Compositions and methods for editing MECP2 transcripts

Abstract

In particular, the present disclosure provides oligonucleotides, compositions and methods that can effect specific editing of target adenosines in target RNA molecules, which are useful for treating, preventing or ameliorating MECP2-associated disorders, diseases and syndromes that may benefit from adenosine modification.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to one or more priority applications, including U.S. Provisional Patent Application No. 63 / 248,524, filed September 26, 2021, and U.S. Provisional Patent Application No. 63 / 341,391, filed May 12, 2022. Each of these priority applications is incorporated herein by reference in its entirety. [Background technology]

[0002] background Oligonucleotides are useful in a variety of applications, including therapeutic, diagnostic, and / or research applications. For example, oligonucleotides targeting various genes may be useful in treating conditions, disorders, or diseases associated with such target genes. Summary of the Invention [Means for solving the problem]

[0003] overview In particular, the present disclosure provides designed oligonucleotides and compositions thereof, wherein the oligonucleotides comprise modifications (e.g., modifications to the nucleobase sugar and / or internucleotide linkage and patterns thereof) as described herein. In some embodiments, the techniques (compounds (e.g., oligonucleotides), compositions, methods, etc.) of the present disclosure (e.g., oligonucleotides, oligonucleotide compositions, methods, etc.) are particularly useful for editing nucleic acids (e.g., site-specific editing in nucleic acids (e.g., editing of targeted adenosines)). In some embodiments, the provided techniques can significantly improve the efficiency of nucleic acid editing (e.g., modifying one or more A residues, such as converting A to I). In some embodiments, the present disclosure provides techniques for editing in RNA (e.g., modifying A residues, e.g., converting A to I). In some embodiments, the present disclosure provides techniques for editing in transcripts (e.g., mRNA) (e.g., modifying A residues, e.g., converting A to I). In particular, the provided technology offers the benefits of utilizing endogenous proteins, such as ADAR (adenosine deaminase acting on RNA) proteins (e.g., ADAR1 and / or ADAR2), to edit nucleic acids (e.g., to modify A (e.g., as a result of a G to A mutation)). Those skilled in the art will understand that such utilization of endogenous proteins may circumvent some challenges and / or provide various advantages compared to those technologies that require delivery of foreign components (e.g., proteins (e.g., engineered to bind to an oligonucleotide (and / or its duplex with a target nucleic acid) that provides a desired activity), nucleic acids encoding proteins, viruses, etc.).

[0004] In some embodiments, the present disclosure provides techniques, e.g., oligonucleotides, compositions, methods, etc., for targeting MECP2. In some embodiments, the provided techniques edit a target adenosine in MECP2. In some embodiments, the provided oligonucleotides form a duplex with an MECP2 transcript. In some embodiments, the target adenosine in the MECP2 transcript is edited by an ADAR polypeptide, e.g., ADAR1, ADAR2, etc.

[0005] In particular, in some embodiments, the provided technology can edit MECP2 mutations in a transcript to provide an edited transcript ("edited MECP2 protein") that encodes an MECP2 protein with improved properties and / or activity compared to the unedited mutant MECP2 protein. In some embodiments, the edited transcript provides one or more improved properties and / or activities compared to the mutant transcript. In some embodiments, the edited transcript provides one or more properties and / or activities equivalent to the wild-type transcript. In some embodiments, the edited MECP2 protein is a wild-type MECP2 protein. In some embodiments, the edited MECP2 protein contains an amino acid residue difference compared to the wild-type MECP2 protein. In some embodiments, the edited MECP2 protein differs from the wild-type MECP2 protein by a single amino acid residue. In some embodiments, the edited MECP2 protein exhibits one or more properties and / or activities equivalent to the wild-type MECP2 protein. In some embodiments, the mutation is a premature stop codon, e.g., R168X, R255X, R270X, or R294X. In some embodiments, the edited MECP2 protein comprises R168W, R255W, R270W, or R294W, and the corresponding mutant MECP2 protein comprises R168X, R255X, R270X, or R294X, respectively.

[0006] In some embodiments, the present disclosure provides methods for preventing or treating conditions, disorders, or diseases associated with MECP2, particularly those associated with R168X, R255X, R270X, and / or R294X mutations in MECP2, comprising administering to a subject susceptible to or suffering from the same an effective amount of a provided oligonucleotide or composition thereof. In some embodiments, the subject has R168X, R255X, R270X, and / or R294X in MECP2. In some embodiments, after treatment, one or more symptoms are ameliorated, progression is slowed, halted, or reversed, and / or one or more functions are improved. In some embodiments, the condition, disorder, or disease is Rett syndrome.

[0007] As described herein, in some embodiments, oligonucleotides of the provided technology include useful sugar modifications and / or patterns thereof (e.g., the presence and / or absence of particular modifications), nucleobase modifications and / or patterns thereof (e.g., the presence and / or absence of particular modifications), internucleotide linkage modifications and / or stereochemistry and / or patterns thereof (e.g., the type, modification, and / or configuration of chiral phosphorus (Rp or Sp), etc.), etc., which, when combined with one or more other structural elements (e.g., additional chemical moieties) described herein, can provide enhanced activity and / or various desirable properties (e.g., enhanced nucleic acid editing efficiency, enhanced selectivity, enhanced stability, enhanced cellular uptake, reduced immune stimulation, reduced toxicity, improved distribution, improved affinity, etc.). In some embodiments, provided oligonucleotides provide enhanced stability, for example, compared to oligonucleotides having a higher percentage of natural RNA sugars utilized for adenosine editing. In some embodiments, provided oligonucleotides provide enhanced activity (e.g., adenosine editing activity). In some embodiments, provided oligonucleotides provide high selectivity, for example, in some embodiments, provided oligonucleotides provide for selective modification of a target adenosine in a target nucleic acid over other adenosines in the same target nucleic acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold or more modification of the target adenosine compared to another adenosine or all other adenosines in the target nucleic acid).

