altmer," e.g., no two consecutive nucleotide units have the same stereochemistry or the same modification at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC.

[0251] Unimer: As used herein, the term "unimer" refers to an oligonucleotide chain whose structural feature pattern characterizes each individual nucleotide unit, in which all nucleotide units in the chain share at least one common structural feature at the internucleotide phosphorus linkage. A common structural feature refers to a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus.

[0252] In some embodiments, the unimer is a "stereounimer," e.g., all nucleotide units have the same stereochemistry at the linkage phosphorus, e.g., all (Sp)-CsAs1GsT, where all the linkages have Sp phosphorus.

[0253] In some embodiments, the unimer is a "P-modified unimer," e.g., all nucleotide units have the same modification at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, where all the internucleotide linkages are phosphorothioate diesters.

[0254] In some embodiments, the unimer is a "linked unimer," e.g., all nucleotide units have the same stereochemistry and the same modification at the linked phosphorus, e.g., all (Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, where all the internucleotide linkages are phosphorothioate diesters with an Sp linked phosphorus.

[0255] Gapmer: As used herein, the term "gapmer" refers to an oligonucleotide chain characterized in that at least one internucleotide phosphorus bond of the oligonucleotide chain is a phosphodiester bond, such as that found in natural DNA or RNA. In some embodiments, one or more internucleotide phosphorus bonds of the oligonucleotide chain are phosphodiester bonds, such as those found in natural DNA or RNA. For example, all (Sp)-CAs1GsT, in which the internucleotide bond between C and A is a phosphodiester bond.

[0256] Skipmer: As used herein, the term "skipmer" refers to a type of gapmer in which every other internucleotide phosphorus bond of the oligonucleotide strand is a phosphodiester bond, such as found in natural DNA or RNA, and every other internucleotide phosphorus bond of the oligonucleotide strand is a modified internucleotide bond, e.g., all (Sp)-AsTCs1GAs2TCs3G.

[0257] For purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, edited by CAS, Handbook of Chemistry and Physics, 67th Edition, 1986-87, inside cover.

[0258] The methods and structures described herein relating to the compounds and compositions of the present invention also apply to pharmaceutically acceptable acid or base addition salts and all stereoisomers of these compounds and compositions. [Brief explanation of the drawings]

[0259] [Figure 1]This figure shows reverse-phase HPLC analysis of rat liver homogenate after incubation. The total amount of remaining oligonucleotide was measured when rat whole liver homogenate was incubated at 37°C for different days. The in vitro metabolic stability of ONT-154 was found to be similar to that of ONT-87, which has a 2'-MOE wing, but both have significantly better stability than the stereorandom 2'-MOE gapmer (ONT-41, mipomersen). The amount of remaining full-length oligomer was measured by reverse-phase HPLC, and the peak area of ​​the peak of interest was normalized to the internal standard.

[0260] [Figure 2] Degradation of various chirally pure analogs of mipomersen (ONT-41) in rat whole liver homogenate was measured. The total amount of remaining oligonucleotide was measured after incubation of rat whole liver homogenate at 37°C for different days. The in vitro metabolic stability of chirally pure diastereomers of the human ApoB sequence ONT-41 (mipomersen) was found to increase with increasing Sp internucleotide linkages. The amount of remaining full-length oligomer was measured by reverse-phase HPLC, and the peak area of ​​the peak of interest was normalized to an internal standard. The compositions used included ONT-41, ONT-75, ONT-77, ONT-80, ONT-87, ONT-88, and ONT-89.

[0261] [Figure 3]Degradation of various chirally pure analogs of the mouse ApoB sequence (ISIS 147764, ONT-83) in rat whole liver homogenate was measured. The total amount of remaining oligonucleotide was measured after incubation of rat whole liver homogenate at 37°C for different days. The in vitro metabolic stability of chirally pure diastereomers of the mouse ApoB sequence (ONT-83, 2'-MOE gapmer, stereorandom phosphorothioate) was found to increase with increasing Sp internucleotide linkage. The amount of remaining full-length oligomer was measured by reverse-phase HPLC, and the peak area of ​​the peak of interest was normalized to an internal standard. The compositions used included ONT-82 through ONT-86.

[0262] [Figure 4] 1 shows the degradation of the mipomersen analog ONT-75 in rat whole liver homogenate over 24 hours. This figure shows the stability of ONT-75 in rat whole liver homogenate.

[0263] [Figure 5] 1 shows the degradation of the mipomersen analog ONT-81 in rat whole liver homogenate over a 24 hour period. This figure shows the stability of ONT-81 in rat whole liver homogenate.

[0264] [Figure 6]This figure shows the duration of knockdown in ONT-87, ONT-88, and ONT-89. Stereoisomers can exhibit significantly different durations of knockdown. ONT-87 produces significantly more durable inhibition than other stereoisomers. The long duration of action of ONT-87 was observed in multiple in vivo studies. ONT-88 demonstrated a similar efficacy and recovery profile to ONT-41 (mipomersen) in certain in vivo studies. Hu ApoB transgenic mice (n=4) were administered a 10 mpk IP bolus (2x / week for 3 weeks). Mice were randomized into groups and administered 10 mg / kg intraperitoneally (IP) based on individual mouse weights measured before dosing on each dosing day (days 1, 4, 8, 11, 15, 18, and 22). Blood was collected by submandibular (cheek) bleeds on days 0, 17, 24, 31, 38, 45, and 52, followed by sacrifice by cardiac puncture and serum processing on day 52. ​​ApoB was measured by ELISA. Highlighted: sustained knockdown after 3 weeks of dosing (72% vs. 35%).

[0265] [Figure 7] Figure 1 shows HPLC profiles measured in human serum demonstrating the differences in metabolic stability of siRNA duplexes containing several Rp, Sp, or stereorandom phosphorothioate linkages. The compositions used include ONT-114, ONT-116, ONT-109, ONT-107, ONT-108, and ONT-106.

[0266] [Figure 8] Effect of stereochemistry on RNase H activity. Oligonucleotides were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1x RNase H buffer. From top to bottom (120 min): ONT-89, ONT-77, ONT-81, ONT-80, ONT-75, ONT-41, ONT-88, ONT-154, ONT-87. ONT-77 / 154 are very close to each other.

[0267] [Figure 9] Analysis of human RNase H1 cleavage of a 20-mer RNA when hybridized with different stereoisomer preparations of phosphorothioate oligonucleotides targeting the same region of human ApoB mRNA. The specific site of cleavage is strongly influenced by the differential stereochemistry. The arrow indicates the cleavage position (cleavage site). Products were analyzed by UPLC / MS. The length of the arrow represents the amount of product present in the reaction mixture, which was determined by the ratio of the UV peak area to the theoretical extinction coefficient of that fragment (the longer the arrow, the more cleavage product was detected). (A) explains the cleavage map symbols. (B) and (C) show the cleavage maps of the oligonucleotides. In the figure, (┬) indicates that both RNase H1 cleavage fragments (5'-phosphate and 5'-OH 3'-OH species) were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that both the 5'-OH and 3'-OH components were detected by mass spectrometry. The compositions used include: ONT-41, ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-88, and ONT-154.

[0268] [Figure 10]Cleavage maps of different oligonucleotide compositions are shown ((A)-((C))). These three sequences target different regions in the FOXO1 mRNA. Each sequence was probed with five different chemistries. Cleavage maps were generated from the reaction mixture obtained after incubating each duplex with RNase H1C in the presence of 1x PBS buffer at 37°C for 30 minutes. Arrows indicate the cleavage sites. The length of the arrow represents the amount of product present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment (the longer the arrow, the more detectable cleavage product). The 5'-phosphate species peak was used for quantification only when 5'-OH and 3'-OH were not detected in the reaction mixture. Cleavage rates were determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the designated time points, the reaction was stopped with 30 mM Na2EDTA. Compositions used include ONT-316, ONT-355, ONT-361, ONT-367, ONT-373, ONT-302, ONT-352, ONT-358, ONT-364, ONT-370, ONT-315, ONT-354, ONT-360, ONT-366 and ONT-372.

