[0001] This application claims priority to United States Provisional 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 entirety of each of which is incorporated herein by reference.

Background

[0002] Oligonucleotides are useful in therapeutic, diagnostic, research and nanomaterials applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) for therapeutics can be limited, for example, because of their instability against extra- and intracellular nucleases and/or their poor cell penetration and distribution. There is a need for new and improved oligonucleotides and oligonucleotide compositions, such as, e.g., new antisense and siRNA oligonucleotides and oligonucleotide compositions.

Summary

[0003] Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugar, base, and/or internucleotidic linkages, and patterns thereof), and/or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotidic linkages), and/or patterns thereof), can have significant impact on properties, e.g., activities, of oligonucleotides. In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements, e.g., controlled chemical modification and/or controlled backbone stereochemistry patterns, provide unexpected properties, including but not limited to those described herein. In some embodiments, the present disclosure demonstrates that combinations of chemical modifications and stereochemistry can provide unexpected, greatly improved properties (e.g., bioactivity, selectivity, etc.). In some embodiments, the present disclosure provides an oligonucleotide composition having a particular sequence of bases, and/or pattern of sugar modifications (e.g., 2’-OMe, 2’-F, 2’-MOE, etc.), and/or pattern or base modifications (e.g., 5-methylcytosine), and/or pattern of backbone modifications (phosphate or phosphorothioate), and/or pattern of

stereochemistry of backbone modifications (e.g., each phosphorothioate is Sp or Rp).

[0004] In some embodiments, modifications of internucleotidic linkages can convert phosphorus atoms in modified linkages into chiral centers. For example, in a phosphorothioate (PS) modification, one of the non-bridging oxygen (O) atoms bonded to a phosphorus (P) atom is replaced with a sulfur (S) atom. A consequence of using PS modification in oligonucleotide synthesis is that it creates a chiral center at phosphorus, which can have either an“Sp” or“Rp” configuration. For instance, a conventional stereorandom PS-modified oligonucleotide composition having 19 PS linkages [e.g., having 20 nucleotides in length, 19 PS modifications, each with two possible stereochemistries (Sp or Rp) at each PS modification] is a random mixture of over 500,000 (219) stereoisomers, each having the same nucleotide sequence (e.g., sequence of bases) but differing in the stereochemistry along their backbones; such a composition is a“stereorandom” oligonucleotide composition. In some embodiments, in contrast to stereorandom compositions, a chirally controlled oligonucleotide composition is a substantially pure preparation of a single oligonucleotide in that 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. In some embodiments, some oligonucleotide compositions are stereopure (i.e., a chirally controlled oligonucleotide composition), wherein the stereochemistry at each PS is defined (Sp or Rp). In some embodiments, in a stereorandom compositions of oligonucleotides, the various oligonucleotides can have the same base sequence, same pattern of sugar modifications (e.g., 2’-OMe, 2’-F, 2’-OME, etc.), same pattern of base modifications (e.g., 5-methylcytosine), and same pattern of backbone modifications (phosphate or PS), but different patterns of backbone chiral centers, and their levels are random from non-stereocontrolled synthesis (not pre-determined as through stereocontrolled synthesis as certain methods exemplified herein using chiral auxilier). A chirally controlled oligonucleotide composition can be selected to have greater desired biological activity (e.g., greater activities, efficiency in RNA interference or RNAse H-mediated pathways, etc.) and decreased undesired activity (e.g., undesired immunogenicity, toxicity, etc.) than a stereorandom preparation of oligonucleotides of the same base sequence. In some embodiments, a chirally controlled oligonucleotide composition is better able to differentiate between a mutant (mu) and a wild-type (wt) HTT sequence (with a single nt difference).

[0005] Among other things, the present disclosure encompasses the recognition that

stereorandom oligonucleotide preparations contain a plurality of distinct chemical entities that differ from one another, e.g., in the stereochemical structure of individual backbone chiral centers within the oligonucleotide chain. Without control of stereochemistry of backbone chiral centers, stereorandom oligonucleotide preparations provide uncontrolled compositions comprising undetermined levels of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence, they are different chemical entities at least due to their different backbone stereochemistry, and they can have, as demonstrated herein, different properties, e.g., bioactivities. Among other things, the present disclosure provides new compositions that are or contain particular stereoisomers of oligonucleotides of interest. In some embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its length, its pattern of backbone linkages, and its pattern of backbone chiral centers. As is understood in the art, in some embodiments, base sequence may refer to the identity and/or modification status of nucleoside residues (e.g., of sugar and/or base components, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) in an oligonucleotide and/or to the hybridization character (i.e., the ability to hybridize with particular complementary residues) of such residues.

[0006] The present disclosure demonstrates, among other things, that individual stereoisomers of a particular oligonucleotide can show different stability and/or activity (e.g., functional and/or toxicity properties) from each other. Moreover, the present disclosure demonstrates that stability and/or activity improvements achieved through inclusion and/or location of particular chiral structures within an oligonucleotide can be comparable to, or even better than those achieved through use of particular backbone linkages, residue modifications, etc. (e.g., through use of certain types of modified phosphates [e.g., phosphorothioate, substituted phosphorothioate, etc.], sugar modifications [e.g., 2’- modifications, etc.], and/or base modifications [e.g., methylation, etc.]).

[0007] Among other things, the present disclosure recognizes that, in some embodiments, properties (e.g., stability and/or activities) of an oligonucleotide can be adjusted by optimizing its pattern of backbone chiral centers, optionally in combination with adjustment/optimization of one or more other features (e.g., linkage pattern, nucleoside modification pattern, etc.) of the oligonucleotide.

[0008] In some embodiments, the present disclosure provides compositions of

oligonucleotides, wherein the oligonucleotides have a common pattern of backbone chiral centers which, unexpectedly, greatly enhances the stability and/or biological activity of the oligonucleotides. In some embodiments, a pattern of backbone chiral centers provides increased stability. In some embodiments, a pattern of backbone chiral centers provides surprisingly increased activity. In some embodiments, a pattern of backbone chiral centers provides increased stability and activity. In some embodiments, when an oligonucleotide is utilized to cleave a nucleic acid polymer, a pattern of backbone chiral centers, surprisingly by itself, changes the cleavage pattern of a target nucleic acid polymer. In some embodiments, a pattern of backbone chiral centers effectively prevents cleavage at secondary sites. In some embodiments, a pattern of backbone chiral centers creates new cleavage sites. In some embodiments, a pattern of backbone chiral centers minimizes the number of cleavage sites. In some embodiments, a pattern of backbone chiral centers minimizes the number of cleavage sites so that a 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, a pattern of backbone chiral centers enhances cleavage efficiency at a cleavage site. In some embodiments, a pattern of backbone chiral centers of the oligonucleotide improves cleavage of a target nucleic acid polymer. In some embodiments, a pattern of backbone chiral centers increases selectivity. In some embodiments, a pattern of backbone chiral centers minimizes off-target effect. In some embodiments, a pattern of backbone chiral centers increase selectivity, e.g., cleavage selectivity between two target sequences differing only by a single nucleotide polymorphism (SNP). In some embodiments, a pattern of backbone chiral centers comprises, comprises one or more repeats of, 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 described herein, m is 1-50; and n is 1-10; and t is 1-50. In some embodiments, a 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, a 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, wherein m > 2. In some embodiments, a 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, a pattern of backbone chiral centers is a sequence comprising at least 5 consecutive (Sp) positions. In some embodiments, a pattern of backbone chiral centers is a sequence comprising at least 8 consecutive (Sp) positions. In some embodiments, a pattern of backbone chiral centers is a

sequence comprising at least 10 consecutive (Sp) positions. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp). In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp) at or adjacent to the position of a SNP. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein the wing on the 5’ end is 1-9 nt long, the core is 1-15 nt long, and the wing on the 3’ end is 1-9 nt long. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein the wing on the 5’ end is 5 nt long, the core is 1-15 nt long, and the wing on the 3’ end is 5 nt long. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein the wing on the 5’ end is 1-9 nt long, the core is 10 nt long, and the wing on the 3’ end is 1-9 nt long. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein the wing on the 5’ end is 5 nt long, the core is 10 nt long, and the wing on the 3’ end is 5 nt long. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein the wing on the 5’ end is 5 nt long, the core is 10 nt long, and the wing on the 3’ end is 5 nt long, and at least one wing comprises a nucleotide with a 2’-OMe modification. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein each wing comprises at least one nucleotide with a 2’-OMe modification. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein each nucleotide in both wings has a 2’-OMe modification. In some embodiments, a pattern of backbone chiral centers is a sequence consisting of all (Sp) with a single (Rp), wherein the molecule has a wing-core-wing format, wherein the wing on the 5’ end is 5 nt long, the core is 10 nt long, and the wing on the 3’ end is 5 nt long, and each nucleotide in each wing has a 2’-OMe modification. In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing format, wherein the wing on the 5' end of the molecule

comprises 4 to 8 nt, each of which has a 2'-OMe modification and wherein the nt at the 5' end of the molecule has a phosphorothioate in the Sp conformation; the core comprises 8 to 12 nt, each of which is DNA (2'-H), wherein each has a phosphorothioate in the Sp position except one nt which has the phosphorothioate in the Rp position; and wherein the wing on the 3' end of the molecule comprises 4 to 8 nt, each of which has a 2'-OMe modification, and wherein the nt at the 3' end of the molecule comprises a phosphorothioate in the Sp conformation. In some embodiments, the oligonucleotide is single-stranded and has a wing-core-wing format, wherein the wing on the 5' end of the molecule comprises 6 nt, each of which has a 2'-OMe modification and wherein the nt at the 5' end of the molecule has a phosphorothioate in the Sp conformation; the core comprises 10 nt, each of which is DNA (2'-H), wherein each has a phosphorothioate in the Sp position except one nt which has the phosphorothioate in the Rp position; and wherein the wing on the 3' end of the molecule comprises 6 nt, each of which has a 2'-OMe modification, and wherein the nt at the 3' end of the molecule comprises a phosphorothioate in the Sp conformation.