[0008] In some embodiments, the present disclosure provides an oligonucleotide comprising a first domain and a second domain, wherein the first domain comprises one or more 2'-F modifications and the second domain comprises one or more sugars without 2'-F modifications. In some embodiments, the provided oligonucleotide comprises one or more chiral modified internucleotide linkages. In some embodiments, the present disclosure provides (a) a first domain; and (b) Second Domain and providing an oligonucleotide comprising: the first domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more sugars that comprise a 2'-F modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the first domain comprise a 2'-F modification; The second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more modified sugars that do not contain a 2'-F modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the second domain do not contain a 2'-F modification.

[0009] In some embodiments, the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain as described herein.

[0010] In some embodiments, the second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more modified sugars that independently comprise a 2'-OR modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the second domain are 2'-OR modified (where R is an optionally substituted C 1~6 In some embodiments, R is methyl. In some embodiments, R is CH2CH2OCH3. As described herein, other sugar modifications may also be utilized in accordance with the present disclosure, optionally in conjunction with the base modifications and / or internucleotide linkage modifications described herein.

[0011] In some embodiments, the base sequence of the provided oligonucleotide is substantially complementary to the base sequence of the target nucleic acid containing the target adenosine. In some embodiments, the provided oligonucleotide contains one or more mismatches (non-Watson-Crick base pairs) when aligned with the target nucleic acid. In some embodiments, the provided oligonucleotide contains one or more wobble sequences (e.g., GU, IA, GA, IU, IC, etc.) when aligned with the target nucleic acid. In some embodiments, the mismatches and / or wobble sequences may assist one or more proteins (e.g., ADAR1, ADAR2, etc.) in recognizing the duplex formed by the provided oligonucleotide and the target nucleic acid. In some embodiments, the provided oligonucleotide forms a duplex with the target nucleic acid. In some embodiments, an ADAR protein recognizes and binds to such a duplex. In some embodiments, the nucleoside opposite the target adenosine is located in the middle of the provided oligonucleotide, e.g., with 5 to 50 nucleosides on the 5' side and 1 to 50 nucleosides on the 3' side. In some embodiments, the 5' side has more nucleosides than the 3' side. In some embodiments, the 5' side has fewer nucleosides than the 3' side. In some embodiments, the 5' side has the same number of nucleosides as the 3' side. In some embodiments, the provided oligonucleotides comprise 15 to 40 (e.g., 15, 20, 25, 30, etc.) consecutive bases of an oligonucleotide set forth in a table. In some embodiments, the base sequence of the provided oligonucleotides is or comprises the base sequence of an oligonucleotide set forth in a table.

[0012] In some embodiments, by utilizing various structural elements (e.g., various modifications, stereochemistries, and patterns thereof), the present disclosure can achieve desirable properties and high activity with short oligonucleotides (e.g., about 20-40, 25-40, 25-35, 26-32, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length). In some embodiments, the provided technology allows for the development of 2'-OR saModified sugar (each R sa independently represents an optionally substituted C 1~6 In some embodiments, each R provides various properties, activities (e.g., A to I editing), advantages, etc. described herein without utilizing a long stretch (e.g., about or at least about 5 consecutive sugars) and / or a high level (e.g., about or at least about 50%) of -OH-4' (where L is an optionally substituted -CH2- (e.g., an LNA sugar, a cEt sugar, etc.)). sa independently represents an optionally substituted C 1~6 In some embodiments, R sa is methyl. In some embodiments, R sa is —CH2CH2OCH3. In some embodiments, each R sa is independently methyl or —CHCHOCH. In some embodiments, each R sa is methyl. In some embodiments, the provided techniques do not utilize a long range and / or high levels of 2'-OMe modified sugars. In some embodiments, the provided techniques do not utilize a long range or high levels of 2'-OMe modified sugars. saIn some embodiments, the long stretch does not utilize modified sugars. In some embodiments, the long stretch is about or at least about 5, 6, 7, 8, 9, or 10 consecutive sugars. In some embodiments, it is about or at least about 6; in some embodiments, it is about or at least about 7; in some embodiments, it is about or at least about 8; in some embodiments, it is about or at least about 9; in some embodiments, it is about or at least about 10. In some embodiments, the high level is about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all nucleosides. In some embodiments, this is about or at least about 50%; in some embodiments, this is about or at least about 55%; in some embodiments, this is about or at least about 60%; in some embodiments, this is about or at least about 65%; in some embodiments, this is about or at least about 70%; in some embodiments, this is about or at least about 75%; in some embodiments, this is about or at least about 80%; in some embodiments, this is about or at least about 85%; in some embodiments, this is about or at least about 90%; in some embodiments, this is about or at least about 95%.