[0269] [Figure 11]Figures (A-B) show cleavage maps of oligonucleotide compositions with different consensus base sequences and lengths. The cleavage maps show a comparison of a stereorandom DNA composition (top panel) with three stereochemically pure, unique oligonucleotide compositions. The data compare the results of a chirality-controlled oligonucleotide composition with two stereorandom phosphorothioate oligonucleotide compositions (ONT-366 and ONT-367) targeting different regions in the FOXO1 mRNA. Each panel shows a comparison of stereorandom DNA (top panel) with three stereochemically pure, unique oligonucleotide preparations. Cleavage maps were generated from the reaction mixtures obtained after incubating each duplex with RNase H1C in the presence of 1x PBS buffer for 30 minutes at 37°C. Arrows indicate the cleavage sites. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined by the ratio of the UV peak area to the theoretical extinction coefficient of that fragment (the longer the arrow, the more detectable cleavage product). The 5'-phosphate species peak was used for quantification only when 5'-OH and 3'-OH were not detected in the reaction mixture. The compositions used included ONT-366, ONT-389, ONT-390, ONT-391, ONT-367, ONT-392, ONT-393, and ONT-394.

[0270] [Figure 12]Effect of stereochemistry on RNase H activity. In two independent experiments, antisense oligonucleotides targeting the same region of FOXO1 mRNA were hybridized to RNA and then incubated with RNase H in the presence of 1x RNase H buffer at 37°C. The disappearance of full-length RNA was measured by RP-HPLC from its peak area at 254 nm. (A) shows the remaining RNA substrate (from top to bottom (60 min): ONT-355, ONT-316, ONT-367, ONT-392, ONT-393, and ONT-394 (at 60 min, ONT-393 and ONT-394 were similar; at 5 min, ONT-393 had a higher remaining RNA substrate (%)). (B) shows the remaining RNA substrate (%) (from top to bottom (60 min): ONT-315, ONT-354, ONT-366, ONT-391, ONT-389, and ONT-390). Cleavage rates were determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the indicated time points, the reaction was stopped with 30 mM Na2EDTA.

[0271] [Figure 13] Figure 1 shows the turnover of antisense oligonucleotides. Duplexes were formed with a concentration of each DNA strand equal to 6 μM and 100 μM of RNA. These duplexes were incubated with 0.02 μM RNase H enzyme, and the disappearance of full-length RNA was measured by RP-HPLC from its peak area at 254 nm. The cleavage rate was determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the specified time points, the reaction was stopped with 30 mM Na2EDTA. From top to bottom (40 min): ONT-316, ONT-367, and ONT-392.

[0272] [Figure 14]Figure 1 shows a cleavage map comparing stereorandom phosphorothioate oligonucleotides with six unique stereochemically pure oligonucleotide preparations targeting the same FOXO1 mRNA region, including ONT-367, ONT-392, ONT-393, ONT-394, ONT-400, ONT-401, and ONT-406.

[0273] [Figure 15] Effect of stereochemistry on RNase H activity. Antisense oligonucleotides were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1x RNase H buffer. A stereochemical dependence on RNase H activity was observed. Similarly, a comparison of ONT-367 (stereorandom DNA) and ONT-316 (5-10-5 2'-MOE gapmer) reveals a strong dependence of compositional chemistry on RNase H activity. From top to bottom (40 min): ONT-316, ONT-421, ONT-367, ONT-392, ONT-394, ONT-415, and ONT-422 (at 40 min, ONT-394 / 415 / 422 are at equivalent levels; at 5 min, the percentage of RNA remaining in the DNA / RNA duplex is ONT-422>ONT-394>ONT-415).

[0274] [Figure 16]Effect of stereochemistry on RNase H activity. Antisense oligonucleotides targeting the same region of FOXO1 mRNA were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1x RNase H buffer. A stereochemical dependence on RNase H activity was observed. From top to bottom (40 minutes): ONT-396, ONT-409, ONT-414, ONT-408 (at 40 minutes, ONT-396 / 409 / 414 / 408 are at the same level), ONT-404, ONT-410, ONT-402 (at 40 minutes, ONT-404 / 410 / 408 are at the same level), ONT-403, ONT-407, ONT-405, ONT-401, ONT-406 and ONT-400 (at 40 minutes, ONT-401 / 405 / 406 / 400 are at the same level).

[0275] [Figure 17] Effect of stereochemistry on RNase H activity. Antisense oligonucleotides targeting the same region of FOXO1 mRNA were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1× RNase H buffer. A stereochemical dependence on RNase H activity was observed. ONT-406 was observed to induce cleavage of double-stranded RNA at a rate slightly greater than that of the phosphodiester oligonucleotide ONT-415. From top to bottom (40 min): ONT-396, ONT-421, ONT-392, ONT-394, ONT-415 ONT-406, and ONT-422 (at 40 min, ONT-394 / 415 / 406 are at equivalent levels; at 5 min, the percentage of RNA remaining in the DNA / RNA duplex is ONT-394>ONT-415>ONT-406).

[0276] [Figure 18]Figure 1 shows exemplary UV chromatograms of RNA cleavage products obtained when RNA (ONT-388) was duplexed with stereorandom DNA (ONT-367) (top) and when stereopure DNA was duplexed with repeating triplet motif-3'-SSR-5' (ONT-394) (bottom). 2.35 min: 7mer; 3.16 min: 8mer and P-6mer; 4.48 min: P-7mer; 5.83 min: P-8mer; 6.88 min: 12mer; 9.32 min: 13mer; 10.13 min: P-11mer; 11.0 min: P-12mer and 14mer; 11.93 min: P-13mer; 13.13 min: P-14mer. Peak assignments for ONT-394 (bottom): 4.55 min: p-7mer; 4.97 min: 10mer; 9.53 min: 13mer.

[0277] [Figure 19] Electrospray ionization spectra of RNA cleavage products: RNA fragments obtained from the duplexes ONT-387, RNA / ONT-354, (7-6-7, DNA-2'-OMe-DNA) (top) and ONT-387, RNA / ONT-315, (5-10-5,2'-MOE gapmer) (bottom) when the duplexes were incubated with RNase H in the presence of 1x RNse H buffer for 30 min.

[0278] [Figure 20] Figure 1 shows UV chromatograms and TICs of ONT-406 and ONT-388 duplexes after 30 min incubation with RNase H.

[0279] [Figure 21] 1 shows exemplary proposed cleavages. The provided chiral controlled oligonucleotide compositions are capable of cleaving targets as shown.

[0280] [Figure 22]Figure 1 shows exemplary allele-specific cleavage targeting mutant huntingtin mRNA. (A) and (B): Exemplary oligonucleotides. (C)-(E): Cleavage maps. (F)-(H): RNA cleavage. Stereorandom and chiral-controlled oligonucleotide compositions were prepared to target single nucleotide polymorphisms for allele-selective suppression of mutant huntingtin. ONT-450 (stereorandom), targeting ONT-453 (muHTT) and ONT-454 (wtHTT), showed minor differences in RNA cleavage and their cleavage maps. Chiral-controlled ONT-451, targeting ONT-453 (muHTT) and ONT-454 (wtHTT) and possessing a selective placement of a 3'-SSR-5' motif within the RNase H recognition site, showed significant differences in RNA cleavage rates. It is noteworthy that in the cleavage maps, the 3'-SSR-5' motif is positioned to direct cleavage between positions 8 and 9, after the mismatch, when read from the 5' end of the RNA. ONT-452, which targets ONT-453 (muHTT) and ONT-454 (wtHTT) and has an alternative placement of the 3'-SSR-5' motif within the RNase H recognition site, showed moderate differences in RNA cleavage rates. The 3'-SSR-5' motif was positioned to direct cleavage at positions 7 and 8, before the mismatch, when read from the 5' end of the RNA. The exemplary data demonstrate that the position of the 3'-SSR-5' motif is important for achieving high discrimination for allele-specific cleavage. All cleavage maps were generated from reaction mixtures obtained after incubating each duplex with RNase H1C for 5 minutes at 37°C in the presence of 1x PBS buffer. Arrows indicate the cleavage sites. (┬) indicates that both fragments, the 5'-phosphate species as well as the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH component was detected by mass spectrometry. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment.Only when 5'-OH and 3'-OH were not detected in the reaction mixture, the 5'-phosphate species peak was used for quantification. The compositions used included ONT-450 to ONT-454.

[0281] [Figure 23] (A)-(C) show exemplary allele-specific cleavage targeting FOXO1 mRNA.