[0009] In some embodiments, the present disclosure recognizes that chemical modifications, such as modifications of nucleosides and internucleotidic linkages, can provide enhanced properties. In some embodiments, the present disclosure demonstrates that combinations of chemical modifications and stereochemistry can provide unexpected, greatly improved properties (e.g., bioactivity, selectivity, etc.). In some embodiments, chemical combinations, such as modifications of sugars, bases, and/or internucleotidic linkages, are combined with stereochemistry patterns, e.g., (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, to provide oligonucleotides and compositions thereof with surprisingly enhanced properties. In some embodiments, a provided oligonucleotide composition is chirally controlled, and comprises a combination of 2’-modification of one or more sugar moieties, one or more natural phosphate linkages, one or more phosphorothioate linkages, and a stereochemistry pattern of (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 a chirally controlled oligonucleotide composition comprising oligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that a predetermined level of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

[0011] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type.

[0012] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that at least about 10% of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

[0013] Among other things, the present disclosure recognizes that combinations of oligonucleotide structural elements (e.g., patterns of chemical modifications, backbone linkages, backbone chiral centers, and/or backbone phosphorus modifications) can provide surprisingly improved properties such as bioactivities. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a predetermined level of oligonucleotides which comprise 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 and optionally comprises one or more chiral internucleotidic linkages;

the core region independently has a length of two or more bases, and independently comprises one or more chiral internucleotidic linkages, and the common core region has:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

[0014] In some embodiments, in an oligonucleotide comprising a wing-core-wing format, a“wing” is a portion of the oligonucleotide on the 5’ or 3’ end of the core, with the “core” (alternatively designated a“gap”) between the two wings. In some embodiments, an oligonucleotide can have a single wing and a single core; in such cases, the wing is on the 5’ or the 3’ end of the oligonucleotide. A wing and core can be defined by any of several structural elements (e.g., modifications or patterns of modifications of sugar, base, backbone or backbone stereochemistry, etc.). In some embodiments, a wing and core is defined by nucleoside modifications, wherein a wing comprises a nucleoside modification that the core region does not have. In some embodiments, oligonucleotides in provided compositions have a wing-core structure of nucleoside modification. In some embodiments, oligonucleotides in provided compositions have a core-wing structure of nucleoside modification. In some embodiments, oligonucleotides in provided compositions have a wing-core-wing structure of nucleoside modification. In some embodiments, a wing and core is defined by modifications of the sugar moieties. In some embodiments, a wing and core is defined by modifications of the base moieties. In some embodiments, each sugar moiety in the wing region has the same 2’-modification which is not found in the core region. In some embodiments, each sugar moiety in the wing region has the same 2’-modification which is different than any sugar modifications in the core region. In some embodiments, each sugar moiety in the wing region has the same 2’-modification, and the core region has no 2’-modifications. In some embodiments, when two or more wings are present, each sugar moiety in a wing region has the same 2’-modification, yet the common 2’-modification in a first wing region can either 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 internucleotidic 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, a wing sugar moiety is modified by a modification that is absent from the core region. In some embodiments, a wing region only has modified internucleotidic linkages at one or both of its ends. In some embodiments, a wing region only has a modified internucleotidic linkage at its 5’-end. In some embodiments, a wing region only has a modified internucleotidic linkage at its 3’-end. In some embodiments, a wing region only has modified internucleotidic linkages at its 5’- and 3’-ends. In some embodiments, a wing is to the 5’-end of a core, and the wing only has a modified internucleotidic linkage at its 5’-end. In some embodiments, a wing is to the 5’-end of a core, and the wing only has a modified internucleotidic linkage at its 3’-end. In some embodiments, a wing is to the 5’-end of a core, and the wing only has modified internucleotidic linkages at both its 5’- and 3’-ends. In some embodiments, a wing is to the 3’-end of a core, and the wing only has a modified internucleotidic linkage at its 5’-end. In some embodiments, a wing is to the 3’-end of a core, and the wing only has a modified internucleotidic linkage at its 3’-end. In some embodiments, a wing is to the 3’-end of a core, and the wing only has modified internucleotidic linkages at both its 5’- and 3’-ends. In some embodiments, the modification(s) to the sugar moiety or internucleotidic linkage or other modifications in one wing can differ from those in another wing.

[0016] In some embodiments, each internucleotidic linkage within a core region is modified. In some embodiments, each internucleotidic linkage within a core region is chiral. In some embodiments, a core region has a pattern of backbone chiral centers of (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, a 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, wherein m > 2. Among other things, the present disclosure demonstrates that, in some embodiments, such patterns can provide or enhance controlled cleavage of a target sequence, e.g., an RNA sequence.

[0017] In some embodiments, oligonucleotides in provided compositions have a common pattern of backbone phosphorus modifications. In some embodiments, a provided composition is an oligonucleotide composition that is chirally controlled in that the composition contains a predetermined level of oligonucleotides of an individual oligonucleotide type, wherein an oligonucleotide type is defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

[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 status of nucleoside residues (e.g., of sugar and/or base components, relative to standard naturally occurring

nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) in the oligonucleotide and/or to the hybridization character (i.e., the ability to hybridize with particular complementary residues) of such residues.

[0019] In some embodiments, a particular oligonucleotide type may be defined by

1A) base identity;

1B) pattern of base modification;

1C) pattern of sugar modification;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

Thus, in some embodiments, oligonucleotides of a particular type may share identical bases but differ in their pattern of base modifications and/or sugar modifications. In some embodiments, oligonucleotides of a particular type may share identical bases and pattern of base modifications (including, e.g., absence of base modification), but differ in pattern of sugar modifications.

[0020] In some embodiments, oligonucleotides of a particular type are chemically identical in that they have the same base sequence (including length), the same pattern of chemical modifications to sugar and base moieties, the same pattern of backbone linkages (e.g., pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., pattern of stereochemistry (Rp/Sp) of chiral internucleotidic linkages), and the same pattern of backbone phosphorus modifications (e.g., pattern of modifications on the internucleotidic phosphorus atom, such as -Sí, and -L-R1 of formula I).

[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 Tables N1, N2, N3, N4 and 8. In some embodiments, the sequence of the oligonucleotide comprises or consists of the sequence of any oligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8. In some embodiments, the sequence of the oligonucleotide in a stereopure (chirally controlled) oligonucleotide composition comprises or consists of the sequence of WV-1092, WVE120101, WV-2603 or WV-2595. In some embodiments, a sequence of an oligonucleotide includes any one or more of: base sequence (including length); pattern of

chemical modifications to sugar and base moieties; pattern of backbone linkages; pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof; pattern of backbone chiral centers; pattern of stereochemistry (Rp/Sp) of chiral internucleotidic linkages; pattern of backbone phosphorus modifications; pattern of modifications on the internucleotidic phosphorus atom, such as -Sí, and -L-R1 of formula I.

[0022] Among other things, the present disclosure recognizes the challenge of stereoselective (rather than stereorandom or racemic) preparation of oligonucleotides. Among other things, the present disclosure provides methods and reagents for stereoselective preparation of oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotidic linkages, and particularly for oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotidic linkages. In some embodiments, in a stereorandom or racemic preparation of oligonucleotides, at least one chiral internucleotidic linkage is formed with less than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 95:5 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 96:4 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 97:3 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 99:1 diastereoselectivity. In some embodiments, diastereoselectivity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5’-nucleoside of the dimer is the same as the nucleoside to the 5’-end of the chiral internucleotidic linkage, and the 3’-nucleoside of the dimer is the same as the nucleoside to the 3’-end of the chiral internucleotidic linkage.

[0023] Among other things, it is surprisingly found that certain provided oligonucleotide compositions achieve unprecedented control of cleavage of target sequences, e.g., cleavage of target RNA by RNase H. In some embodiments, the present disclosure demonstrates that precise control of chemical and stereochemical attributes of oligonucleotides achieves improved activity of oligonucleotide preparations as compared with otherwise comparable preparations for which stereochemical attributes are not controlled. Among other things, the present disclosure specifically demonstrates improved rate, degree, and or specificity of cleavage of nucleic acid targets to which provided oligonucleotides hybridize.

[0024] In some embodiments, the present disclosure provides various uses of oligonucleotide compositions. Among other things, the present disclosure demonstrates that by controlling structural elements of oligonucleotides, such as base sequence, chemical modifications, stereochemistry, etc., properties of oligonucleotides can be greatly improved. For example, in some embodiments, the present disclosure provides methods for highly selective suppression of transcripts of a target nucleic acid sequence. In some embodiments, the present disclosure provides methods for treating a subject by suppressing transcripts 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 enhanced activity and/or selectivity when suppressing a transcript of a target sequence. In some embodiments, the present disclosure provides methods for designing and/or preparing oligonucleotide compositions which provide allele-specific suppression of a transcript from a target nucleic acid sequence.

[0025] In some embodiments, the present disclosure provides a method for controlled cleavage of a nucleic acid polymer, the method comprising steps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises a target sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length, wherein the common base sequence is or comprises a sequence that is complementary to a target sequence found in the nucleic acid polymer;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the particular base sequence and length, for

oligonucleotides of the particular oligonucleotide type.

[0026] In some embodiments, the present disclosure provides a method for altering a cleavage pattern observed when a nucleic acid polymer whose nucleotide sequence includes a target sequence is contacted with a reference oligonucleotide composition that comprises oligonucleotides having a particular base sequence and length, which particular base sequence is or comprises a sequence that is complementary to the target sequence, the method comprising: contacting the nucleic acid polymer with a chirally controlled oligonucleotide

composition of oligonucleotides having the particular base sequence and length, which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the particular base sequence and length, for

oligonucleotides of a single oligonucleotide type characterized by:

1) the particular base sequence and length;

2) a particular pattern of backbone linkages; and

3) a particular pattern of backbone chiral centers.

[0027] In some embodiments, the present disclosure provides a method for suppression of a transcript from a target nucleic acid sequence for which one or more similar nucleic acid sequences exist within a population, each of the target and similar sequences contains a specific nucleotide characteristic sequence element that defines the target sequence relative to the similar sequences, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acid sequence with an oligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that is complementary to the characteristic sequence element that defines the target nucleic acid sequence, the composition being characterized in that, when it is contacted with a system comprising transcripts of both the target nucleic acid sequence and a similar nucleic acid sequences, transcripts of the target nucleic acid sequence are suppressed at a greater level than a level of suppression observed for a similar nucleic acid sequence.