[0013] In some embodiments, provided oligonucleotides comprise a modified nucleobase. In some embodiments, the modified nucleobase facilitates modification of the target adenosine. In some embodiments, the nucleobase opposite the target adenine maintains interaction with an enzyme (e.g., ADAR) compared to when U is present, while interacting less strongly with the target adenosine (e.g., forming fewer hydrogen bonds) compared to U. In some embodiments, the opposite nucleobase and / or its associated sugar provide particular flexibility (e.g., compared to U) to facilitate modification of the target adenosine by an enzyme, e.g., ADAR1, ADAR2, etc. In some embodiments, the nucleoside opposite the target adenosine, e.g., N0, is C. In some embodiments, it does not base pair with A. In some embodiments, it does not base pair with A, such as T or U. In some embodiments, it base pairs with G or I. In some embodiments, the nucleobase immediately 5' or 3' of the opposite nucleobase (relative to the target adenosine), e.g., I and derivatives thereof, enhances modification of the target adenosine. In particular, the present disclosure recognizes that such nucleobases may provide less steric hindrance than G when a duplex of a provided oligonucleotide and its target nucleic acid interacts with a modifying enzyme (e.g., ADAR1 or ADAR2). In some embodiments, the base sequence of the oligonucleotide is selected (e.g., when multiple adenosine residues are suitable targets) and / or designed (e.g., through the use of various nucleobases described herein) such that steric hindrance can be reduced or eliminated (e.g., the adjacent nucleoside opposite the target A is not G).

[0014] A variety of internucleotide linkages can be utilized in the oligonucleotides provided in accordance with the present disclosure. In some embodiments, the oligonucleotide comprises one or more types of internucleotide linkage. In some embodiments, the oligonucleotide comprises two or more types of internucleotide linkage. In some embodiments, the oligonucleotide comprises at least three types of internucleotide linkage. In some embodiments, the linkage contains a linked phosphorus atom where the oxygen atom is linked to an oxygen atom that is not linked to or part of the backbone sugar (a "PO linkage," e.g., a natural phosphate linkage). In some embodiments, the linkage contains a linked phosphorus atom where the oxygen atom is linked to an oxygen atom that is not linked to or part of the backbone sugar (a "PS linkage," e.g., a phosphorothioate internucleotide linkage). In some embodiments, the linkage contains a linked phosphorus atom where the sulfur atom is linked to a sulfur atom that is not linked to or part of the backbone sugar (a "PS linkage," e.g., a phosphorothioate internucleotide linkage). In some embodiments, the linkage contains a linked phosphorus atom where the nitrogen atom is linked to a nitrogen atom that is not linked to or part of the backbone sugar (a "PN linkage," e.g., n001). In some embodiments, the oligonucleotide contains one or more PS linkages. In some embodiments, the oligonucleotide contains one or more PO linkages. In some embodiments, the oligonucleotide contains one or more PN linkages. In some embodiments, the oligonucleotide contains one or more PS and one or more PO linkages. In some embodiments, the oligonucleotide contains one or more PS and one or more PN linkages. In some embodiments, the oligonucleotide comprises one or more PS, one or more PN, and one or more PO linkages.

[0015] In some embodiments, the first domain comprises one or more PO linkages, one or more PS linkages, and one or more PN linkages. In some embodiments, the first subdomain comprises one or more PO linkages, one or more PS linkages, and / or one or more PN linkages. In some embodiments, the first subdomain comprises one or more PO linkages. In some embodiments, the first subdomain comprises one or more natural phosphate linkages. In some embodiments, the second subdomain comprises one or more modified internucleotide linkages. In some embodiments, each internucleotide linkage attached to a nucleoside of the second subdomain is independently a modified internucleotide linkage. In some embodiments, each internucleotide linkage attached to a nucleoside of the second subdomain is independently a PS or PN linkage. In some embodiments, the third subdomain comprises one or more PO linkages, one or more PS linkages, and / or one or more PN linkages. In some embodiments, the third subdomain comprises one or more PO linkages. In some embodiments, the third subdomain comprises one or more PO linkages. In some embodiments, the third subdomain comprises one or more natural phosphate linkages. In some embodiments, the third subdomain comprises one or more PS linkages. In some embodiments, the third subdomain comprises one or more PN linkages. In some embodiments, the third subdomain comprises one or more PO linkages, one or more PS linkages, and one or more PN linkages. In some embodiments, the first internucleotide linkage of the first domain or oligonucleotide is a PN linkage. In some embodiments, the last internucleotide linkage of the third subdomain or oligonucleotide is a PN linkage. In some embodiments, the natural DNA sugar is linked to a modified internucleotide linkage. In some embodiments, the natural DNA sugar is linked to a PN or PS internucleotide linkage. In some embodiments, each natural DNA sugar in an oligonucleotide or portion thereof (e.g., first domain, first subdomain, second subdomain, third subdomain, etc.) is independently linked to a modified internucleotide linkage. In some embodiments, each natural DNA sugar is independently linked to a PN or PS internucleotide linkage.In some embodiments, the natural RNA sugar is linked to a modified internucleotide linkage. In some embodiments, the natural RNA sugar is linked to a PN or PS internucleotide linkage. In some embodiments, each natural RNA sugar in an oligonucleotide or portion thereof (e.g., first domain, first subdomain, second subdomain, third subdomain, etc.) is independently linked to a modified internucleotide linkage. In some embodiments, each natural RNA sugar is independently linked to a PN or PS internucleotide linkage.

[0016] In some embodiments, the 2'-F modified sugar is linked to a modified internucleotide linkage. In some embodiments, the 2'-F modified sugar is linked to a PN or PS internucleotide linkage. In some embodiments, each 2'-F modified sugar in an oligonucleotide or portion thereof (e.g., the first domain, first subdomain, second subdomain, third subdomain, etc.) is independently linked to a modified internucleotide linkage. In some embodiments, each 2'-F modified sugar is independently linked to a PN or PS internucleotide linkage. In some embodiments, each PO linkage is independently a natural phosphate linkage. In some embodiments, each PS linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, one or more PN linkages are independently a non-negatively charged internucleotide linkage. In some embodiments, one or more PN linkages are independently a natural internucleotide linkage. In some embodiments, one or more PN linkages are independently a phosphorylguanidine linkage. In some embodiments, each PN linkage is independently a phosphorylguanidine linkage. In some embodiments, one or more PN bonds are independently n001. In some embodiments, each PN bond is independently n001.