[0282] [Figure 24] Figure 1 shows in vitro dose-response silencing of ApoB mRNA after treatment with ApoB oligonucleotides. Stereochemically pure diastereomers with and without the 2'-MOE wing show similar effects to ONT-41 (mipomersen). The compositions used include ONT-87, ONT-41, and ONT-154.

[0283] [Figure 25] This figure shows a comparison of RNase H cleavage maps (A) and RNA cleavage rates (B) for a stereorandom (ONT-367) and chiral-controlled oligonucleotide compositions (ONT-421 (all Sp) and ONT-455 (all Rp)) and DNA (ONT-415). These sequences target the same region in the FOXO1 mRNA. Cleavage maps were generated from the reaction mixture obtained after incubating each duplex with RNase H1C in the presence of 1x PBS buffer at 37°C for 5 minutes. Arrows indicate the cleavage sites. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment. The 5'-phosphate species peak was used for quantification only when 5'-OH and 3'-OH were not detected in the reaction mixture. Cleavage rates were determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the designated time points, the reaction is stopped with 30 mM Na2EDTA.

[0284] [Figure 26] This figure shows a comparison of cleavage maps of sequences containing one Rp with a change in position starting from the 3'-end of the DNA. The compositions used include ONT-396 through ONT-414. These sequences target the same region in the FOXO1 mRNA. The cleavage maps were generated from the reaction mixtures obtained after incubating each duplex with RNase H1C in 1X RNase H buffer at 37°C for 5 minutes. The arrows indicate the cleavage sites. The length of the arrows represents the amount of metabolite present in the reaction mixture, which was determined by the ratio of the UV peak area to the theoretical extinction coefficient of that fragment. The 5'-phosphate species peak was used for quantification only when 5'-OH 3'-OH was not detected in the reaction mixture.

[0285] [Figure 27](A) Comparison of RNase H cleavage rates of stereopure oligonucleotides (ONT-406), (ONT-401), (ONT-404), and (ONT-408). All four sequences are stereopure phosphorothioates with a single Rp linkage. These sequences target the same region in the FOXO1 mRNA. All duplexes were incubated with RNase H1C in the presence of 1× RNase H buffer at 37°C. At designated time points, the reaction was stopped with 30 mM Na2EDTA. Cleavage rates were determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. ONT-406 and ONT-401 were found to have superior cleavage rates. (B) Correlation between % RNA cleaved in the RNase H assay (10 μM oligonucleotide) and % mRNA knockdown in the in vitro assay (20 nM oligonucleotide). All sequences target the same region of the FOXO1 mRNA. The amount of remaining RNA was determined by the UV peak area of ​​the RNA when normalized to DNA in the same reaction mixture. All cleavage maps described above were generated from the reaction mixture obtained after incubating each duplex with RNase H1C for 5 minutes at 37°C in the presence of 1X RNase H buffer. All sequences from ONT-396 to ONT-414 contain a single Rp phosphorothioate, and these sequences differ in the position of the Rp. ONT-421 (all Sp) phosphorothioates were inactive in in vitro assays. This is related to the slow rate of RNA cleavage in RNase H assays when ONT-421 is duplexed with complementary RNA.

[0286] [Figure 28]Figure 1 shows a 2-day serum stability assay in rat serum of single Rp walk PS DNA (ONT-396-ONT-414), stereorandom PS DNA (ONT-367), all Sp PS DNA (ONT-421), and all Rp PS DNA (ONT-455). Note that ONT-396 and ONT-455 degraded at the time of testing. The compositions used include ONT-396 through ONT-414, ONT-397, ONT-421, and ONT-455.

[0287] [Figure 29]Figure 1 shows exemplary oligonucleotides containing hemimers. (A): Cleavage map. (B): RNA cleavage assay. (C): FOXO1 mRNA knockdown. ONT-440, ONT-441, and ONT-367 were used. In some embodiments, the introduction of 2'-modifications at the 5'-end of the sequence increases the stability of binding to target RNA while maintaining RNase H activity. ONT-367 (stereorandom phosphorothioate DNA) and ONT-440 (5-15, 2'-F-DNA) have similar cleavage maps and similar RNA cleavage rates in RNase H assays (10 μM oligonucleotide). In some embodiments, the ONT-440 (5-11, 2'-F-DNA) sequence may have better cell permeability. In some embodiments, asymmetric 2'-modifications provide a Tm advantage while maintaining RNase H activity. The introduction of an RSS motif in hemimers may further improve the efficiency of RNase H. Cleavage maps were generated from the reaction mixtures obtained after incubating each duplex with ribonuclease H1C in the presence of 1X RNase H buffer at 37°C for 5 minutes. Arrows indicate the cleavage sites. (┬) indicates that both fragments, the 5'-phosphate species and the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH component was detected by mass spectrometry. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined by the ratio of the UV peak area to the theoretical extinction coefficient of that fragment. The 5'-phosphate species peak was used for quantification only if the 5'-OH 3'-OH species was not detected in the reaction mixture.

[0288] [Figure 30]Figure 1 shows exemplary mass spectrometry data for a cleavage assay. Top: Data for ONT-367: 2.35 min: 7-mer; 3.16 min: 8-mer and P-6-mer; 4.58 min: P-7-mer; 5.91 min: P-8-mer; 7.19 min: 12-mer; 9.55 min: 13-mer; 10.13 min: P-11-mer; 11.14 min: P-12-mer and 14-mer; 12.11 min: P-13-mer; 13.29 min: P-14-mer; 14.80 min: full-length RNA (ONT-388) and 18.33 min: stereorandom DNA (ONT-367). Bottom: Data for ONT-406: 4.72 min: p-rArUrGrGrCrUrA, 5'-phosphorylated 7-mer RNA; 9.46 min: 5'-rGrUrGrArGrCrArGrCrUrGrCrA, 5'-OH 3'-OH 13-mer RNA; 16.45 min: full-length RNA (ONT-388); 19.48 min and 19.49 min: stereopure DNA (ONT-406).

[0289] [Figure 31] Example RNA cleavage rates. The duplex was incubated with RNase H1C in the presence of 1x RNase H buffer at 37°C. At designated time points, the reaction was stopped by adding 30mM Na2EDTA. The cleavage rate was determined by measuring the amount of full-length RNA remaining in the reaction mixture. The compositions used included: WV-944, WV-945, WV-936, WV-904, WV-937, WV-905, WV-938, WV-906, WV-939, WV-907, WV-940, WV-908, WV-941, and WV-909.

[0290] [Figure 32]AN: RNA cleavage rates in RNase H assays of specific compositions targeting rs362307. Some of these compositions are stereorandom, and some are chiral-controlled. The compositions used include: WV-1085, WV-1086, WV-1087, WV-1088, WV-1089, WV-1090, WV-1091, WV-1092, WV-905, WV-944, WV-945, WV-911, WV-917, WV-931, WV-937, and WV-1497.

[0291] [Figure 33] A: Example cleavage map. The cleavage map was generated from the reaction mixture obtained after incubating each duplex with RNase H1C in the presence of 1x RNase H buffer at 37°C for 5 minutes. B: Legend. Arrows indicate the cleavage site. (┬) indicates that both fragments, the 5'-phosphate species and the 3'-OH species, were identified. (┌) indicates that only the 5'-OH 3'-OH species was detected, and (┐) indicates that only the 5'-phosphate component was detected. The length of the arrow represents the amount of fragment present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment. The 5'-phosphate species peak was used for quantification only if the 5'-OH 3'-OH fragment was not detected in the reaction mixture. The compositions used include: WV-944, WV-945, WV-904, WV-905, WV-906, WV-907, WV-908 and WV-909.

[0292] [Figure 34]Figure 3 shows an example cleavage map. Figure 3 shows an example cleavage map. Cleavage maps were generated from the resulting reaction mixtures after incubating each duplex with RNase H1C in the presence of 1x RNase H buffer at 37°C for 30 minutes. For legend, see Figure 33. The compositions used include: WV-944, WV-945, WV-936, WV-937, WV-938, WV-939, WV-940, WV-941, WV-1085, WV-1086, WV-1087, WV-1088, WV-1089, WV-1090, WV-1091, and WV-1092.