[0028] In some embodiments, the present disclosure provides a method for suppression of a transcript from a target nucleic acid sequence for which one or more similar nucleic acid sequences exist within a population, each of the target and similar sequences contains a specific nucleotide characteristic sequence element that defines the target sequence relative to the similar sequences, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acid sequence with an oligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

wherein the common base sequence is or comprises a sequence that is complementary to the characteristic sequence element that defines the target nucleic acid sequence, the composition being characterized in that, when it is contacted with a system comprising transcripts of both the target nucleic acid sequence and a similar nucleic acid sequences, transcripts of the target nucleic acid sequence are suppressed at a greater level than a level of suppression observed for a similar nucleic acid sequence.

[0029] In some embodiments, transcripts of the target nucleic acid sequence are suppressed at a greater level than a level of suppression observed for any one of the similar nucleic acid sequence.

[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 a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acid sequence with an oligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system comprising transcripts of both the target allele and another allele of the same nucleic acid sequence, transcripts of the particular allele are suppressed at a greater level than a level of suppression observed for 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 a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acid sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of the particular

oligonucleotide type is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system comprising transcripts of both the target allele and another allele of the same nucleic acid sequence, transcripts of the particular allele are suppressed at a greater level than a level of suppression observed for 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 a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target gene, the method comprising steps of:

contacting a sample comprising transcripts of the target gene with an oligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system comprising transcripts of both the target allele and another allele of the same gene, transcripts of the particular allele are suppressed at a level at least 2 fold greater than a level of suppression observed for 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 a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target gene, the method comprising steps of:

contacting a sample comprising transcripts of the target gene with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of the particular

oligonucleotide type is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system comprising transcripts of both the target allele and another allele of the same gene, transcripts of the particular allele are suppressed at a level at least 2 fold greater than a level of suppression observed for 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 a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that

defines the allele relative to other alleles of the same target gene, the method comprising steps of: contacting a sample comprising transcripts of the target gene with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of the particular oligonucleotide type is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system expressing transcripts of both the target allele and another allele of the same gene, transcripts of the particular allele are suppressed at a level at least 2 fold greater than a level of suppression observed for 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 a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acid sequence with an oligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system comprising transcripts of the same target nucleic acid sequence, it shows suppression of transcripts of the particular allele at a level that is: a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele of the same nucleic acid

sequence; or

c) both greater than when the composition is absent, and greater than a level of

suppression observed for another allele of the same nucleic acid sequence.

[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 a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acid sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of the particular

oligonucleotide type is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system comprising transcripts of the same target nucleic acid sequence, it shows suppression of transcripts of the particular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele of the same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than a level of

suppression observed for another allele of the same nucleic acid sequence.

[0037] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target gene, the method comprising steps of: contacting a sample comprising transcripts of the target gene with an oligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system expressing transcripts of the target gene, it shows suppression of expression of transcripts of the particular allele at a level that is:

a) at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed for another allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent, and at least 2 fold greater than a level of suppression observed for another allele of the same gene.

[0038] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target gene, the method comprising steps of: contacting a sample comprising transcripts of the target gene with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of the particular

oligonucleotide type is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system expressing transcripts of the target gene, it shows suppression of expression of transcripts of the particular allele at a level that is:

a) at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed for another allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent, and at least 2 fold greater than a level of suppression observed for another allele of the same gene.

[0039] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same target gene, the method comprising steps of: contacting a sample comprising transcripts of the target gene with an oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being

characterized in that, when it is contacted with a system expressing transcripts of the target gene, it shows suppression of expression of transcripts of the particular allele at a level that is:

a) at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed for another allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent, and at least 2 fold greater than a level of suppression observed for another allele of the same gene.

[0040] In some embodiments, the present disclosure provides a method for allele-specific suppression of a transcript from a target gene for which a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that

defines the allele relative to other alleles of the same target gene, the method comprising steps of: contacting a sample comprising transcripts of the target gene with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of the particular oligonucleotide type is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele, the composition being characterized in that, when it is contacted with a system expressing transcripts of the target gene, it shows suppression of expression of transcripts of the particular allele at a level that is:

a) at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed for another allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular allele are detected in amounts that are 2 fold lower when the composition is present relative to when it is absent, and at least 2 fold greater than a level of suppression observed for another allele of the same gene.

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

[0042] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide composition comprising oligonucleotides of a particular sequence, which composition provides selective suppression of a transcript of a target sequence, comprising providing a chirally controlled oligonucleotide composition comprising oligonucleotides of a

particular oligonucleotide type characterized by:

1) a common base sequence which is the same as the particular sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which pattern comprises (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, wherein:

m is 1-50;

n is 1-10;

t is 1-50; and

each Np is independent Rp or Sp.

[0043] In general, activities of oligonucleotide compositions as described herein can be assessed using any appropriate assay. Relative activities for different compositions (e.g., stereocontrolled vs non-stereocontrolled, and/or different stereocontrolled compositions) are typically desirably determined in the same assay, in some embodiments substantially simultaneously and in some embodiments with reference to historical results.

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

[0045] For example, certain assays that may be useful in the assessment of one or more features (e.g., rate, extent, and/or selectivity of cleavage) of RNase H cleavage may include an assay as described in any assay 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 a base sequence can impact properties of oligonucleotides. The present disclosure demonstrates that chemical and stereochemical modifications, combined with designed base sequences, can provide oligonucleotide compositions with unexpectedly improved properties (e.g., surprisingly higher activity, and/or selectivity, etc.). In some embodiments, oligonucleotides having a common base sequence complementary to a characteristic sequence element of a target nucleic acid sequence provide better activity compared to another common base sequence complementary to the characteristic sequence element of a target nucleic acid sequence. In some embodiments,

oligonucleotides having a common base sequence complementary to a characteristic sequence element of a target nucleic acid sequence provide better selectivity compared to another common base sequence complementary to the characteristic sequence element of a target nucleic acid sequence.

[0047] In some embodiments, a composition of oligonucleotides having a common base sequence complementary to a characteristic sequence element of a target nucleic acid sequence, when compared to another composition of oligonucleotides having another common base sequence complementary to the characteristic sequence element of the target nucleic acid sequence, provides higher cleavage rate of a transcript from the target nucleic acid sequence, and/or a cleavage pattern which has only one major cleavage site, and the major cleavage site is within or close to the nucleotide characteristic sequence. In some embodiments, a composition of oligonucleotides having a complementary common base sequence, when compared to another composition of oligonucleotides having another complementary common base sequence, provide higher cleavage rate of a transcript from the target nucleic acid sequence, and a cleavage pattern which has only one major cleavage site, and the major cleavage site is within or close to a nucleotide characteristic sequence. In some embodiments, greater than 50%, 60%, 70%, 80% or 90% of cleavage occurs at the one major cleavage site, for example, when measured by a suitable method, e.g., an RNase H assay. In some embodiments, a composition of oligonucleotides having a complementary common base sequence, when compared to another composition of oligonucleotides having another complementary common base sequence, provides higher cleavage rate of a transcript from the target nucleic acid sequence, and a cleavage pattern which has only one major cleavage site, and the major cleavage site is within or close to a mutation or a SNP that defines the target sequence relative to other similar sequences. In some embodiments, a mutation is a point mutation. In some embodiments, a major cleavage site is next to a mutation or a SNP that defines the target sequence relative to other similar sequences. In some embodiments, each common base sequence is 100% complementary to the characteristic sequence element of the target nucleic acid sequence. In some embodiments, a major cleavage site is within less than 5, 4, 3, or 1 internucleotidic linkage from a mutation or a SNP that defines the target sequence relative to other similar sequences. In some embodiments, a major cleavage site is within less than 5, 4, 3, or 1 internucleotidic linkage from a mutation or a SNP that defines the target sequence relative to other similar sequences, and is within less than 5, 4, 3, or 1

internucleotidic linkage from a cleavage site when a stereorandom composition of oligonucleotides having the same common sequence, and/or a composition of DNA oligonucleotides having the same common sequence, is used. In some embodiments, a major cleavage site is a cleavage site when a stereorandom composition of oligonucleotides having the same common sequence is used. In some embodiments, a major cleavage site is a major cleavage site when a stereorandom composition of oligonucleotides having the same common sequence is used. In some embodiments, a major cleavage site is a cleavage site when a composition of DNA oligonucleotides having the same common sequence is used. In some embodiments, a major cleavage site is a major cleavage site when a composition of DNA oligonucleotides having the same common sequence is used.

[0048] In some embodiments, when comparing effects of a first and a second common base sequences, 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, a 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, a stereorandom composition is a composition of oligonucleotides having a common base sequence, a common pattern of nucleoside modifications, wherein each internucleotidic linkage is phosphorothioate. In some embodiments, when comparing effects of a first and a second common base sequences, a chirally controlled oligonucleotide composition of oligonucleotides having a first common base sequence may be compared to a chirally controlled oligonucleotide composition of oligonucleotides having a second common base sequence. In some embodiments, oligonucleotides in a chirally 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 internucleotidic linkage is phosphorothioate.

[0049] In some embodiments, oligonucleotide compositions and technologies 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 a Huntington’s disease-associated allele of a particular target sequence, relative to one or more (e.g., all non-Huntington’s disease-associated) other alleles of the sequence.

[0050] Huntington's disease is an inherited disease that can cause progressive degeneration of nerve cells in the brain and affect a subject’s motor and cognitive abilities. In some embodiments, Huntington’s disease is an autosomal dominant disorder. In some embodiments, it is caused by mutations in the Huntingtin gene. Normal HTT gene contains 10 to 35 CAG tri-nucleotide repeats. People with 40 or more repeats often develop the disorder. In some embodiments, the expanded CAG segment on the first exon of HTT gene leads to the production of an abnormally long version of the Huntingtin protein (expanded polyglutamine tract) which is cut into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal functions of these cells. Warby et al. (Am J Hum Genet.2009, 84(3), 351– 366) reported many SNPs that are associated with disease chromosomes and have stronger linkage associations with CAG expansion than those reported before. Many SNPs highly associated with CAG expansion do not segregate independently and are in Linkage Disequilibrium with each other. Among other things, the present disclosure recognizes that strong association between specific SNPs and CAG expanded chromosomes provides an attractive therapeutic opportunity for the treatment of Huntington Disease, e.g., through antisense therapy. Furthermore, the association of specific SNPs combined with high rates of heterozygosity in HD patients provides suitable targets for allele-specific knockdown of the mutant gene product. For example references, see Liu et al. Journal of Huntington’s Disease 2, 2013, 491–500; Aronin, Neil and Pfister, Edith WO 2010/118263 A1; Pfister et al. Current Biology 2009,19, 774–778.