[0017] In some embodiments, the oligonucleotides of the present disclosure contain a modified internucleotide linkage (i.e., an internucleotide linkage that is not a natural phosphate linkage). In some embodiments, the linking phosphorus of the modified internucleotide linkage (e.g., a chiral internucleotide linkage) is chiral and can exist in different configurations (Rp and Sp). For example, in a phosphorothioate internucleotide linkage (-OP(O)(SH)-O-, which can exist as a natural phosphate linkage and in various salt forms), the linking phosphorus can be either Rp or Sp. Thus, conventional oligonucleotide compositions of oligonucleotides containing chiral linking phosphorus are mixtures of multiple stereoisomers. For example, conventional compositions prepared without chiral control of the linking phosphorus center of the oligonucleotide (e.g., phosphorothioate internucleotide linkage, n001 linkage, etc.) contain up to 2 N It can be a mixture of 1,000 stereoisomers (N is the number of chiral bonded phosphorus centers). When there are 10 such chiral bonded phosphorus centers (N=10), it can be a mixture of up to 1,000 (2 10 ) stereoisomers; when there are 20 such chiral bonded phosphorus centers, it can have up to 1 million (2 20) stereoisomers. Structurally, these stereoisomers may share the same configuration or be identical except for different stereochemistry along the backbone chiral center of the chiral linking phosphorus atom, but in various instances may have dramatically different activities and / or properties. In some embodiments, such oligonucleotide compositions are referred to as stereoirregular oligonucleotide compositions. In contrast to such stereoirregular oligonucleotide compositions, many compositions of the present disclosure are chiral-controlled oligonucleotide compositions, in which one or more or all of the selected configurations of the chiral linking phosphorus centers, Rp or Sp, are independently enriched compared to stereoirregular oligonucleotide compositions. In some embodiments, one or more or all of the selected configurations of the linking phosphorus centers are independently enriched to a level as described herein (e.g., about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%). In particular, the incorporation of modified internucleotide linkages, particularly through control of the stereochemistry at the linking phosphorus center (enriching one configuration at such controlled centers compared to stereoirregular oligonucleotide preparations), can significantly improve properties (e.g., stability) and / or activity (e.g., adenosine-modifying activity (e.g., converting adenosine to inosine)). In some embodiments, provided oligonucleotides have significantly higher stereochemical purity compared to stereoirregular preparations. In some embodiments, provided oligonucleotides are chiral controlled.

[0018] In some embodiments, oligonucleotides of the present disclosure include one or more chiral internucleotide linkages (e.g., phosphorothioate internucleotide linkages) in which the linked phosphorus is chiral. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50%-100%, 60%-100%) of all internucleotide linkages in the oligonucleotide are chiral. In some embodiments, at least one internucleotide linkage is a chiral internucleotide linkage. In some embodiments, at least one internucleotide linkage is a chiral internucleotide linkage. In some embodiments, at least one internucleotide linkage is a natural phosphate linkage. In some embodiments, each internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, at least one chiral internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, at least one chiral internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, at least one chiral internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, one or more chiral internucleotide linkages are independently non-negatively charged or neutral. In some embodiments, one or more chiral internucleotide linkages are independently phosphorylguanidine internucleotide linkages. In some embodiments, one or more chiral internucleotide linkages are independently chiral controlled. In some embodiments, each chiral internucleotide linkage is independently chiral controlled. In some embodiments, one or more chiral internucleotide linkages are not chiral controlled. In some embodiments, each phosphorothioate internucleotide linkage is independently chiral controlled.In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a phosphorylguanidine internucleotide linkage. In some embodiments, a phosphorylguanidine internucleotide linkage is nOOl. In some embodiments, a phosphorylguanidine internucleotide linkage is nOOl. In some embodiments, each non-negatively charged internucleotide linkage is nOOl. In some embodiments, each neutral internucleotide linkage is nOOl. The bound phosphorus can be either Rp or Sp. In some embodiments, at least one bound phosphorus is Rp. In some embodiments, at least one bound phosphorus is Sp. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50% to 100%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%) of all chiral internucleotide linkages in the oligonucleotide are chiral. % to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 60% to 95%, 70% to 95%, 75% to 95%, 80% to 95%, 85% to 95%, 90% to 95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% etc.) or all chiral internucleotide linkages are Sp.In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50% to 100%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%) of all phosphorothioate internucleotide linkages in the oligonucleotide. Among these, at least 100%, 70% to 100%, 75% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 60% to 95%, 70% to 95%, 75% to 95%, 80% to 95%, 85% to 95%, 90% to 95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50% to ...100%, 75% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 60% to 95%, 70% to 100%, 75% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 60% to 95%, 70% to 100%, In some embodiments, at least 80% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 85% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 90% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 95% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 96% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 97% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 98% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, all phosphorothioate internucleotide linkages are Sp. In some embodiments, no more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive phosphorothioate internucleotide linkages are Rp.In some embodiments, no more than three consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than four consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than five consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than six consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than seven consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than eight consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than nine consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than ten consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, phosphorothioate internucleotide linkages of consecutive Rp are not utilized in a moiety where a majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all of the sugars are natural DNA and / or RNA and / or 2'-F modified sugars. In some embodiments, when phosphorothioate internucleotide linkages of consecutive Rp are utilized, one or more or a majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all of such internucleotide linkages are independently linked to sugars that may improve stability. In some embodiments, when phosphorothioate internucleotide linkages of consecutive Rp are utilized, one or more, or a majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or higher), or all of such internucleotide linkages are independently bicyclic sugars or 2'-OR modified sugars (where R is optionally substituted C). 1~6In some embodiments, when phosphorothioate internucleotide linkages of consecutive Rp are utilized, one or more, or a majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or higher), or all of such internucleotide linkages are independently linked to a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each 2'-OR-modified sugar is independently a 2'-OMe-modified sugar or a 2'-MOE-modified sugar. In some embodiments, each 2'-OR-modified sugar is independently a 2'-OMe-modified sugar. In some embodiments, each 2'-OR-modified sugar is independently a 2'-MOE-modified sugar.