[0293] [Figure 35]

[0033] Figure 33 is an example cleavage map. For legend, see Figure 33. Compositions used include: WV-944, WV-945, WV-905, WV-911, WV-917, WV-931, and WV-937.

[0294] [Figure 36] Total ion chromatograms of the RNase H cleavage reaction of WV-937 when duplexed with WT HTT RNA (WV-944, top panel) or mu HTT RNA (WV-945, bottom panel). After 30 min, the enzymatic reaction was stopped with disodium EDTA. RNase H cleavage products were chromatographically separated and analyzed using an Agilent 1290 UPLC coupled to an Agilent 6230 MS-TOF mass spectrometer. High-mass-accuracy, high-resolution MS spectra of each identified peak were extracted and deconvoluted. Metabolite identification, with cleavage sites determined, was performed by comparing the deconvoluted average mass with the masses of predicted RNA metabolites.

[0295] [Figure 37] 1 is an illustration of a luciferase reporter-based screen.

[0296] [Figures 38A-38I]Figures 38A-38I and Figures 39A-39G show the activity of various HTT oligonucleotides. Dose-response curves for HTT silencing in a reporter-based assay in COS7 cells after transfection of ASOs targeting the rs362331_T or rs2530595_T SNP. The addition of a stereopure design improves ASO specificity without significantly compromising efficacy (calculated IC50 indicated). Data represent two independent experiments. Lines represent fitted curves, and error bars represent standard deviation. The positions of the SNPs are indicated on the figures. The compositions tested in Figure 38 include: WV-2067, WV-2416, WV-2069, WV-2417, WV-2072, WV-2418, WV-2076, WV-2419, WV-2605, WV-2589, WV-2606, WV-2590, WV-2607, WV-2591, WV-2608, WV-2592, WV-2609, WV-2593, WV-2610, WV-2594, WV-2611, WV-2595, WV -2612, WV-2596, WV-2611, WV-2595, WV-2671, WV-2672, WV-2673, WV-2675, WV-2674, WV-2613, WV-2597, WV-2614, WV-259 8, WV-2615, WV-2599, WV-2616, WV-2600, WV-2617, WV-2601, WV-2618, WV-2602, WV-2619, WV-2603, WV-2620 and WV-2604.

[0297] [Figures 39A-39G] Dose-response curves for HTT silencing in a reporter-based assay in COS7 cells after transfection of ASOs. Tested compositions include: WVE120101, WV-1092, WV-1497, WV-2619, WV-2603, WV-2611, and WV-2595. IC50 data are also shown.

[0298] [Figures 40A-40D]Liquid chromatographic and mass spectral data for oligonucleotides: WV1092.22 (WV-1092), WV2595.01 (WV-2595), and WV2603.01 (WV-2603), where the subscripts (01), (02), 01, 02, 22, etc., represent batch numbers.

[0299] [Figure 41] FIG. 1 shows liquid chromatographic and mass spectral data for oligonucleotides: WV-1510, WV-2378 and WV-2380. DETAILED DESCRIPTION OF THE INVENTION

[0300] Synthetic oligonucleotides provide useful molecular tools for a wide variety of applications. For example, oligonucleotides are useful in therapeutics, diagnostics, research, and novel nanomaterial applications. The use of natural nucleic acids (e.g., unmodified DNA or RNA) is limited by their susceptibility to endo- and exonucleases. Therefore, to circumvent these shortcomings, various synthetic equivalents have been developed. These include synthetic oligonucleotides containing backbone modifications that make these molecules less susceptible to degradation. From a structural perspective, such modifications to internucleotide phosphate linkages introduce chirality. It has been shown that certain properties of oligonucleotides can be influenced by the arrangement of phosphorus atoms forming the oligonucleotide backbone. For example, in vitro studies have shown that properties of antisense nucleotides, such as binding affinity, specific binding sequences with complementary RNA, and stability against nucleases, are particularly influenced by backbone chirality (e.g., the arrangement of phosphorus atoms).

[0301] Among other things, the present disclosure encompasses the recognition that oligonucleotide components, such as base sequence, chemical modifications (e.g., sugar, base, and / or internucleotide linkage modifications and their patterns), and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or their patterns), can significantly impact the properties, e.g., activity, of the oligonucleotide. In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising controlled components, e.g., oligonucleotides with controlled chemical modifications and / or controlled backbone stereochemical patterns, result in unexpected properties, including, but not limited to, those described herein. In some embodiments, the present disclosure provides oligonucleotide compositions comprising predetermined levels of individual oligonucleotide types that are chemically identical, e.g., oligonucleotides having the same base sequence, the same pattern of nucleoside modifications (modifications to the sugar and base moieties, if present), the same pattern of backbone chiral centers, and the same pattern of backbone phosphorus modifications.

[0302] In particular, the present disclosure encompasses the recognition that stereorandom oligonucleotide preparations contain multiple unique chemical entities that differ from one another, for example, in the stereochemical configuration of individual backbone chiral centers within the oligonucleotide chain. Without control of the stereochemistry of the backbone chiral centers, stereorandom oligonucleotide preparations result in uncontrolled compositions containing undefined levels of oligonucleotide stereoisomers. These stereoisomers may have the same base sequence, but due at least to their different backbone stereochemistry, they are different chemical entities and, as demonstrated herein, can have different properties, e.g., biological activity. Stereopure (or "chiral-controlled") oligonucleotide compositions or preparations can have improved biological activity compared to otherwise identical stereorandom oligonucleotide preparations (e.g., both the stereopure and stereorandom versions have the same base sequence, base modification pattern, sugar modification, etc.). For example, the stereorandom oligonucleotide WV-1497 composition and the stereopure oligonucleotide WV-1092 composition both have the same sequence of bases and the same pattern of sugar modifications and backbone linkages, differing only in stereochemistry. However, at higher concentrations, there was a marked difference in the ability of the stereopure WV-1092 and stereorandom WV-1497 compositions to distinguish between wild-type and mutant HTT (which differ by only one nt). At high concentrations, both significantly knocked down mutant HTT, which is desirable; however, while stereopure WV-1092 showed only a slight knockdown of wild-type HTT, WV-1497 showed a much greater knockdown of wild-type HTT, which may be less desirable in some cases.

[0303] Both the chiral oligonucleotide compositions, WVE120101 and WV-1092, were able to distinguish between the wt and mutant versions of SNP rs362307 (which differ by one nt); both WVE120101 and WV-1092 significantly knocked down the mutant allele, but the wt did not, while the stereorandom version, WV-1497, was unable to significantly distinguish between the wt and mutant alleles (see Figure 39D). The modified sequences of WVE120101 and WV-1092 are identical.

[0304] The chiral oligonucleotide composition of WV-2595 was able to distinguish between the C and T alleles of SNP rs2530595 (which also differ by only one nt). Unlike the stereorandom oligonucleotide composition of WV-2611, which could not significantly distinguish between the alleles, the stereopure WV-2595 significantly knocked down the T allele but not the C allele (see Figure 39F). The sequence of WV-2595 contains a 5'-mG fragment with specific stereochemical information. * mGmGmUmC * C * T * C * C * C * C * A * C * A * G * mAmGmGmG * mA-3' or 5'-mG * SmGmGmUmC * SC * ST * SC * SC * SC * SC * SA * SC * RA * SG * SmAmGmGmG * SmA-3'.

[0305] The stereopure oligonucleotide composition of WV-2603 was able to distinguish between the C and T alleles of SNP rs362331 (which also differ by only one nt). Unlike the stereorandom oligonucleotide composition of WV-2619, which could not significantly distinguish between the alleles, stereopure WV-2603 significantly knocked down the T allele but not the C allele (see Figures 39A, 39B, 39C, and 39E). The sequence of WV-2603 contains a 5'-mG residue with specific stereochemical information. * mUmGmCmA * C * A * C * A * G * T * A * G * A * T * mGmAmGmG * mG-3' or 5'-mG * SmUmGmCmA * SC * SA * SC * SA * SG * ST * SA * SG * RA * ST * SmGmAmGmG * SmG-3'.