[0051] In some embodiments, a targeted SNP of the present disclosure has high frequency of heterozygosity in HD and has a particular variant associated with the mutant HTT allele. In some embodiments, a SNP is rs362307. In some embodiments, a SNP is rs7685686. In some embodiments, a SNP may not be linked but may have a high heterozygous frequency. In some embodiments, a SNP is rs362268 (3’-UTR region). In some embodiments, a SNP is rs362306 (3’-UTR region). In some embodiments, a SNP is rs2530595. In some embodiments, a SNP is rs362331.

[0052] In some embodiments, a provided method for treating or preventing Huntington's disease in a subject, comprising administering to the subject a provided oligonucleotide compositions. In some embodiments, a provided method for treating or preventing Huntington's disease in a subject, comprising administering to the subject a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type.

[0053] In some embodiments, a provided method ameliorates a symptom of Huntington's disease. In some embodiments, a provided method slows onset of Huntington's disease. In some embodiments, a provided method slows progression of Huntington's disease.

[0054] In some embodiments, the present disclosure provides methods for identifying patients for a given oligonucleotide composition. In some embodiments, the present disclosure provides methods for patient stratification. In some embodiments, a provided method comprises identifying a mutation and/or SNP associated with a disease-causing allele. For example, in some embodiments, a provided method comprises identifying in a subject a SNP associated with expanded CAG repeats that are associated with or causing Huntington’s disease.

[0055] In some embodiments, a subject has a SNP in the subject’s Huntingtin gene. In some embodiments, a subject has a SNP, wherein one allele is mutant Huntingtin associated with expanded CAG repeats. In some embodiments, a subject has a SNP selected from rs362307, rs7685686, rs362268, rs2530595, rs362331, or rs362306. In some embodiments, oligonucleotides of a provided composition have a sequence complementary to a sequence comprising a SNP from the disease-causing allele (mutant), and the composition selectively suppresses expression from the diseasing-causing allele.

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

embodiments, a sequence is selected from the sequence of WV-1092, WVE120101, WV-2603 or WV-2595. In some embodiments, provided oligonucleotides are of the type defined by WV-1092, WVE120101, WV-2603 or WV-2595. In some embodiments, provided oligonucleotides are of the type defined by WV-1092. In some embodiments, provided oligonucleotides are of the type defined by WVE120101. In some embodiments, provided oligonucleotides are of the type defined by WV-2603. In some embodiments, provided oligonucleotides are of the type defined by WV-2595.

[0057] In some embodiments, provided oligonucleotide compositions comprises a lipid and an oligonucleotide. In some embodiments, a lipid is conjugated to an 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, alpha-linolenic acid, gamma-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, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid, and dilinoleyl.

[0059] In some embodiments, a composition comprises an oligonucleotide and a lipid selected from:

.

[0060] In some embodiments, a composition comprises an oligonucleotide and a lipid, wherein the lipid comprises a C10-C40 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C1-4 aliphatic group.

[0061] In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides, which share:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone phosphorus modifications;

wherein one or more oligonucleotides of the plurality are individually conjugated to a lipid.

[0062] In some embodiments, a chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides, which share:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone phosphorus modifications;

wherein:

the composition is chirally controlled in that the plurality of oligonucleotides share the same stereochemistry at one or more chiral internucleotidic linkages;

one or more oligonucleotides of the plurality are individually conjugated to a lipid; and one or more oligonucleotides of the plurality are optionally and individually conjugated to a targeting compound or moiety.

[0063] In some embodiments, a method of delivering an oligonucleotide to a cell or tissue in a human subject, comprises:

(a) Providing a composition of any one of the embodiments described herein; and

(b) Administering the composition to the human subject such that the oligonucleotide is delivered to a cell or tissue in the subject.

[0064] In some embodiments, a method for delivering an oligonucleotide to a cell or tissue comprises 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 of modulating the level of a transcript or gene product of a gene in a cell, the method comprises the step of 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 comprises 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 in a mammal comprises preparing a composition according to any one of the embodiments described herein and administering the composition to the mammal.

[0068] In some embodiments, a method of treating a disease that is caused by the over-expression of one or several proteins in a cell or tissue in a subject, said method comprises the administration of a composition according to any one of the embodiments described herein to the subject.

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

[0070] In some embodiments, a method for generating an immune response in a subject, said method comprises the administration of a composition according to any one of the

embodiments described herein to the subject, wherein the biologically active compound is an immunomodulating nucleic acid.

[0071] In some embodiments, a method for treating a sign and/or symptom of Huntington'sDisease by providing a composition of any one of the embodiments described herein and administering the composition to the subject.

[0072] In some embodiments, a method of modulating the amount of RNaseH-mediated cleavage in a cell, the method comprises the step of 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 steps of providing a composition comprises the agent a lipid, and administering the composition to the subject, wherein the agent is any agent disclosed herein, and wherein the lipid is any lipid disclosed herein.

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

[0075] In some embodiments, a lipid comprises an optionally substituted C10-C40 saturated or partially unsaturated aliphatic chain.

[0076] In some embodiments, a lipid comprises an optionally substituted C10-C40 linear, saturated or partially unsaturated, aliphatic chain.

[0077] In some embodiments, a lipid comprises a C10-C40 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C1-4 aliphatic group.

[0078] In some embodiments, a lipid comprises an unsubstituted C10-C40 linear, saturated or partially unsaturated, aliphatic chain.

[0079] In some embodiments, a lipid comprises no more than one optionally substituted C10-C40 linear, saturated or partially unsaturated, aliphatic chain.

[0080] In some embodiments, a lipid comprises two or more optionally substituted C10-C40 linear, saturated or partially unsaturated, aliphatic chain.

[0081] In some embodiments, a lipid comprises no tricyclic or polycyclic moiety.

[0082] In some embodiments, a lipid has the structure of R1-COOH, wherein R1 is an optionally substituted C10-C40 saturated or partially unsaturated aliphatic chain.

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

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

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

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

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

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

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

[0090] In some embodiments, a lipid is conjugated to an oligonucleotide through a linker.

[0091] In some embodiments, one or more oligonucleotides of the plurality are independently conjugated to a targeting compound or moiety.

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

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

[0094] In some embodiments, oligonucleotides of the plurality share the same chemical modification patterns.

[0095] In some embodiments, oligonucleotides of the plurality share the same chemical modification patterns comprises one or more base modifications.

[0096] In some embodiments, oligonucleotides of the plurality share the same chemical modification patterns comprises one or more sugar modifications.

[0097] In some embodiments, a common base sequence is capable of hybridizing with a transcript in a cell, which transcript contains a mutation that is linked to Huntington's Disease, or whose level, activity and/or distribution is linked to Huntington's Disease.

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

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

[00100] In some embodiments, an oligonucleotide is an oligonucleotide which participates in RNaseH-mediated cleavage of a mutant Huntingtin gene mRNA.

[00101] In some embodiments, a disease or disorder is Huntington's Disease.

[00102] In some embodiments, a lipid comprises an optionally substituted, C10-C80 saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1–C6 alkylene, C1–C6 alkenylene, , a C1–C6 heteroaliphatic moiety, -C(Rc)2–,–Cy–,–O–,– S–, –S–S–, -N(Rc)–, -C(O)–, –C(S)–, –C(NRc)–, –C(O)N(Rc)–, -N(Rc)C(O)N(Rc)-, – N(Rc)C(O)–,–N(Rc)C(O)O–, -OC(O)N(Rc)-,–S(O)–,–S(O)2–, -S(O)2N(Rc)–, -N(Rc)S(O)2–,– SC(O)–,–C(O)S–,–OC(O)–, and -C(O)O–, wherein each variable is independently as defined and described herein.

[00103] In some embodiments, a lipid comprises an optionally substituted C10-C80 saturated or partially unsaturated, aliphatic chain.

[00104] In some embodiments, a lipid comprises an optionally substituted C10-C80 linear, saturated or partially unsaturated, aliphatic chain.

[00105] In some embodiments, a lipid comprises an optionally substituted C10-C60 saturated or partially unsaturated, aliphatic chain.

[00106] In some embodiments, a lipid comprises an optionally substituted C10-C60 linear, saturated or partially unsaturated, aliphatic chain.

[00107] In some embodiments, a lipid comprises an optionally substituted C10-C40 saturated or partially unsaturated, aliphatic chain.

[00108] In some embodiments, a lipid comprises an optionally substituted C10-C40 linear, saturated or partially unsaturated, aliphatic chain.

[00109] In some embodiments, a lipid comprises an optionally substituted, C10-C60 saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1–C6 alkylene, C1–C6 alkenylene, , a C1–C6 heteroaliphatic moiety, -C(Rc)2–,–Cy–,–O–,– S–, –S–S–, -N(Rc)–, -C(O)–, –C(S)–, –C(NRc)–, –C(O)N(Rc)–, -N(Rc)C(O)N(Rc)-, – N(Rc)C(O)–,–N(Rc)C(O)O–, -OC(O)N(Rc)-,–S(O)–,–S(O)2–, -S(O)2N(Rc)–, -N(Rc)S(O)2–,– SC(O)–,–C(O)S–,–OC(O)–, and -C(O)O–, wherein each variable is independently as defined and described herein.

[00110] In some embodiments, a lipid comprises an optionally substituted C10-C80 saturated or partially unsaturated, aliphatic chain.

[00111] In some embodiments, a lipid comprises an optionally substituted C10-C60 linear, saturated or partially unsaturated, aliphatic chain.

[00112] In some embodiments, a lipid comprises an optionally substituted C10-C40 linear, saturated or partially unsaturated, aliphatic chain.