[0019] In some embodiments, the stereochemistry of one or more chiral internucleotide linkages of a provided oligonucleotide is controlled in the composition. In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides, the plurality of oligonucleotides having a common base sequence and one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, chiral internucleotide linkages ("chiral-controlled internucleotide linkages") share the same configuration of the independent linking phosphorus (e.g., all Rp or all Sp with respect to the chiral linking phosphorus). In some embodiments, they share the same stereochemistry at their respective chiral linking phosphorus. In some embodiments, multiple oligonucleotides share the same configuration. In some embodiments, multiple oligonucleotides are structurally identical except for the internucleotide linkage. In some embodiments, multiple oligonucleotides are structurally identical. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all of the oligonucleotides in the composition, or all of the oligonucleotides sharing a common base sequence, share the pattern of chiral centers in the backbones of the plurality of oligonucleotides. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all of the oligonucleotides in the composition, or all of the oligonucleotides sharing a common base sequence, are a plurality of oligonucleotides.

[0020] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition, or all oligonucleotides having the same base sequence, or all oligonucleotides having the same base sequence and sugar and base modifications, or all oligonucleotides of the same composition, have one or more chiral internucleotide linkages (e.g., about 1-50, ... 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of chiral internucleotide linkages independently share a linking phosphorus of the same configuration (e.g., all Rp or all Sp with respect to the chiral linking phosphorus). In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all of the oligonucleotides in the composition, or all of the oligonucleotides having the same base sequence, or all of the oligonucleotides having the same base sequence and sugar and base modifications, or all of the oligonucleotides of the same composition, are in one or more forms of the oligonucleotide (e.g., an acid form, a salt form (e.g., a pharmaceutically acceptable salt form; as will be understood by one of skill in the art, if the oligonucleotide is a salt, other salt forms of the corresponding acid or base form of the oligonucleotide), etc.).

[0021] In some embodiments, chiral controlled oligonucleotide compositions offer several advantages (e.g., higher stability, activity, etc.) compared to corresponding stereoirregular oligonucleotide compositions. In some embodiments, chiral controlled oligonucleotide compositions provide high levels of adenosine modifying (e.g., A to I conversion) activity with various isoforms of ADAR proteins (e.g., the p150 and p110 forms of ADAR1), while corresponding stereoirregular compositions provide high levels of adenosine modifying (e.g., A to I conversion) activity only with specific isoforms of ADAR proteins (e.g., the p150 isoform of ADAR1).

[0022] In some embodiments, provided oligonucleotides comprise an additional moiety (e.g., a targeting moiety, a carbohydrate moiety, etc.). In some embodiments, the additional moiety is or comprises a ligand for the asialoglycoprotein receptor. In some embodiments, the additional moiety is or comprises GalNAc or a derivative thereof. In particular, the additional moiety may facilitate delivery to a particular target site (e.g., a cell, tissue, organ, etc. (e.g., a site comprising a receptor that interacts with the additional moiety)). In some embodiments, the additional moiety facilitates delivery to the liver.

[0023] In some embodiments, the present disclosure provides techniques for preparing oligonucleotides and compositions thereof, particularly chiral controlled oligonucleotide compositions. In some embodiments, the provided oligonucleotides and compositions thereof are of high purity. In some embodiments, the oligonucleotides of the present disclosure are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically pure at the linkage phosphorus of the chiral internucleotide linkage. In some embodiments, the oligonucleotides of the present disclosure are stereoselectively prepared and are substantially free of stereoisomers. In some embodiments, provided compositions include a plurality of oligonucleotides sharing the same base sequence with the same pattern of stereochemistry at the chiral linking phosphorus (e.g., each chiral linking phosphorus independently comprises one or more of Rp and / or Sp, where Rp or Sp), at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same base sequence with the plurality of oligonucleotides share the same pattern of stereochemistry at the chiral linking phosphorus, or are the plurality of oligonucleotides. In some embodiments, provided compositions include a plurality of oligonucleotides sharing the same base sequence with the same pattern of stereochemistry at the chiral linking phosphorus, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same configuration with the plurality of oligonucleotides share the same pattern of stereochemistry at the chiral linking phosphorus, or are the plurality of oligonucleotides.

[0024] In some embodiments, the present disclosure describes useful techniques for evaluating oligonucleotides and compositions thereof. For example, various techniques of the present disclosure are useful for evaluating adenosine modification. As will be understood by those skilled in the art, in some embodiments, adenosine modification / editing can be evaluated, optionally in light of the presence of other components (e.g., ADAR proteins) in a modification system (e.g., an in vitro system, an ex vivo system, a cell, a tissue, an organ, an organism, a subject, etc.), through sequencing, mass spectrometry, evaluation (e.g., levels, activity, etc.) of products (e.g., RNA, protein, etc.) of modified nucleic acids (e.g., adenosine of a target nucleic acid is converted to inosine). Those skilled in the art will understand that an oligonucleotide that results in adenosine modification of a target nucleic acid can also provide a modified nucleic acid (e.g., adenosine of a target nucleic acid is converted to I) and one or more of its products (e.g., mRNA, protein, etc.). Certain useful techniques are described in the Examples.