[0306] In some embodiments, the sequence of the oligonucleotides in the stereopure (chiral controlled) oligonucleotide composition comprises or consists of the sequence of any oligonucleotide disclosed herein. In some embodiments, the sequence of the oligonucleotides in the stereopure (chiral controlled) oligonucleotide composition comprises or consists of the sequence of any oligonucleotide selected from Table N1, Table N2, Table N3, Table N4, and Table 8. In some embodiments, the sequence of the oligonucleotides in the stereopure (chiral controlled) oligonucleotide composition comprises or consists of the sequence of any oligonucleotide selected from Table N1A, Table N2A, Table N3A, Table N4A, and Table 8. In some embodiments, the sequence of the oligonucleotides in the stereopure (chiral controlled) oligonucleotide composition comprises or consists of the sequence of WV-1092, WVE120101, WV-2603, or WV-2595.

[0307] Each oligonucleotide described herein containing an HTT sequence represents an HTT oligonucleotide that has been designed, constructed, and tested in various assays, and in some embodiments, in one or more in vitro assays. Each HTT oligonucleotide listed in any of Tables N1A, N2A, N3A, N4A, and 8, or described elsewhere herein, has been designed, constructed, and tested in various assays, and in some embodiments, in one or more in vitro assays. For example, the HTT oligonucleotides described herein have been tested in a dual-luciferase reporter assay. In some embodiments, the HTT oligonucleotides have been tested in one or more other assays described herein and / or techniques according to the present disclosure. In some embodiments, HTT oligonucleotides found to be particularly effective in the dual-luciferase assay have been tested in additional in vitro and in vivo assays according to the present disclosure.

[0308] In some embodiments, the sequences of the oligonucleotides in the stereopure (chiral controlled) oligonucleotide composition are determined by the base sequence (including length); the pattern of chemical modifications to the sugar and base moieties; the pattern of backbone linkages; the pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof; the pattern of backbone chiral centers; the pattern of chiral internucleotide linkage stereochemistry (Rp / Sp); the pattern of backbone phosphorus modifications; -S - and -LR of formula I 1 The internucleotide may include any one or more of the patterns of modification to the phosphorus atom such as:

[0309] Among other things, the present disclosure provides novel compositions that are or include specific stereoisomers of the subject oligonucleotides. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, its length, its backbone bond pattern, and its backbone chiral center pattern. As understood in the art, in some embodiments, the base sequence may refer to the identity and / or modification state of the nucleoside residues (e.g., sugar moieties and / or base moieties, relative to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) within the oligonucleotide, and / or the hybridization properties (i.e., ability to hybridize with specific complementary residues) of such residues. In some embodiments, the oligonucleotides in the provided compositions include sugar modifications, e.g., 2'-modifications, e.g., of the wing regions. In some embodiments, the oligonucleotides in the provided compositions include a middle region, e.g., a core region, that lacks sugar modifications.

[0310] The present disclosure demonstrates, inter alia, that individual stereoisomers of a particular oligonucleotide may exhibit different stability and / or activity (e.g., functional and / or toxicological properties) from one another. Furthermore, the present disclosure demonstrates that the improved stability and / or activity achieved by the inclusion and / or location of a particular chiral structure within an oligonucleotide may be comparable to or even better than the improved stability and / or activity achieved by the use of particular backbone linkages, residue modifications, etc. (e.g., by the use of particular types of modified phosphates [e.g., phosphorothioates, substituted phosphorothioates, etc.], sugar modifications [e.g., 2'-modifications, etc.], and / or base modifications [e.g., methylation, etc.]).

[0311] In particular, the present disclosure recognizes that, in some embodiments, the properties (e.g., stability and / or activity) of an oligonucleotide can be adjusted by optimizing the pattern of its backbone chiral centers, optionally in combination with adjusting / optimizing one or more other features of the oligonucleotide (e.g., linkage pattern, nucleoside modification pattern, etc.). In some embodiments, the present disclosure provides oligonucleotide compositions in which the oligonucleotide comprises nucleoside modifications, chiral internucleotide linkages, and natural phosphate linkages. For example, WV-1092 comprises 2'-OMe modifications, phosphate linkages in its 5'- and 3'-wing regions, and phosphorothioate linkages in its core region.

[0312] In some embodiments, the present disclosure demonstrates that the improved stability achieved by the inclusion and / or location of particular chiral structures within an oligonucleotide can be comparable to, or even better than, the improved stability achieved by the use of modified backbone linkages, bases, and / or sugars (e.g., by the use of particular types of modified phosphate esters, 2'-modifications, base modifications, etc.). In some embodiments, the present disclosure also demonstrates that the improved activity achieved by the inclusion and / or location of particular chiral structures within an oligonucleotide can be comparable to, or even better than, the improved activity achieved by the use of modified backbone linkages, bases, and / or sugars (e.g., by the use of particular types of modified phosphate esters, 2'-modifications, base modifications, etc.).

[0313] In some embodiments, the inclusion and / or location of specific chiral linkages within an oligonucleotide can surprisingly alter the cleavage pattern of a nucleic acid polymer when such an oligonucleotide is utilized to cleave the nucleic acid polymer. For example, in some embodiments, the pattern of backbone chiral centers results in unexpectedly high cleavage efficiency of a target nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers results in new cleavage sites. In some embodiments, the pattern of backbone chiral centers results in fewer cleavage sites, for example, by blocking certain existing cleavage sites. Even more unexpectedly, in some embodiments, the pattern of backbone chiral centers results in cleavage of a target nucleic acid polymer at only one site within the sequence complementary to the oligonucleotide utilized for cleavage. In some embodiments, even higher cleavage efficiency is achieved by selecting a pattern of backbone chiral centers that minimizes the number of cleavage sites.

[0314] In some embodiments, the present disclosure provides oligonucleotide compositions, wherein the oligonucleotides have a common pattern of backbone chiral centers that unexpectedly and significantly improves the stability and / or biological activity of the oligonucleotides. In some embodiments, the pattern of backbone chiral centers results in increased stability. In some embodiments, the pattern of backbone chiral centers results in surprisingly increased activity. In some embodiments, the pattern of backbone chiral centers results in increased stability and activity. In some embodiments, when the oligonucleotides are utilized to cleave nucleic acid polymers, the pattern of backbone chiral centers surprisingly alone alters the cleavage pattern of the target nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers effectively prevents cleavage at secondary sites. In some embodiments, the pattern of backbone chiral centers creates new cleavage sites. In some embodiments, the pattern of backbone chiral centers minimizes the number of cleavage sites. In some embodiments, the pattern of backbone chiral centers minimizes the number of cleavage sites so that the target nucleic acid polymer is cleaved at only one site within the sequence of the target nucleic acid polymer that is complementary to the oligonucleotide (e.g., cleavage at other sites cannot be easily detected by a specific method; in some embodiments, greater than 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of cleavage occurs at such sites). In some embodiments, the pattern of backbone chiral centers improves cleavage efficiency at the cleavage sites. In some embodiments, the pattern of backbone chiral centers of the oligonucleotide improves cleavage of the target nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers increases selectivity. In some embodiments, the pattern of backbone chiral centers minimizes off-target effects. In some embodiments, the pattern of backbone chiral centers increases selectivity, e.g., cleavage selectivity between two target sequences that differ only by a single nucleotide polymorphism (SNP).In some embodiments, the pattern of backbone chiral centers increases cleavage at the cleavage site of a stereorandom oligonucleotide composition or a DNA oligonucleotide composition. In some embodiments, the pattern of backbone chiral centers increases cleavage at the primary cleavage site of a stereorandom oligonucleotide composition or a DNA oligonucleotide composition. In some embodiments, such a site is the primary cleavage site of an oligonucleotide having a pattern of backbone chiral centers. In some embodiments, a site is considered a primary site if it is the site with the most, second, third, fourth, or fifth most frequent cleavage, or the site at which more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cleavage occurs. In some embodiments, the pattern of backbone chiral centers is (Sp). m (Rp) n , (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m In some embodiments, the pattern of backbone chiral centers comprises or is (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m In some embodiments, the pattern of backbone chiral centers comprises or is (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) mwherein n is 1, t>1 and m>2. In some embodiments, m>3. In some embodiments, m>4.