[00113] In some embodiments, a lipid comprises an optionally substituted, C10-C40 saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1–C6 alkylene, C1–C6 alkenylene, , a C1–C6 heteroaliphatic moiety, -C(Rc)2–,–Cy–,–O–,– S–, –S–S–, -N(Rc)–, -C(O)–, –C(S)–, –C(NRc)–, –C(O)N(Rc)–, -N(Rc)C(O)N(Rc)-, – N(Rc)C(O)–,–N(Rc)C(O)O–, -OC(O)N(Rc)-,–S(O)–,–S(O)2–, -S(O)2N(Rc)–, -N(Rc)S(O)2–,– SC(O)–,–C(O)S–,–OC(O)–, and -C(O)O–, wherein each variable is independently as defined and described herein.

[00114] In some embodiments, a lipid comprises an optionally substituted C10-C40 saturated or partially unsaturated, aliphatic chain.

[00115] In some embodiments, a lipid comprises an optionally substituted C10-C40 linear, saturated or partially unsaturated, aliphatic chain.

[00116] In some embodiments, a composition further comprises one or more additional components selected from: a polynucleotide, carbonic anhydrase inhibitor, a dye, an intercalating agent, an acridine, a cross-linker, psoralene, mitomycin C, a porphyrin, TPPC4, texaphyrin, Sapphyrin, a polycyclic aromatic hydrocarbon phenazine, dihydrophenazine, an artificial endonuclease, a chelating agent, EDTA, an alkylating agent, a phosphate, an amino, a mercapto, a PEG, PEG-40K, MPEG, [MPEG]2, a polyamino, an alkyl, a substituted alkyl, a radiolabeled marker, an enzyme, a hapten biotin, a transport/absorption facilitator, aspirin, vitamin E, folic acid, a synthetic ribonuclease, a protein, a glycoprotein, a peptide, a molecule having a specific affinity for a co-ligand, an antibody, a hormone, a hormone receptor, a non-peptidic species, a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, selectivity agent, or a drug. In some embodiments, a composition further comprises one or more additional components selected from: a polynucleotide, carbonic anhydrase inhibitor, a dye, an intercalating agent, an acridine, a cross-linker, psoralene, mitomycin C, a porphyrin, TPPC4, texaphyrin, Sapphyrin, a polycyclic aromatic hydrocarbon phenazine, dihydrophenazine, an artificial endonuclease, a chelating agent, EDTA, an alkylating agent, a phosphate, an amino, a mercapto, a PEG, PEG-40K, MPEG,

[MPEG]2, a polyamino, an alkyl, a substituted alkyl, a radiolabeled marker, an enzyme, a hapten biotin, a transport/absorption facilitator, aspirin, vitamin E, folic acid, a synthetic ribonuclease, a protein, a glycoprotein, a peptide, a molecule having a specific affinity for a co-ligand, an antibody, a hormone, a hormone receptor, a non-peptidic species, a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, or a drug.

[00117] 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, a selectivity agent binds specifically to one or more neurotransmitter transporters selected from the group consisting of a dopamine transporter (DAT), a serotonin transporter (SERT), and a norepinephrine transporter (NET). In some embodiments, a selectivity agent is selected from the group consisting of a dopamine reuptake inhibitor (DRI), a selective serotonin reuptake inhibitor (SSRI), a noradrenaline reuptake inhibitor (NRI), a norepinephrine-dopamine reuptake inhibitor (NDRI), and a serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI). In some embodiments, a selectivity agent is selected from the group consisting of a triple reuptake inhibitor, a noradrenaline dopamine double reuptake inhibitor, a serotonin single reuptake inhibitor, a noradrenaline single reuptake inhibitor, and a dopamine single reuptake inhibitor. In some embodiments, a selectivity agent is selected from the group consisting of a dopamine reuptake inhibitor (DRI), a Norepinephrine-Dopamine Reuptake Inhibitor (NDRI) and a serotonin-Norepinephrine-Dopamine Reuptake Inhibitor (SNDRI). In some embodiments, a selectivity agent is selected from the selectivity agents which are described in U.S. Pat. Nos. 9,084,825; and 9,193,969; and WO2011131693, WO2014064258.

[00118] In some embodiments, a lipid comprises a C10-C80 linear, saturated or partially unsaturated, aliphatic chain.

[00119] In some embodiments, a composition further comprises a linker linking the oligonucleotide and the lipid, wherein the linker is selected from: an uncharged linker; a charged linker; a linker comprises an alkyl; a linker comprises a phosphate; a branched linker; an unbranched linker; a linker comprises at least one cleavage group; a linker comprises at least one redox cleavage group; a linker comprises at least one phosphate-based cleavage group; a linker

comprises at least one acid-cleavage group; a linker comprises at least one ester-based cleavage group; a linker comprises at least one peptide-based cleavage group.

[00120] In some embodiments, an oligonucleotide comprises or consists of or is an oligonucleotide or oligonucleotide composition or chirally controlled oligonucleotide composition.

[00121] In some embodiments, an oligonucleotide comprises or consists of or is an oligonucleotide composition or chirally controlled oligonucleotide composition, wherein the sequence of the oligonucleotide comprises or consists of the sequence of any oligonucleotide described herein.

[00122] In some embodiments, an oligonucleotide comprises or consists of or is an oligonucleotide composition or chirally controlled oligonucleotide composition, wherein the sequence of the oligonucleotide comprises or consists of the sequence of any oligonucleotide listed in Table 4.

[00123] In some embodiments, an oligonucleotide comprises or consists of or is an oligonucleotide composition or chirally controlled oligonucleotide composition, wherein the sequence of the oligonucleotide comprises or consists of the sequence of a splice-switching oligonucleotide.

[00124] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is a chirally controlled oligonucleotide composition.

[00125] The composition or method of any of the embodiments described herein, wherein the disease or disorder is Huntington's Disease.

[00126] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is capable of participating in RNaseH-mediated cleavage of a mutant Huntingtin gene mRNA.

[00127] The composition or method of any of the embodiments described herein, wherein the oligonucleotide comprises, consists of or is the sequence of any oligonucleotide disclosed herein.

[00128] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is capable of differentiating between a wild-type and a mutant Huntingtin allele.

[00129] The composition or method of any of the embodiments described herein, wherein the oligonucleotide is capable of participating in RNaseH-mediated cleavage of a mutant Huntingtin gene mRNA.

[00130] The composition or method of any of the embodiments described herein, wherein the oligonucleotide comprises, consists of or is the sequence of any oligonucleotide disclosed in Table 4.

[00131] In some embodiments, an oligonucleotide comprises or consists of or is an oligonucleotide or oligonucleotide composition or chirally 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.

[00132] In some embodiments, a sequence of an oligonucleotide includes any one or more of: base sequence (including length); pattern of chemical modifications to sugar and base moieties; pattern of backbone linkages; pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof; pattern of backbone chiral centers; pattern of stereochemistry (Rp/Sp) of chiral internucleotidic linkages; pattern of backbone phosphorus modifications; pattern of modifications on the internucleotidic phosphorus atom, such as -Sí, and -L-R1 of formula I.

[00133] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type, wherein the oligonucleotides target a mutant Huntingtin gene, and the length is from about 10 to about 50 nucleotides, wherein the backbone linkages comprise at least one phosphorothioate, and wherein the pattern of backbone chiral centers comprises at least one chiral center in a Rp conformation and at least one chiral center in a Sp conformation.

[00134] In some embodiments, the present disclosure provides a method for cleavage of a nucleic acid having a base sequence comprising a target sequence, the method comprising steps of:

(a) contacting a nucleic acid having a base sequence comprising a target sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length, wherein the common base sequence is or comprises a sequence that is complementary to the target sequence in the nucleic acid; 2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the particular base sequence and length, for

oligonucleotides of the particular oligonucleotide type, wherein the oligonucleotide targets a mutant Huntingtin gene, and the length is from about 10 to about 50 nucleotides, wherein the backbone linkages comprise at least one phosphorothioate, and wherein the pattern of backbone chiral centers comprises at least one chiral center in a Rp conformation and at least one chiral center in a Sp conformation.

[00135] In some embodiments, the present disclosure provides a method for cleavage of a nucleic acid having a base sequence comprising a target sequence, the method comprising steps of:

(a) contacting a nucleic acid having a base sequence comprising a target sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length, wherein the common base sequence is or comprises a sequence that is complementary to the target sequence in the nucleic acid; 2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the particular base sequence and length, for

oligonucleotides of the particular oligonucleotide type, wherein the oligonucleotide targets a mutant Huntingtin gene, and the length is from about 10 to about 50 nucleotides, wherein the

backbone linkages comprise at least one phosphorothioate, and wherein the pattern of backbone chiral centers comprises at least one chiral center in a Rp conformation and at least one chiral center in a Sp conformation; and

(b) cleavage of the nucleic acid mediated by a RNAseH or RNA interference mechanism.

[00136] In some embodiments, a provided composition further comprises a selectivity agent selected from: the group of compounds which binds specifically to one or more neurotransmitter transporters selected from the group consisting of a dopamine transporter (DAT), a serotonin transporter (SERT), and a norepinephrine transporter (NET); the group consisting of a dopamine reuptake inhibitor (DRI), a selective serotonin reuptake inhibitor (SSRI), a noradrenaline reuptake inhibitor (NRI), a norepinephrine-dopamine reuptake inhibitor (NDRI), and a serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI); the group consisting of a triple reuptake inhibitor, a noradrenaline dopamine double reuptake inhibitor, a serotonin single reuptake inhibitor, a noradrenaline single reuptake inhibitor, and a dopamine single reuptake inhibitor; and the group consisting of a dopamine reuptake inhibitor (DRI), a Norepinephrine-Dopamine Reuptake Inhibitor (NDRI) and a serotonin-Norepinephrine-Dopamine Reuptake Inhibitor (SNDRI).