[0025] As described herein, the oligonucleotides and compositions of the present disclosure may be provided / utilized in a variety of forms. In some embodiments, the present disclosure provides oligonucleotides in one or more forms (e.g., an acid form (e.g., a native phosphate linkage exists as -(P(O)(OH)-O- and a phosphorothioate internucleotide linkage exists as -O(P(O)(SH)-O-), a base form, a salt form (e.g., a native phosphate linkage exists as a salt form (e.g., a sodium salt (-O(P(O)(O - Na + )-O-), and phosphorothioate internucleotide linkages exist in salt form (e.g., sodium salt (-O(P(O)(S - Na +As will be appreciated by those skilled in the art, oligonucleotides may exist in various salt forms and solutions (e.g., various aqueous buffer systems), including pharmaceutically acceptable salts, and the cation may dissociate from the anion. In some embodiments, the present disclosure provides pharmaceutical compositions comprising a provided oligonucleotide and / or one or more pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is a chiral controlled oligonucleotide composition.

[0026] The provided technology can be utilized for a variety of purposes. For example, those skilled in the art will understand that the provided technology is useful for many purposes, including modifying adenosines, e.g., correcting G to A mutations, modulating the level and / or activity of particular nucleic acids and / or their encoded products (e.g., decreasing or increasing the level of a protein by introducing a G / I modification at A, decreasing or increasing the activity of a protein by introducing a G / I modification at A in a transcript encoding such a protein), modulating splicing, modulating translation (e.g., modulating translation start and / or stop sites by introducing a G / I modification at A), modulating interactions (e.g., increasing or decreasing interactions of proteins, nucleic acids, small molecules, carbohydrates, lipids, etc. with proteins, nucleic acids, etc.), etc.

[0027] In some embodiments, the present disclosure provides techniques for preventing or treating conditions, disorders, or diseases that are susceptible to adenosine modifications (e.g., A to I or G conversion). As will be understood by one of skill in the art, I can perform one or more functions of G, for example, in base pairing, translation, etc. In some embodiments, a G to A mutation can be corrected via an A to I conversion so that one or more products (e.g., proteins) of the G form of the nucleic acid can be produced. In some embodiments, the present disclosure provides techniques for preventing or treating conditions, disorders, or diseases associated with the mutation, comprising administering to a subject susceptible to or suffering from the mutation a provided oligonucleotide or composition thereof, wherein the oligonucleotide or composition can edit the mutation. In some embodiments, the present disclosure provides techniques for preventing or treating conditions, disorders, or diseases associated with a G to A mutation, comprising administering to a subject susceptible to or suffering from the mutation a provided oligonucleotide or composition thereof, wherein the oligonucleotide or composition can modify the A. In some embodiments, the provided techniques modify the A in a transcript (e.g., an RNA transcript). In some embodiments, A is converted to I. In some embodiments, during translation, the protein synthesis machinery reads I as G. In some embodiments, a G / I type encodes one or more proteins having one or more higher desired activities and / or one or more better desired properties compared to that encoded by its corresponding A type. In some embodiments, a G / I type provides higher levels of one or more proteins having one or more higher desired activities and / or one or more better desired properties compared to its corresponding A type. In some embodiments, a product encoded by a G / I type is structurally different (e.g., a longer, in some embodiments, full-length protein) from that encoded by its corresponding A type. In some embodiments, a G / I type provides a structurally identical product (e.g., protein) compared to its corresponding A type, but the G / I type provides such product at a more desirable level. [Brief explanation of the drawings]