[0315] In some embodiments, the present disclosure recognizes that chemical modifications, such as modifications of nucleosides and internucleotide linkages, can improve properties. In some embodiments, the present disclosure demonstrates that a combination of chemical modifications and stereochemistry can result in unexpected and significantly improved properties (e.g., biological activity, selectivity, etc.). In some embodiments, the combination of chemical modifications, such as modifications of sugars, bases, and / or internucleotide linkages, can result in stereochemical patterns, e.g., (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m In some embodiments, the oligonucleotide compositions provided are chiral and comprise one or more 2'-modifications of the sugar moiety, one or more natural phosphate linkages, one or more phosphorothioate linkages, and (Rp) n (Sp) m , (Np) t (Rp) n (Sp) m or (Sp) t (Rp) n (Sp) m (wherein m>2). In some embodiments, n is 1, t>1 and m>2. In some embodiments, m>3. In some embodiments, m>4.

[0316] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers wherein the composition is a substantially pure preparation of a single oligonucleotide, wherein a predetermined level of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0317] In some embodiments, the common base sequence and length may be referred to as a common base sequence. In some embodiments, oligonucleotides having a common base sequence may have the same pattern of nucleoside modifications, such as sugar modifications, base modifications, etc. In some embodiments, the nucleoside modification pattern may be represented by a combination of positions and modifications. For example, in WV-1092, the nucleoside modification pattern is 5x2'-OMe (2'-OMe modifications to the sugar moiety)-DNA (no 2'-modification to the sugar moiety)-5x2'-OMe from the 5'-end to the 3'-end. In some embodiments, the backbone linkage pattern includes the position and type of each internucleotide linkage (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.). In some embodiments, an oligonucleotide can have a specified pattern of backbone linkages. In some embodiments, an oligonucleotide is n P.S.- n PO- n P.S.- n PO- nThe PS has a backbone linkage pattern of PS, where PO is phosphate (phosphorodiester), PS is phosphorothioate, and n is 1-15, and each occurrence of n can be the same or different. In some embodiments, at least one n is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, at least one n of PS is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the n of PS between two POs is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is greater than 5. In some embodiments, n is greater than 6. In some embodiments, n is greater than 7. In some embodiments, n is greater than 8. In some embodiments, n is greater than 9. In some embodiments, n is greater than 10. In some embodiments, n is greater than 11. In some embodiments, n is greater than 12. In some embodiments, n is greater than 13. In some embodiments, n is greater than 14. In some embodiments, n is greater than 15. In some embodiments, the oligonucleotide has a backbone linkage pattern of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS (meaning 1-5 phosphorothioates, 1-7 phosphates, 5-15 phosphorothioates, 1-7 phosphates, and 1-5 phosphorothioates). In some embodiments, the oligonucleotide is 1PS-3PO-11PS-3PO-1PS (1 phosphorothioate, 3 phosphates, 11 phosphorothioates, 3 phosphates and 1 phosphorothioate, or, this means PS1PO3PS 11PO3PS1). For example, in WV-1092, the backbone linkage pattern is 1PS-3PO-11PS-3PO-1PS from the 5'-end to the 3'-end. In some embodiments, the oligonucleotide has a backbone linkage pattern of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS (wherein each PS is Sp except for one Rp). In some embodiments, the oligonucleotide has a backbone linkage pattern of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS (wherein each PS is Sp except for one PS at any position from the 5th to the 15th PS is Rp). In some embodiments, an oligonucleotide has a backbone linkage pattern of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS (wherein each PS is Sp except for the 10th PS, counting from the 5' end, which is Rp). In some embodiments, an oligonucleotide has a backbone linkage pattern of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS (wherein each PS is Sp except for the 9th PS, counting from the 5' end, which is Rp). In some embodiments, an oligonucleotide has a backbone linkage pattern of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS (wherein each PS is Sp except for the 11th PS, counting from the 5' end, which is Rp). The pattern of backbone chiral centers of an oligonucleotide can be specified by the combination of 5' to 3' bond phosphorus stereochemistry (Rp / Sp). For example, WV-1092 has a 1S-3PO(phosphate)-8S-1R-2S-3PO-1S pattern, and WV-937 has a 12S-1R-6S pattern. In some embodiments, when describing the pattern of backbone chiral centers, all non-chiral bonds (e.g., PO) may be omitted. As exemplified above, the position of the non-chiral bond may be derived, for example, from the pattern of backbone bonds. Any sequence disclosed herein can be combined with any pattern of backbone bonds and / or any pattern of backbone chiral centers disclosed herein.Base sequences, backbone bond patterns, stereochemical patterns (eg, Rp or Sp), base modification patterns, backbone chiral center patterns, etc. are shown in the 5' to 3' direction unless otherwise noted.

[0318] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; wherein the composition is chirally controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0319] An exemplary substantially racemic preparation of oligonucleotides is a phosphorothioate oligonucleotide prepared by a process well known in the art, which involves sulfurizing a phosphite triester using either tetraethylthiuram disulfide (TETD) or 3H-1,2-bensodithiol-3-one 1,1-dioxide (BDTD) in the commonly used phosphoramidite oligonucleotide synthesis. In some embodiments, a substantially racemic preparation of oligonucleotides results in a substantially racemic oligonucleotide composition (or an oligonucleotide composition without chiral control).

[0320] As will be understood by those skilled in the art, stereorandom or racemic preparations of oligonucleotides are typically prepared by non-stereoselective and / or low-stereoselective coupling of nucleotide monomers without the use of any chiral auxiliary, chiral modifying reagent, and / or chiral catalyst. In some embodiments, in a substantially racemic (or non-chiral controlled) preparation of oligonucleotides, all or most of the coupling steps are not chirally controlled, in that the coupling steps are not specifically performed to enhance stereoselectivity. An example of a substantially racemic preparation of oligonucleotides is a phosphorothioate oligonucleotide prepared by a process well known in the art, in which a phosphite triester is sulfurized using either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) in commonly used phosphoramidite oligonucleotide synthesis. In some embodiments, a substantially racemic preparation of oligonucleotides results in a substantially racemic oligonucleotide composition (or a non-chirally controlled oligonucleotide composition). In some embodiments, at least one coupling of nucleotide monomers has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least two couplings of nucleotide monomers have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least three couplings of nucleotide monomers have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1.In some embodiments, the coupling of at least four of the nucleotide monomers has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, the coupling of at least five of the nucleotide monomers has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least one internucleotide linkage has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least two internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least three internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least four internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a stereorandom or racemic preparation, at least five internucleotide linkages have a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 60:40.In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 70:30. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 80:20. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 91:9. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 92:8. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 93:7. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 94:6. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 95:5. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 96:4. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 97:3. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 98:2. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 99:1. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 91:9. In some embodiments, at least one coupling has a diastereoselectivity of less than about 90:10. In some embodiments, at least two couplings have a diastereoselectivity of less than about 90:10. In some embodiments, at least three couplings have a diastereoselectivity of less than about 90:10. In some embodiments, at least four couplings have a diastereoselectivity of less than about 90:10.In some embodiments, at least five couplings have a diastereoselectivity of less than about 90:10. In some embodiments, at least one internucleotide linkage has a diastereoselectivity of less than about 90:10. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least three internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least four internucleotide linkages have a diastereoselectivity of less than about 90:10. In some embodiments, at least five internucleotide linkages have a diastereoselectivity of less than about 90:10.

[0321] As will be appreciated by those skilled in the art, in some embodiments, the diastereoselectivity of coupling or conjugation can be assessed by the diastereoselectivity of dimer formation under the same or equivalent conditions, where the dimers have the same 5'- and 3'-nucleosides and internucleotide linkages. For example, WV-1092 mG * SmGmCmAmC * SA * SA * S G * SG * SG * SC * SA * SC * RA * SG * SmAmCmUmU * The diastereoselectivity of the underlined coupling or bond in SmC can be assessed by coupling two G moieties under the same or equivalent conditions (e.g., monomers, chiral auxiliaries, solvents, activators, temperature, etc.).

[0322] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers wherein the composition is a substantially pure preparation of a single oligonucleotide, wherein at least about 10% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0323] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein the compositions are enriched for oligonucleotides of a single oligonucleotide type, relative to a substantially racemic preparation of the same oligonucleotide. In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein the compositions are enriched for oligonucleotides of a single oligonucleotide type, relative to a substantially racemic preparation of the same oligonucleotide: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers The present invention provides chiral controlled oligonucleotide compositions of oligonucleotides in which the compositions are enriched for oligonucleotides of a single oligonucleotide type that share a common nucleotide sequence.