[00137] In some embodiments, a provided composition comprises oligonucleotides wherein the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of the oligonucleotides comprises or consists of the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of any of any oligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

[00138] In some embodiments, a provided composition comprises oligonucleotides wherein the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of the oligonucleotides comprises or consists of the base sequence, and pattern of backbone linkages, and/or pattern of backbone chiral centers of any of any oligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

[00139] In some embodiments, a provided composition comprises oligonucleotides wherein the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of the oligonucleotides comprises or consists of the base sequence, and pattern of backbone linkages, and pattern of backbone chiral centers of any of any oligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

Definitions

[00140] Aliphatic: The term“aliphatic” or“aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic or polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "carbocycle"“cycloaliphatic” or“cycloalkyl”), that has a single point of attachment to the rest of the molecule. In some embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms. Unless otherwise specified, aliphatic groups contain 1-10 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments,“cycloaliphatic” (or“carbocycle” or“cycloalkyl”) refers to a monocyclic or bicyclic C3-C10 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. In some embodiments,“cycloaliphatic” (or“carbocycle” or “cycloalkyl”) refers to a monocyclic C3–C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[00141] Alkylene: The term“alkylene” refers to a bivalent alkyl group. An“alkylene chain” is a polymethylene group, i.e.,–(CH2)n–, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

[00142] Alkenylene: The term“alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

[00143] Animal: As used herein, the term“animal” refers to any member of the animal kingdom. In some embodiments,“animal” refers to humans, at any stage of development. In some embodiments,“animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, a genetically-engineered animal, and/or a clone.

[00144] Approximately: As used herein, the terms“approximately” or“about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). In some embodiments, use of the term“about” in reference to dosages means r 5 mg/kg/day.

[00145] 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 systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term“aryl” may be used interchangeably with the term“aryl ring.” In certain embodiments of the present disclosure, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term“aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non–aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[00146] Characteristic portion: As used herein, the phrase a“characteristic portion” of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. Each such continuous stretch generally will contain at least two amino acids. Furthermore, those of ordinary skill in the art will appreciate that typically at least 5, 10, 15, 20 or more amino acids are required to be characteristic of a protein. In general, a characteristic portion is one that, in addition to the sequence identity specified above, shares at least one functional characteristic with the relevant intact protein.

[00147] Characteristic sequence: A“characteristic 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 ordinary skill in the art to define members of the family.

[00148] Characteristic structural element: The term“characteristic structural element” refers to a distinctive structural element (e.g., core structure, collection of pendant moieties, sequence element, etc) that is found in all members of a family of polypeptides, small molecules, or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.

[00149] Comparable: The term“comparable” is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In some embodiments, comparable sets of conditions or circumstances are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of conditions are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under the different sets of conditions or circumstances are caused by or indicative of the variation in those features that are varied.

[00150] Dosing regimen: As used herein, a“dosing regimen” or“therapeutic regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regime comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.

[00151] Equivalent agents: Those of ordinary skill in the art, reading the present disclosure, will appreciate that the scope of useful agents in the context of the present disclosure is not limited to those specifically mentioned or exemplified herein. In particular, those skilled in the art will recognize that active agents typically have a structure that consists of a core and attached pendant moieties, and furthermore will appreciate that simple variations of such core and/or pendant moieties may not significantly alter activity of the agent. For example, in some embodiments, substitution of one or more pendant moieties with groups of comparable three-dimensional structure and/or chemical reactivity characteristics may generate a substituted compound or portion equivalent to a parent reference compound or portion. In some embodiments, addition or removal of one or more pendant moieties may generate a substituted compound equivalent to a parent reference compound. In some embodiments, alteration of core structure, for example by addition or removal of a small number of bonds (typically not more than 5, 4, 3, 2, or 1 bonds, and often only a single bond) may generate a substituted compound equivalent to a parent reference compound. In many embodiments, equivalent compounds may be prepared by methods illustrated in general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional or provided synthesis procedures. In these reactions, it is also possible to make use of variants, which are in themselves known, but are not mentioned here.

[00152] Equivalent Dosage: The term“equivalent dosage” is used herein to compare dosages of different pharmaceutically active agents that effect the same biological result. Dosages of two different agents are considered to be“equivalent” to one another in accordance with the present disclosure if they achieve a comparable level or extent of the biological result. In some embodiments, equivalent dosages of different pharmaceutical agents for use in accordance with the present disclosure are determined using in vitro and/or in vivo assays as described herein. In some embodiments, one or more lysosomal activating agents for use in accordance with the present disclosure is utilized at a dose equivalent to a dose of a reference lysosomal activating agent; in some such embodiments, the reference lysosomal activating agent for such purpose is selected from the group consisting of small molecule allosteric activators (e.g., pyrazolpyrimidines), imminosugars (e.g., isofagomine), antioxidants (e.g., n-acetyl-cysteine), and regulators of cellular trafficking (e.g., Rab1a polypeptide).

[00153] Heteroaliphatic: The term“heteroaliphatic” refers to an aliphatic group wherein

one or more units selected from C, CH, CH2, or CH3 are independently replaced by a heteroatom. In some embodiments, a heteroaliphatic group is heteroalkyl. In some embodiments, a heteroaliphatic group is heteroalkenyl.

[00154] 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 S electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms“heteroaryl” and“heteroar–,” as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, 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. A heteroaryl group may be mono– or bicyclic. The term“heteroaryl” may be used interchangeably with the terms“heteroaryl ring,”“heteroaryl group,” or“heteroaromatic,” any of which terms include rings that are optionally substituted. The term“heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[00155] Heteroatom: The term“heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, boron, selenium, or silicon (including, any oxidized form of nitrogen, boron, selenium, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)).

[00156] 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 7–10–membered bicyclic heterocyclic moiety that is either

saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term“nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0–3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4–dihydro–2H–pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N–substituted pyrrolidinyl).

[00157] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, 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 also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H–indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono– or bicyclic. The term“heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[00158] Intraperitoneal: The phrases“intraperitoneal administration” and“administered intraperitonealy” as used herein have their art-understood meaning referring to administration of a compound or composition into the peritoneum of a subject.

[00159] In vitro: As used herein, the term“in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant, and/or microbe).

[00160] In vivo: As used herein, the term“in vivo” refers to events that occur within an organism (e.g., animal, plant, and/or microbe).

[00161] Lower alkyl: The term“lower alkyl” refers to a C1-4 straight or branched alkyl group. Example lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[00162] Lower haloalkyl: The term“lower haloalkyl” refers to a C1-4 straight or branched

alkyl group that is substituted with one or more halogen atoms.

[00163] Optionally substituted: As described herein, compounds of the disclosure may contain“optionally substituted” moieties. In general, the term“substituted,” whether preceded by the term“optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an“optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term“stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[00164] Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen; -(CH2)0-4Rq; -(CH2)0-4ORq; -O(CH2)0-4R°, -O-(CH2)0-4C(O)OR°; -(CH2)0-4CH(ORq)2; -(CH2)0-4SRq; -(CH2)0-4Ph, which may be substituted with R°; -(CH2)0-4O(CH2)0í1Ph which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)0-4O(CH2)0í1-pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)0-4N(Rq)2; -(CH2)0-4N(Rq)C(O)Rq; -N(Rq)C(S)Rq; -(CH2)0-4N(Rq)C(O)NRq2; -N(Rq)C(S)NRq2; -(CH2)0-4N(Rq)C(O)ORq; -N(Rq)N(Rq)C(O)Rq; -N(Rq)N(Rq)C(O)NRq2; -N(Rq)N(Rq)C(O)ORq; -(CH2)0-4C(O)Rq; -C(S)Rq; -(CH2)0-4C(O)ORq; -(CH2)0-4C(O)SRq; -(CH2)0-4C(O)OSiRq3; -(CH2)0-4OC(O)Rq; -OC(O)(CH2)0-4SR, -SC(S)SR°; -(CH2)0-4SC(O)Rq; -(CH2)0-4C(O)NRq2; -C(S)NRq2; -C(S)SR°; -SC(S)SR°, -(CH2)0-4OC(O)NRq2; -C(O)N(ORq)Rq; -C(O)C(O)Rq; -C(O)CH2C(O)Rq; -C(NORq)Rq; -(CH2)0-4SSRq; -(CH2)0-4S(O)2Rq; -(CH2)0-4S(O)2ORq; -(CH2)0-4OS(O)2Rq; -S(O)2NRq2; -(CH2)0-4S(O)Rq; -N(Rq)S(O)2NRq2; -N(Rq)S(O)2Rq; -N(ORq)Rq; -C(NH)NRq2; -P(O)2Rq; -P(O)Rq2; -OP(O)Rq2; -OP(O)(ORq)2; -SiRq3; -(C1-4 straight or branched alkylene)O-N(Rq)2; or -(C1-4 straight or branched alkylene)C(O)O-N(Rq)2, wherein each Rq may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2Ph, -O(CH2)0í1Ph, -CH2-(5-6 membered heteroaryl ring), or a 5-6

membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of Rq, taken together with their intervening atom(s), form a 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[00165] Suitable monovalent substituents on Rq (or the ring formed by taking two independent occurrences of Rq together with their intervening atoms), are independently

is unsubstituted or where preceded by“halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, -CH2Ph, -O(CH2)0í1Ph, 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 Rq include =O and =S.

[00166] Suitable divalent substituents on a saturated carbon atom of an“optionally

occurrence of R* is selected from hydrogen, C1–6 aliphatic which may be substituted as defined below, or an unsubstituted 5–6–membered saturated, partially unsaturated, or aryl ring having 0– 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an“optionally substituted” group include:–O(CR*

2)2–3O–, wherein each independent occurrence of R* is selected from hydrogen, C1–6 aliphatic which may be substituted as defined below, or an unsubstituted 5–6–membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[00167] Suitable substituents on the aliphatic group of R* include halogen,

or -NO2, wherein each Rz is unsubstituted or where preceded by“halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2Ph, -O(CH2)0í1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[00168] Suitable substituents on a substitutable nitrogen of an“optionally substituted”

independently hydrogen, C1–6 aliphatic which may be substituted as defined below, unsubstituted –OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl mono– or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[00169] Suitable substituents on the aliphatic group of R† are independently halogen,

or -NO2, wherein each Rz is unsubstituted or where preceded by“halo” is substituted only with one or more halogens, and is independently 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.

[00170] Oral: The phrases“oral administration” and“administered orally” as used herein have their art-understood meaning referring to administration by mouth of a compound or composition.