[0028] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various minimally spaced nucleoside modifications, can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2. Cells were also transfected with the MECP2-GFP R168X mutant construct and (A) ADAR1-p110 or (B) ADAR2 construct. RNA editing was measured by Sanger sequencing (n=2 biological replicates, * indicates n=1). "WV-" may not be included in the ID (e.g., "42125" is WV-42125). [Figure 2] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various minimally spaced nucleoside modifications, can provide the desired activity. HEK293T cells were transfected with oligonucleotide compositions targeting the premature TGA stop codon in MECP2. Cells were also transfected with (A) ADAR1-p150 and MECP2-GFP R255X mutant constructs, (B) ADAR1-p150 and MECP2-GFP R270X mutant constructs, and (C) ADAR2 and MECP2-GFP R270X mutant constructs. RNA editing was measured by Sanger sequencing (n=2 biological replicates, * indicates n=1). [Figure 3]The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various minimally spaced nucleoside modifications, can provide the desired activity. HEK293T cells were transfected with oligonucleotides targeting the premature TGA stop codons in MECP2 and ADAR1-p150. Cells were also transfected with (A) the MECP2-GFP R168X mutant construct, (B) the MECP2-GFP R255X mutant construct, and (C) the MECP2-GFP R270X mutant construct. RNA editing was measured by Sanger sequencing (n=2 biological replicates, * indicates n=1 sample). [Figure 4] The provided technology can produce edited proteins. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2, ADAR1-p150, and the MECP2-GFP R168X mutant construct. Cells were lysed two days after treatment and analyzed by Western blotting using an MECP2 antibody. Detected bands include endogenous MECP2 and the edited MECP2-GFP fusion protein (S1, S2, and S3 represent biological sample replicates). [Figure 5]The provided techniques can produce edited proteins with desired properties and activities. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2, MECP2-GFP R168X mutant constructs, and either ADAR1-p150, ADAR1-p110, or ADAR2. (A) RNA editing was measured by Sanger sequencing (n=1 biological replicate). (B) Proteins were generated from MECP2 R168X editing. Two days after oligonucleotide treatment, cells were lysed for nuclear fraction analysis by Western blotting using GFP and MECP2 antibodies. Detected bands include endogenous MECP2 and the edited MECP2-GFP fusion protein. Histone H3 was used as a loading control for the nuclear fraction. [Figure 6] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various nucleosides opposite the target adenosine and adjacent nucleosides, can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and the MECP2-GFP R168X mutant construct. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates, * indicates n=1). [Figure 7]The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various nucleosides opposite the target adenosine and / or adjacent nucleoside, can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and the MECP2-GFP R168X mutant construct. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates, * indicates n=1). [Figure 8] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various nucleosides opposite the target adenosine and / or adjacent nucleoside, can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and the MECP2-GFP R168X mutant construct. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates, * indicates n=1). [Figure 9] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various nucleosides opposite the target adenosine and / or adjacent nucleoside, can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and the MECP2-GFP R168X mutant construct. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates, * indicates n=1 sample). [Figure 10] The provided technology can provide editing. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). In some embodiments, the nucleoside opposite the target adenosine at positions 24, 25, or 26, or 23, 24, 25, 26, or 22, 23, 24, or 25, provides higher editing levels compared to other positions. [Figure 11] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns thereof can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 12] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns thereof can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 13]The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry and patterns, and various shortest distances between nucleobases (e.g., N-1) can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n = 2 biological replicates). [Figure 14] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various nucleosides opposite the target adenosine and / or adjacent nucleosides, can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and the MECP2-GFP R168X mutant construct. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 15] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns thereof can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). In some embodiments, the nucleoside opposite the target adenosine at position 22, 23, 24, or 25 provides higher editing levels compared to other positions. [Figure 16] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns thereof can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). In some embodiments, the nucleoside opposite the target adenosine at position 22, 23, 24, 25, or 26 provides higher editing levels compared to other positions. [Figure 17] The provided technology can provide editing. In some embodiments, oligonucleotides with and without CpG chemical modifications can provide editing. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 18] The provided technology can provide editing. As demonstrated, oligonucleotides containing various sugar, base, and / or linkage modifications, stereochemistry, and patterns, including various nucleosides opposite the target adenosine, can provide the desired activity. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and the MECP2-GFP R168X mutant construct. Cells were also transfected with (A) ADAR1-P150, (B) ADAR1-P110, and (C) ADAR2. RNA editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 19]The provided technology can provide editing. As demonstrated, oligonucleotides can provide high editing levels with or without non-targeting oligonucleotides. In some embodiments, the presence of certain non-targeting oligonucleotides increases editing levels. HEK293T cells were transfected with the indicated oligonucleotide compositions targeting the premature TGA stop codon in MECP2 and MECP2-GFP R168X mutant constructs at a dose of 12.5 nM (approximately 1.25 μg / well). Cells were also transfected with ADAR1-p110 plasmid. After 48 hours, cells were harvested and RNA editing was measured by Sanger sequencing (n=2 biological replicates). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 20] The provided technology can provide editing. In some embodiments, chemical modification of the nucleoside opposite the target adenosine and / or various shortest distances can provide the desired activity. In some embodiments, chemical modification of the nucleoside opposite the target adenosine and / or various shortest distances can provide increased levels of editing. Oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at either 10 μM or 3.3 μM doses as indicated. A-to-G editing was measured by amplicon sequencing 5 days after treatment (n=2 biological replicates, NA=sample not analyzed). The X-axis denotes oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45115" is WV-45115)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 21]The provided techniques can provide editing. In some embodiments, various minimally spaced nucleosides and editing site locations can provide the desired activity. Oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at doses of either 10 μM or 3.3 μM as indicated. A-to-G editing was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates, NA = sample not analyzed). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45131" is WV-45131)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 22] The provided technology can provide editing. In some embodiments, various chemical modifications and internucleotide linkages can provide the desired activity. Oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at doses of either 10 μM or 3.3 μM as indicated. A-to-G editing was measured by amplicon sequencing 5 days after treatment (n=2 biological replicates). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45096" is WV-45096)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 23]The provided technology can provide editing. In some embodiments, varying positions of the PN internucleotide linkage can provide editing. In some embodiments, varying positions of the PN internucleotide linkage can provide increased levels of editing. The oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=4 biological replicates). The X-axis refers to the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 24] The provided technology can provide editing. In some embodiments, various chemical modifications can provide the desired activity. In some embodiments, various chemical modifications can provide increased levels of editing. The oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=2 biological replicates). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 25]The provided technology can provide editing. In some embodiments, varying the position of the PO internucleotide linkage can provide the desired activity. In some embodiments, varying the position of the PO internucleotide linkage can provide increased levels of editing. The oligonucleotides all have the same sequence targeting the premature TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=2 biological replicates). The X-axis refers to the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 26] The provided technology can provide editing. In some embodiments, various chemical modifications can provide the desired activity. In some embodiments, various chemical modifications can provide increased levels of editing. The oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=2 biological replicates). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 27]The provided technology can provide editing. In some embodiments, varying stereochemistry or patterns can provide the desired activity. In some embodiments, varying stereochemistry or patterns can provide increased levels of editing. Oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=2 biological replicates). The X-axis refers to the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 28] The provided technology can provide editing. In some embodiments, various chemical modifications, internucleotide linkages, stereochemistry, and patterns, as well as the location of the editing site, can provide the desired activity. The oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=2 biological replicates). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 29]The provided technology can provide editing. In some embodiments, various positions of the wobble can provide the desired activity. The oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=2 biological replicates). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. [Figure 30] The provided technology can provide editing. In some embodiments, various positions of mismatches can provide the desired activity. The oligonucleotide compositions targeted all premature TGA stop codons within the MECP2 coding sequence. Patient-derived (MECP2R168X) cortical neurons were treated with the indicated compositions via gymnosis uptake at a dose of 10 μM. A-to-G editing was measured by amplicon sequencing 6 days after treatment (n=2 biological replicates). The X-axis represents the oligonucleotide composition ("XV-" may not be included in the composition ID (e.g., "45129" is WV-45129)), while the Y-axis represents the percentage of editing. Error bars represent the standard error of the mean. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of Specific Embodiments The techniques of the present disclosure may be more readily understood by reference to the following detailed description of specific embodiments.