[0324] In some embodiments, the present disclosure provides: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) common patterns of backbone chiral centers; wherein the composition is chirally controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0325] In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have the same structure.

[0326] In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides of an oligonucleotide type are identical.

[0327] In some embodiments, the chiral controlled oligonucleotide composition is a substantially pure preparation of an oligonucleotide type, wherein any oligonucleotides in the composition that are not of the oligonucleotide type are impurities form the preparation process of said oligonucleotide type (optionally after certain purification procedures).

[0328] In some embodiments, at least about 20% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 25% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 30% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 35% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 40% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 45% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 50% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 55% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 60% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 65% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 70% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, at least about 75% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 80% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 85% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 90% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 92% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 94% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 95% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, more than about 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, the purity of the chiral-controlled oligonucleotide composition of oligonucleotides can be expressed as the percentage of oligonucleotides in the composition that have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0329] In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are identical.

[0330] In some embodiments, the oligonucleotides in the provided compositions have a common pattern of backbone phosphorus modifications. In some embodiments, the common base sequence is a base sequence of an oligonucleotide type. In some embodiments, the provided compositions are chiral controlled oligonucleotide compositions in that they contain predetermined levels of oligonucleotides of individual oligonucleotide types, where the oligonucleotide types are: 1) Nucleotide sequence; 2) pattern of skeletal bonding; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification is defined as:

[0331] As noted above and understood in the art, in some embodiments, the base sequence of an oligonucleotide may refer to the identity and / or modification state of the nucleoside residues (e.g., sugar and / or base moieties, relative to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) within the oligonucleotide, and / or the hybridization properties of such residues (i.e., the ability to hybridize with specific complementary residues).

[0332] In some embodiments, a particular oligonucleotide type is 1A) base identity; 1B) base modification patterns; 1C) Glycosylation patterns; 2) pattern of skeletal bonding; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification It may be defined by: Thus, in some embodiments, oligonucleotides of a particular type may share the same bases but differ in their patterns of base and / or sugar modifications, hi some embodiments, oligonucleotides of a particular type may share the same bases and patterns of base modifications (including, for example, the absence of base modifications), but differ in their patterns of sugar modifications.

[0333] In some embodiments, oligonucleotides of a particular type have the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same pattern of backbone linkages (e.g., natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., the pattern of chiral internucleotide linkage stereochemistry (Rp / Sp)), and the same pattern of backbone phosphorus modifications (e.g., -S - and -LR of formula I 1They are identical in that they have a pattern of internucleotide modifications to the phosphorus atom, such as α- and β-nucleotides.

[0334] In some embodiments, the purity of a chiral controlled oligonucleotide composition of an oligonucleotide type is expressed as the percentage of oligonucleotides in the composition that are of that oligonucleotide type. In some embodiments, at least about 10% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 20% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 30% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 40% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 50% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 60% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 70% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 80% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 90% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 92% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type, hi some embodiments, at least about 94% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type.In some embodiments, at least about 95% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 96% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 97% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 98% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 99% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type.

[0335] In some embodiments, the purity of a chiral controlled oligonucleotide composition can be controlled by the stereoselectivity of each coupling step in its preparation process. In some embodiments, the coupling step has 60% stereoselectivity (e.g., diastereoselectivity) (60% of the new internucleotide linkages formed by the coupling step have the desired stereochemistry). After such a coupling step, the new internucleotide linkages formed may be said to have 60% purity. In some embodiments, each coupling step has at least 60% stereoselectivity. In some embodiments, each coupling step has at least 70% stereoselectivity. In some embodiments, each coupling step has at least 80% stereoselectivity. In some embodiments, each coupling step has at least 85% stereoselectivity. In some embodiments, each coupling step has at least 90% stereoselectivity. In some embodiments, each coupling step has at least 91% stereoselectivity. In some embodiments, each coupling step has at least 92% stereoselectivity. In some embodiments, each coupling step has at least 93% stereoselectivity. In some embodiments, each coupling step has at least 94% stereoselectivity. In some embodiments, each coupling step has at least 95% stereoselectivity. In some embodiments, each coupling step has at least 96% stereoselectivity. In some embodiments, each coupling step has at least 97% stereoselectivity. In some embodiments, each coupling step has at least 98% stereoselectivity. In some embodiments, each coupling step has at least 99% stereoselectivity. In some embodiments, each coupling step has substantially 100% stereoselectivity. In some embodiments, the coupling step has substantially 100% stereoselectivity, wherein all products of the coupling step, detectable by analytical methods (e.g., NMR, HPLC, etc.), have the desired stereoselectivity.

[0336] Among other things, the present disclosure recognizes that combinations of oligonucleotide building blocks (e.g., patterns of chemical modifications, backbone linkages, backbone chiral centers and / or backbone phosphorus modifications) can confer properties such as surprisingly improved biological activity.

[0337] In some embodiments, the present disclosure provides oligonucleotide compositions comprising predetermined levels of oligonucleotides comprising one or more wing regions and a common core region, wherein: each wing region independently has a length of two or more bases and independently optionally contains one or more chiral internucleotide linkages; The core regions independently have a length of two or more bases and independently contain one or more chiral internucleotide linkages, and the common core region comprises: 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers It has.

[0338] In some embodiments, the wing regions comprise structural features not present in the core region. In some embodiments, the wings and core can be defined by any component, such as base modification (e.g., methylated / unmethylated, 1-methylated / 2-methylated, etc.), sugar modification (e.g., modified / unmodified, 2'-modified / another type of modification, one type of 2'-modification / another type of 2'-modification, etc.), backbone linkage type (e.g., phosphate / phosphorothioate, phosphorothioate / substituted phosphorothioate, etc.), backbone chiral center stereochemistry (e.g., all Sp / all Rp, (SpRp) repeat / all Rp, etc.), backbone phosphorus modification type (e.g., s1 / s2, s1 / s3, etc.), etc.

[0339] In some embodiments, the wings and core are defined by nucleoside modifications, where the wings include nucleoside modifications that the core region does not have. In some embodiments, the wings and core are defined by sugar modifications, where the wings include sugar modifications that the core region does not have. 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 example sugar modifications are described in this disclosure.

[0340] In some embodiments, the oligonucleotides in the provided compositions have a wing-core structure (hemimer). In some embodiments, the oligonucleotides in the provided compositions have a wing-core structure with nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a core-wing structure (another type of hemimer). In some embodiments, the oligonucleotides in the provided compositions have a core-wing structure with nucleoside modifications. In some embodiments, the oligonucleotides in the provided compositions have a wing-core-wing structure (gapmer). In some embodiments, the oligonucleotides in the provided compositions have a wing-core-wing structure with nucleoside modifications. In some embodiments, the wings and core are defined by modifications of the sugar moieties. In some embodiments, the wings and core are defined by modifications of the base moieties. In some embodiments, each sugar moiety in the wing region has the same 2'-modification that is not found in the core region. In some embodiments, each sugar moiety in the wing region has the same 2'-modification that is different from any sugar modification in the core region. In some embodiments, the core region has no sugar modifications. In some embodiments, each sugar moiety in the wing region has the same 2'-modification, and the core region has no 2'-modification. In some embodiments, when more than one wing is present, each wing is defined by its own modification. In some embodiments, each wing has its own characteristic sugar modification. In some embodiments, each wing has the same characteristic sugar modification that distinguishes it from the core. In some embodiments, each wing sugar moiety has the same modification. In some embodiments, each wing sugar moiety has the same 2'-modification. In some embodiments, each sugar moiety within a wing region has the same 2'-modification, but the common 2'-modification within a first wing region can be the same or different from the common 2'-modification within a second wing region. In some embodiments, each sugar moiety within a wing region has the same 2'-modification, and the common 2'-modification within a first wing region is the same as the common 2'-modification within a second wing region.In some embodiments, each sugar moiety within a wing region has the same 2'-modification, and the common 2'-modification within a first wing region is different from the common 2'-modification within a second wing region.