[00171] Parenteral: The phrases “parenteral administration” and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[00172] Partially unsaturated: As used herein, the term“partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term“partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[00173] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[00174] Pharmaceutically acceptable: As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00175] Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, 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; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[00176] Pharmaceutically acceptable salt: The term“pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salt include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. [00177] Prodrug: A general, a“prodrug,” as that term is used herein and as is 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 involves removal of at least one“prodrug moiety” so that the active agent is formed. Various forms of“prodrugs” are known in the art. For examples of such prodrug moieties, see:

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).

[00178] As with other compounds described herein, prodrugs may be provided in any of a variety of forms, e.g., crystal forms, salt forms etc. In some embodiments, prodrugs are provided as pharmaceutically acceptable salts thereof.

[00179] Protecting group: The term“protecting group,” as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Also included are those protecting groups specially adapted for nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al.06/2012, the entirety of Chapter 2 is incorporated herein by reference. Suitable amino–protecting groups include methyl carbamate, ethyl carbamante, 9–fluorenylmethyl carbamate (Fmoc), 9–(2–sulfo)fluorenylmethyl carbamate, 9– (2,7–dibromo)fluoroenylmethyl carbamate, 2,7–di–t–butyl–[9–(10,10–dioxo–10,10,10,10– tetrahydrothioxanthyl)]methyl carbamate (DBD–Tmoc), 4–methoxyphenacyl carbamate (Phenoc), 2,2,2–trichloroethyl carbamate (Troc), 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–isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4–nitrocinnamyl carbamate (Noc), 8–quinolyl carbamate, N–hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p– methoxybenzyl carbamate (Moz), p–nitobenzyl carbamate, p–bromobenzyl carbamate, p– chlorobenzyl carbamate, 2,4–dichlorobenzyl carbamate, 4–methylsulfinylbenzyl carbamate (Msz), 9–anthrylmethyl carbamate, diphenylmethyl carbamate, 2–methylthioethyl carbamate, 2– methylsulfonylethyl carbamate, 2–(p–toluenesulfonyl)ethyl carbamate, [2–(1,3– dithianyl)]methyl carbamate (Dmoc), 4–methylthiophenyl carbamate (Mtpc), 2,4– dimethylthiophenyl carbamate (Bmpc), 2–phosphonioethyl carbamate (Peoc), 2– triphenylphosphonioisopropyl carbamate (Ppoc), 1,1–dimethyl–2–cyanoethyl carbamate, m– chloro–p–acyloxybenzyl carbamate, p–(dihydroxyboryl)benzyl carbamate, 5– benzisoxazolylmethyl carbamate, 2–(trifluoromethyl)–6–chromonylmethyl carbamate (Tcroc), m–nitrophenyl carbamate, 3,5–dimethoxybenzyl carbamate, o–nitrobenzyl carbamate, 3,4– dimethoxy–6–nitrobenzyl carbamate, phenyl(o–nitrophenyl)methyl carbamate, phenothiazinyl– (10)–carbonyl derivative, N’–p–toluenesulfonylaminocarbonyl derivative, N’– phenylaminothiocarbonyl derivative, t–amyl carbamate, S–benzyl thiocarbamate, p–cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p–decyloxybenzyl carbamate, 2,2–dimethoxycarbonylvinyl carbamate, o–(N,N–dimethylcarboxamido)benzyl carbamate, 1,1–dimethyl–3–(N,N– dimethylcarboxamido)propyl carbamate, 1,1–dimethylpropynyl carbamate, di(2–pyridyl)methyl carbamate, 2–furanylmethyl carbamate, 2–iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p–(p’–methoxyphenylazo)benzyl carbamate, 1– methylcyclobutyl carbamate, 1–methylcyclohexyl carbamate, 1–methyl–1–cyclopropylmethyl carbamate, 1–methyl–1–(3,5–dimethoxyphenyl)ethyl carbamate, 1–methyl–1–(p– phenylazophenyl)ethyl carbamate, 1–methyl–1–phenylethyl carbamate, 1–methyl–1–(4– pyridyl)ethyl carbamate, phenyl carbamate, p–(phenylazo)benzyl carbamate, 2,4,6–tri–t– butylphenyl carbamate, 4–(trimethylammonium)benzyl carbamate, 2,4,6–trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3–phenylpropanamide, picolinamide, 3–pyridylcarboxamide, N– benzoylphenylalanyl derivative, benzamide, p–phenylbenzamide, o–nitophenylacetamide, o– nitrophenoxyacetamide, acetoacetamide, (N’–dithiobenzyloxycarbonylamino)acetamide, 3–(p– hydroxyphenyl)propanamide, 3–(o–nitrophenyl)propanamide, 2–methyl–2–(o– nitrophenoxy)propanamide, 2–methyl–2–(o–phenylazophenoxy)propanamide, 4– chlorobutanamide, 3–methyl–3–nitrobutanamide, o–nitrocinnamide, N–acetylmethionine derivative, o–nitrobenzamide, o–(benzoyloxymethyl)benzamide, 4,5–diphenyl–3–oxazolin–2– one, N–phthalimide, N–dithiasuccinimide (Dts), N–2,3–diphenylmaleimide, N–2,5– dimethylpyrrole, N–1,1,4,4–tetramethyldisilylazacyclopentane adduct (STABASE), 5– substituted 1,3–dimethyl–1,3,5–triazacyclohexan–2–one, 5–substituted 1,3–dibenzyl–1,3,5– triazacyclohexan–2–one, 1–substituted 3,5–dinitro–4–pyridone, N–methylamine, N–allylamine, N–[2–(trimethylsilyl)ethoxy]methylamine (SEM), N–3–acetoxypropylamine, N–(1–isopropyl–4– nitro–2–oxo–3–pyroolin–3–yl)amine, quaternary ammonium salts, N–benzylamine, N–di(4– methoxyphenyl)methylamine, N–5–dibenzosuberylamine, N–triphenylmethylamine (Tr), N–[(4– methoxyphenyl)diphenylmethyl]amine (MMTr), N–9–phenylfluorenylamine (PhF), N–2,7– dichloro–9–fluorenylmethyleneamine, N–ferrocenylmethylamino (Fcm), N–2–picolylamino N’– oxide, N–1,1–dimethylthiomethyleneamine, N–benzylideneamine, N–p– methoxybenzylideneamine, N–diphenylmethyleneamine, N–[(2– pyridyl)mesityl]methyleneamine, N–(N’,N’–dimethylaminomethylene)amine, N,N’– isopropylidenediamine, N–p–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 derivative, N–diphenylborinic acid derivative, N–[phenyl(pentacarbonylchromium– or tungsten)carbonyl]amine, N–copper chelate, N–zinc chelate, N–nitroamine, N–nitrosoamine, 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–nitropyridinesulfenamide (Npys), p–toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,–trimethyl–4– methoxybenzenesulfonamide (Mtr), 2,4,6–trimethoxybenzenesulfonamide (Mtb), 2,6–dimethyl– 4–methoxybenzenesulfonamide (Pme), 2,3,5,6–tetramethyl–4–methoxybenzenesulfonamide (Mte), 4–methoxybenzenesulfonamide (Mbs), 2,4,6–trimethylbenzenesulfonamide (Mts), 2,6– dimethoxy–4–methylbenzenesulfonamide (iMds), 2,2,5,7,8–pentamethylchroman–6– sulfonamide (Pmc), methanesulfonamide (Ms), ȕ–trimethylsilylethanesulfonamide (SES), 9– anthracenesulfonamide, 4–(4’,8’–dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[00180] Suitably protected carboxylic acids further include, but are not limited to, silyl–, alkyl–, alkenyl–, aryl–, and arylalkyl–protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t–butyldimethylsilyl, t–butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p– methoxybenzyl, 3,4–dimethoxybenzyl, trityl, t–butyl, 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.

[00181] Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t–butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p–methoxybenzyloxymethyl (PMBM), (4–methoxyphenoxy)methyl (p–AOM), guaiacolmethyl (GUM), t–butoxymethyl, 4–pentenyloxymethyl (POM), siloxymethyl, 2–methoxyethoxymethyl (MEM), 2,2,2–trichloroethoxymethyl, bis(2– chloroethoxy)methyl, 2–(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3– bromotetrahydropyranyl, tetrahydrothiopyranyl, 1–methoxycyclohexyl, 4– methoxytetrahydropyranyl (MTHP), 4–methoxytetrahydrothiopyranyl, 4– methoxytetrahydrothiopyranyl S,S–dioxide, 1–[(2–chloro–4–methyl)phenyl]–4– methoxypiperidin–4–yl (CTMP), 1,4–dioxan–2–yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a–octahydro–7,8,8–trimethyl–4,7–methanobenzofuran–2–yl, 1–ethoxyethyl, 1– (2–chloroethoxy)ethyl, 1–methyl–1–methoxyethyl, 1–methyl–1–benzyloxyethyl, 1–methyl–1– benzyloxy–2–fluoroethyl, 2,2,2–trichloroethyl, 2–trimethylsilylethyl, 2–(phenylselenyl)ethyl, t– butyl, allyl, p–chlorophenyl, p–methoxyphenyl, 2,4–dinitrophenyl, benzyl, p–methoxybenzyl, 3,4–dimethoxybenzyl, o–nitrobenzyl, p–nitrobenzyl, p–halobenzyl, 2,6–dichlorobenzyl, p–

cyanobenzyl, p–phenylbenzyl, 2–picolyl, 4–picolyl, 3–methyl–2–picolyl N–oxido, diphenylmethyl, p,p’–dinitrobenzhydryl, 5–dibenzosuberyl, triphenylmethyl, Į– 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–(imidazol–1–yl)bis(4’,4’’–dimethoxyphenyl)methyl, 1,1– bis(4–methoxyphenyl)–1’–pyrenylmethyl, 9–anthryl, 9–(9–phenyl)xanthenyl, 9–(9–phenyl–10– oxo)anthryl, 1,3–benzodithiolan–2–yl, benzisothiazolyl S,S–dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t–butyldimethylsilyl (TBDMS), t– butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri–p–xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t–butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p–chlorophenoxyacetate, 3–phenylpropionate, 4–oxopentanoate (levulinate), 4,4–(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4– methoxycrotonate, benzoate, p–phenylbenzoate, 2,4,6–trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9–fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2– trichloroethyl carbonate (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– napththyl carbonate, methyl dithiocarbonate, 2–iodobenzoate, 4–azidobutyrate, 4–nitro–4– methylpentanoate, o–(dibromomethyl)benzoate, 2–formylbenzenesulfonate, 2– (methylthiomethoxy)ethyl, 4–(methylthiomethoxy)butyrate, 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, monosuccinoate, (E)–2–methyl–2–butenoate, o– (methoxycarbonyl)benzoate, Į–naphthoate, nitrate, alkyl N,N,N’,N’– tetramethylphosphorodiamidate, alkyl N–phenylcarbamate, borate, dimethylphosphinothioyl, 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, 3,4–dimethoxybenzylidene acetal, 2–nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1– methoxyethylidene ortho ester, 1–ethoxyethylidine ortho ester, 1,2–dimethoxyethylidene ortho ester, Į–methoxybenzylidene ortho ester, 1–(N,N–dimethylamino)ethylidene derivative, Į– (N,N’–dimethylamino)benzylidene derivative, 2–oxacyclopentylidene ortho ester, di–t– butylsilylene group (DTBS), 1,3–(1,1,3,3–tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra–t–butoxydisiloxane–1,3–diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

[00182] In some embodiments, a 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, trifiuoroacetyl, 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)xanthine-9-y1 (MOX). In some embodiments, each of the hydroxyl protecting groups is, independently selected from acetyl, benzyl, t- butyldimethylsilyl, t-butyldiphenylsilyl and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4'-dimethoxytrityl group.

[00183] In some embodiments, a phosphorus protecting group is a group attached to the

Claims

1. A chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type, wherein the oligonucleotides target a mutant Huntingtin gene, and the length is from about 10 to about 50 nucleotides, wherein the backbone linkages comprise at least one phosphorothioate, and wherein the pattern of backbone chiral centers comprises at least one Rp chiral center and at least one Sp chiral center.

2. A chirally controlled oligonucleotide composition comprising oligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that a predetermined level of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers; or

a chirally controlled oligonucleotide composition comprising oligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that at least about 10% of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

3. The composition of claim 1, wherein the oligonucleotides comprise 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 and optionally comprises one or more chiral internucleotidic linkages; and

the core region independently has a length of two or more bases and independently comprises one or more chiral internucleotidic linkages.

4. The composition of claim 1, wherein oligonucleotides of the oligonucleotide type comprises at least one wing region and a core region, wherein:

each wing region independently has a length of two or more bases, and independently and optionally comprises one or more chiral internucleotidic linkages;

the core region independently has a length of two or more bases, and independently comprises one or more chiral internucleotidic linkages; and

wherein at least one nucleotide in a wing region differs from at least one nucleotide of the core region, wherein the difference is in one or more of:

1) backbone linkage;

2) pattern of backbone chiral centers;

3) sugar modification.

5. The composition of claim 1, wherein oligonucleotides of the same oligonucleotide type have identical structure.

6. The composition of claim 1, wherein the oligonucleotides comprise one or more natural

phosphate linkages
and one or more phosphorothioate linkages.

7. The composition of claim 1, wherein the oligonucleotides comprise a structure of wing-core-wing.

8. The composition claim 7, wherein a wing comprises a chiral internucleotidic linkage and

a natural phosphate linkage

9. The composition of claim 8, wherein the core comprises one or more phosphorothioate linkages.

10. The composition of claim 6, wherein each of the oligonucleotides comprises a modified sugar moiety.

11. The composition of claim 10, wherein the modified sugar moiety comprises a high-affinity sugar modification.

12. The composition of claim 10, wherein the modified sugar moiety has a 2’-modification. 13. The composition of claim 10, wherein the modified sugar moiety comprises a bicyclic sugar modification.

14. The composition of claim 10, wherein the modified sugar moiety comprises a 2’-modification, wherein a 2’-modification is 2’-OR1, wherein R1 is optionally substituted C1-6 alkyl. 15. The composition of claim 10, wherein the modified sugar moiety comprises a 2’-modification, wherein a 2’-modification is 2’-MOE.

16. The composition of claim 10, wherein the modified sugar moiety comprises a 2’-modification, wherein a 2’-modification is 2’-OMe.

17. The composition of claim 10, wherein the modified sugar moiety comprises a 2’-modification, wherein the 2’-modification is S-cEt.

18. The composition of claim 10, wherein the modified sugar moiety comprises a 2’-modification, wherein the 2’-modification is FANA.

19. The composition of claim 10, wherein the modified sugar moiety comprises a 2’-modification, wherein the 2’-modification is FRNA.

20. The composition of claim 10, wherein the modified sugar moiety has a 5’-modification. 21. The composition of claim 1 or 2, wherein the oligonucleotides comprise one or more natural phosphate linkages, and a pattern of backbone chiral centers comprising (Sp)t(Rp)n(Sp)m, wherein t is 2-10, n is 1, and m is 2-10, and at least one of t and m is greater than 5.

22. The composition of claim 6, wherein the oligonucleotides comprise a pattern of backbone chiral centers comprising SSR, RSS, SSRSS, SSRSSR, RSSSRSRRRS, RSSSSSSSSS,

SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR,

RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR,

RRSSRSRRRR, RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS,

RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, or

SSSSRRRRSR.

23. The composition of claim 22, wherein the oligonucleotides target a mutant Huntingtin gene comprising a single nucleotide polymorphism (SNP).

24. The composition of claim 23, wherein the single nucleotide polymorphism is selected from rs362307, rs7685686, rs362268, rs2530595, rs362331, and rs362306.

25. The composition of claim 1, wherein the oligonucleotides have a structure selected from Tables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595 and WV-2603.

26. The composition of claim 1, wherein the oligonucleotides are WV-1092.

27. The composition of claim 1, wherein the oligonucleotides are WV-2595.

28. The composition of claim 1, wherein the oligonucleotides are WV-2603.

29. A method for controlled cleavage of a nucleic acid polymer, the method comprising: contacting a nucleic acid polymer whose nucleotide sequence comprises a target sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length, wherein the common base sequence is or comprises a sequence that is complementary to a target sequence found in the nucleic acid polymer;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the particular base sequence and length, for

oligonucleotides of the particular oligonucleotide type.

30. A method for cleavage of a nucleic acid having a base sequence comprising a target sequence, the method comprising steps of:

(a) contacting a nucleic acid having a base sequence comprising a target sequence with a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length, wherein the common base sequence is or comprises a sequence that is complementary to the target sequence in the nucleic acid; 2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the particular base sequence and length, for

oligonucleotides of the particular oligonucleotide type, wherein the oligonucleotide targets a mutant Huntingtin gene, and the length is from about 10 to about 50 nucleotides, wherein the backbone linkages comprise at least one phosphorothioate, and wherein the pattern of backbone

chiral centers comprises at least one chiral center in a Rp conformation and at least one chiral center in a Sp conformation; and

(b) cleavage of the nucleic acid mediated by a RNAseH or RNA interference mechanism. 31. The method of claim 30, wherein the method is performed in vitro or in vivo.

32. The composition of claim 1, or the method of claim 30, wherein the composition further comprises one or more additional components selected from: a polynucleotide, carbonic anhydrase inhibitor, a dye, an intercalating agent, an acridine, a cross-linker, psoralene, mitomycin C, a porphyrin, TPPC4, texaphyrin, Sapphyrin, a polycyclic aromatic hydrocarbon phenazine, dihydrophenazine, an artificial endonuclease, a chelating agent, EDTA, an alkylating agent, a phosphate, an amino, a mercapto, a PEG, PEG-40K, MPEG, [MPEG]2, a polyamino, an alkyl, a substituted alkyl, a radiolabeled marker, an enzyme, a hapten biotin, a

transport/absorption facilitator, aspirin, vitamin E, folic acid, a synthetic ribonuclease, a protein, a glycoprotein, a peptide, a molecule having a specific affinity for a co-ligand, an antibody, a hormone, a hormone receptor, a non-peptidic species, a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, or a drug.

33. The composition of claim 1, or the method of claim 20, wherein the oligonucleotides are capable of participating in RNaseH-mediated cleavage of a mutant Huntingtin gene mRNA. 34. The composition of claim 1, or the method of claim 20, wherein the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of the oligonucleotides comprises or consists of the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of any of any oligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

35. The composition of claim 1, or the method of claim 20, wherein the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of the oligonucleotides comprises or consists of the base sequence, and pattern of backbone linkages, and/or pattern of backbone chiral centers of any of any oligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

36. The composition of claim 1, or the method of claim 20, wherein the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of the oligonucleotides comprises or consists of the base sequence, and pattern of backbone linkages, and pattern of

backbone chiral centers of any of any oligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

37. The composition of claim 1, or the method of claim 20, wherein the base sequence, pattern of backbone linkages and pattern of backbone chiral centers of the oligonucleotides comprises or consists of the base sequence, pattern of backbone linkages and/or pattern of backbone chiral centers of any of WV-1092, WV-2595, and WV-2603.

38. A composition comprising the composition of claim 1 and a selectivity agent selected from: the group of compounds which binds specifically to one or more neurotransmitter transporters selected from the group consisting of a dopamine transporter (DAT), a serotonin transporter (SERT), and a norepinephrine transporter (NET); the group consisting of a dopamine reuptake inhibitor (DRI), a selective serotonin reuptake inhibitor (SSRI), a noradrenaline reuptake inhibitor (NRI), a norepinephrine-dopamine reuptake inhibitor (NDRI), and a serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI); the group consisting of a triple reuptake inhibitor, a noradrenaline dopamine double reuptake inhibitor, a serotonin single reuptake inhibitor, a noradrenaline single reuptake inhibitor, and a dopamine single reuptake inhibitor; and the group consisting of a dopamine reuptake inhibitor (DRI), a Norepinephrine-Dopamine Reuptake Inhibitor (NDRI) and a serotonin-Norepinephrine-Dopamine Reuptake Inhibitor (SNDRI).

39. A method for preventing and/or treating Huntington’s disease in a subject, comprising administering to the subject a composition of claim 1.

40. The composition of any one of the preceding claims, further comprising artificial cerebrospinal fluid.

41. An oligonucleotide, an oligonucleotide composition, or a method selected from embodiments 1-606.