[0341] In some embodiments, the chirality-controlled (and / or stereochemically pure) preparations provided are antisense oligonucleotides (e.g., chiromersen). In some embodiments, the chirality-controlled (and / or stereochemically pure) preparations provided are siRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions provided are of oligonucleotides that may be antisense oligonucleotides, antagomirs, microRNAs, pre-microRNs, anti-mirs, supermirs, ribozymes, Ul adapters, RNA activators, RNAi agents, decoy oligonucleotides, triplex-forming oligonucleotides, aptamers, or adjuvants. In some embodiments, the chirality-controlled oligonucleotide compositions are of antisense oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of antagomir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of microRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of pre-microRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of anti-mir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of supermir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide composition is a ribozyme oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is a Ul adaptor oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an RNA activator oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an RNAi agent oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is a decoy oligonucleotide.In some embodiments, the chirality-controlled oligonucleotide composition is a triplex-forming oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an aptamer oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an adjuvant oligonucleotide.

[0342] In some embodiments, chirally controlled (and / or stereochemically pure) preparations provided are of oligonucleotides containing one or more modified backbone linkages, bases, and / or sugars.

[0343] In some embodiments, provided oligonucleotides contain one or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain two or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain three or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain four or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain five or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain five or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain six or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 7 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 8 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 9 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 10 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 11 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 12 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 13 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 14 or more chiral modified phosphate linkages.In some embodiments, the oligonucleotide types provided contain 15 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 16 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 17 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 18 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 19 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 20 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 21 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 22 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 23 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 24 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 25 or more chiral modified phosphate linkages.

[0344] In some embodiments, provided oligonucleotides contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral modified phosphate linkages. Exemplary such chiral modified phosphate linkages are described above and herein. In some embodiments, provided oligonucleotides contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral modified phosphate linkages in the Sp configuration.

[0345] In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 80% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 85% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 90% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 91% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 92% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 93% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 94% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 95% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 96% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 97% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 98% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 99% stereochemical purity.

[0346] In some embodiments, the chiral modified phosphate linkage is a chiral phosphorothioate linkage, i.e., a phosphorothioate internucleotide linkage. In some embodiments, the provided oligonucleotide contains at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral phosphorothioate internucleotide linkages. In some embodiments, all chiral modified phosphate linkages are chiral phosphorothioate internucleotide linkages. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide linkages of the provided oligonucleotide are in the Sp configuration. In some embodiments, at least about 10% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 20% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 30% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 40% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 50% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 60% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 70% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 80% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Sp configuration.In some embodiments, at least about 90% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 95% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 10% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 20% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 30% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 40% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 50% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 60% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 70% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 80% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 90% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 95% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration.In some embodiments, less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 10% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 20% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 30% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 40% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 50% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 60% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 70% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 80% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 90% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 95% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, a provided oligonucleotide has only one Rp chiral phosphorothioate internucleotide linkage. In some embodiments, a provided oligonucleotide has only one Rp chiral phosphorothioate internucleotide linkage, where all internucleotide linkages are chiral phosphorothioate internucleotide linkages.In some embodiments, the chiral phosphorothioate internucleotide linkages are chiral phosphorothioate diester linkages. In some embodiments, the chiral phosphorothioate internucleotide linkages are each independently a chiral phosphorothioate diester linkage. In some embodiments, the internucleotide linkages are each independently a chiral phosphorothioate diester linkage. In some embodiments, the internucleotide linkages are each independently a chiral phosphorothioate diester linkage and only one linkage is Rp.

[0347] In some embodiments, the chiral (and / or stereochemically pure) preparations provided are of oligonucleotides containing one or more modified bases. In some embodiments, the chiral (and / or stereochemically pure) preparations provided are of oligonucleotides that do not contain modified bases. Exemplary such modified bases are described above and herein.

[0348] In some embodiments, the oligonucleotides of the provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 1 natural phosphate linkage. In some embodiments, the oligonucleotides of the provided compositions comprise at least 2 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 3 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 4 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 5 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 6 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 7 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 8 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 9 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions contain at least 10 natural phosphate linkages.

[0349] In some embodiments, the oligonucleotides of the provided compositions contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise one natural phosphate linkage. In some embodiments, the oligonucleotides of the provided compositions comprise two natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise three natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise four natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise five natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise six natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise seven natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise eight natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise nine natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise ten natural phosphate linkages.

[0350] In some embodiments, the oligonucleotides of the provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 2 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 3 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 4 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 5 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 6 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 7 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 8 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise at least 9 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions contain at least 10 consecutive natural phosphate linkages.

[0351] In some embodiments, the oligonucleotides of the provided compositions comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 2 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 3 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 4 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 5 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 6 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 7 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 8 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 9 consecutive natural phosphate linkages. In some embodiments, the oligonucleotides of the provided compositions comprise 10 consecutive natural phosphate linkages.

[0352] In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 8 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 9 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 10 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 11 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 12 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 13 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 14 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 15 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 16 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 17 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 18 bases.In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 19 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 20 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 21 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 22 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 23 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 24 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 25 bases. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of oligonucleotides having a common base sequence of at least 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 bases.

[0353] In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides containing one or more residues modified at the sugar moiety. In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides containing one or more residues modified at the 2'-position of the sugar moiety (referred to herein as "2'-modified"). Examples of such modifications are described above and herein and include, but are not limited to, 2'-OMe, 2'-MOE, 2'-LNA, 2'-F, FRNA, FANA, S-cEt, and the like. In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides containing one or more residues that are 2'-modified. For example, in some embodiments, provided oligonucleotides contain one or more residues that are 2'-O-methoxyethyl (2'-MOE)-modified residues. In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides that do not contain any 2'-modifications. In some embodiments, the chiral (and / or stereochemically pure) preparations provided are oligonucleotides that do not contain any 2'-MOE residues. That is, in some embodiments, the provided oligonucleotides are not MOE-modified. Other exemplary sugar modifications are described in this disclosure.

[0354] In some embodiments, provided oligonucleotides are of the general motif of wing-core or core-wing (hemimers, also generally referred to herein as XY or YX, respectively). In some embodiments, provided oligonucleotides are of the general motif of wing-core-wing (gapmers, also generally referred to herein as XYX). ​​In some embodiments, each wing independently comprises one or more residues with a particular modification that is absent from the core "Y" portion. In some embodiments, each wing independently comprises one or more residues with a particular nucleoside modification that is absent from the core "Y" portion. In some embodiments, each wing independently comprises one or more residues with a particular base modification that is absent from the core "Y" portion. In some embodiments, each wing independently comprises one or more residues with a particular sugar modification that is absent from the core "Y" portion. Example sugar modifications are well known in the art. In some embodiments, the sugar modification is a modification selected from those described in U.S. Pat. No. 9,006,198, which are incorporated herein by reference. Other exemplary sugar modifications are described in this disclosure. In some embodiments, each wing comprises one or more residues with a 2'-modification that is not present in the core moiety. In some embodiments, the 2'-modification is a 2'-OR. 1 (In the formula, R 1 are as defined and described in this disclosure.

[0355] In some embodiments, provided oligonucleotides have a wing-core motif designated as XY, or a core-wing motif designated as YX, where the residues in the "X" portion are sugar-modified residues of a particular type and the residues in the core "Y" portion are not sugar-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core-wing motif designated as XYX, where the residues in each "X" portion are sugar-modified residues of a particular type and the residues in the core "Y" portion are not sugar-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core motif designated as XY, or a core-wing motif designated as YX, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core motif designated as XY, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a core-wing motif designated YX, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core-wing motif designated XYX, where the residues in each "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are not 2'-modified residues of the same particular type. In some embodiments, provided oligonucleotides have a wing-core motif designated XY, where the residues in the "X" portion are 2'-modified residues of a particular type and the residues in the core "Y" portion are 2'-deoxyribonucleosides. In some embodiments, provided oligonucleotides have a core-wing motif designated YX, where the residues in the "X" portion are 2'-modified residues of a particular type and the resi...

Claims

[Claim 1] 1) consensus sequence and length; 2) a common pattern of backbone bonds; and 3) common patterns of backbone chiral centers; A chiral controlled oligonucleotide composition comprising an oligonucleotide of a specific oligonucleotide type characterized by: The composition is chiral controlled in that it is enriched for oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, the oligonucleotide targets a mutant huntingtin gene, and the length is from about 10 to about 50 nucleotides, the backbone linkages include at least one phosphorothioate, and the pattern of backbone chiral centers includes at least one Rp chiral center and at least one Sp chiral center. composition.

Images