[0030] definition As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS system, Handbook of Chemistry and Physics, 75th Edition. Furthermore, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5th Edition, Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001.

[0031] As used herein in this disclosure, unless otherwise clear from the context, (i) the term "a" or "an" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising," "including," "including" (whether or not used in conjunction with "but not limited to") and "including" (whether or not used in conjunction with "but not limited to") may be understood to encompass the itemized elements or steps, whether presented by themselves or presented with one or more additional elements or steps; (iv) the term "another" may be understood to mean at least an additional / second one or more; (v) the terms "about" and "approximately" may be understood to allow for standard variations as understood by one of ordinary skill in the art; and (vi) when ranges are given, the endpoints are included.

[0032] Unless otherwise specified, descriptions of oligonucleotides and their elements (e.g., base sequence, sugar modifications, internucleotide linkages, stereochemistry of the linking phosphorus, their patterns, etc.) are in 5' to 3' order. As one of skill in the art will understand, in some embodiments, oligonucleotides can be provided and / or utilized in salt form (particularly pharmaceutically acceptable salt forms, e.g., sodium salts). As one of skill in the art will also understand, in some embodiments, individual oligonucleotides within a composition can be considered to be of the same constitution and / or structure even within such a composition (e.g., a liquid composition), and a particular such oligonucleotide may be in a different salt form at a particular moment (and may be dissolved, or may exist as an anionic form when the oligonucleotide chain is in, for example, a liquid composition). For example, one of skill in the art will understand that at a given pH, individual internucleotide bonds along an oligonucleotide chain can be in the acid (H) form or one of several possible salt forms (e.g., sodium salts or salts of different cations depending on the ions that may be present in the preparation or composition), and that the acid form (e.g., all cations, if present, are H) is not necessarily a pharmaceutically acceptable salt form. + It will be understood that so long as the nucleotide sequence (replaced by ) is of the same composition and / or structure, such individual oligonucleotides may be considered to be of the same composition and / or structure, as appropriate.

[0033] Aliphatic: As used herein, "aliphatic" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated (but not aromatic) units, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more unsaturated (but not aromatic) units, or a combination thereof. In some embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0034] Alkenyl: As used herein, the term "alkenyl" refers to an aliphatic group, as defined herein, having one or more double bonds.

[0035] Alkyl: As used herein, the term "alkyl" has its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight-chain or branched-chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C for a straight chain). 20 , C2 to C for branched chains20 ) or about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure, and such rings are monocyclic, bicyclic or polycyclic, or have about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group can be a lower alkyl group, wherein the lower alkyl group has from 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0036] Alkynyl: As used herein, the term "alkynyl" refers to an aliphatic group, as defined herein, having one or more triple bonds.

[0037] Analog: The term "analog" includes any chemical moiety that is structurally different from a reference chemical moiety or class of moieties, but that can perform at least one function of such reference chemical moiety or class of moieties. Non-limiting examples include a nucleotide analog that is structurally different from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog that is structurally different from a nucleobase but performs at least one function of a nucleobase, etc.

[0038] 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, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0039] Aryl: The term "aryl," used alone or as part of a larger moiety of "aralkyl," "aralkoxy," or "aryloxyalkyl," as used herein, refers to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, wherein at least one ring in these systems is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 14 ring members, wherein at least one ring in these systems is aromatic, and wherein each ring in these systems contains 3 to 7 ring members. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, an aryl group is a biaryl group. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl, and the like, which may bear one or more substituents. As used herein, also included within the scope of the term "aryl" are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0040] Characteristic portion: As used herein, the term "characteristic portion," in its broadest sense, refers to a portion of a substance whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion found in the substance and related substances that share the particular characteristic, attribute, or activity, but not in those that do not share the particular characteristic, attribute, or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is one that contains a contiguous stretch of amino acids or a set of contiguous stretches of amino acids that together are characteristic of the protein or polypeptide. In some embodiments, each such contiguous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. Generally, a characteristic portion of a substance (e.g., a protein, antibody, etc.) is one that shares at least one functional characteristic with the related intact substance, in addition to the sequence and / or structural identity specified above. In some embodiments, a characteristic portion may be biologically active.

[0041] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical assignment of the chiral linking phosphorus at a chiral internucleotide linkage within an oligonucleotide. As used herein, a chiral internucleotide linkage is an internucleotide linkage in which the linking phosphorus is chiral. In some embodiments, the control is achieved by a chiral element not present in the sugar and base moieties of the oligonucleotide; for example, in some embodiments, the control is achieved by the use of one or more chiral auxiliary agents during oligonucleotide preparation, which are often part of the chiral phosphoramidite used during oligonucleotide preparation. In contrast to chiral control, those skilled in the art will understand that conventional oligonucleotide synthesis without the use of a chiral auxiliary agent cannot control the stereochemistry at the chiral internucleotide linkage when such conventional oligonucleotide synthesis is used to form the chiral internucleotide linkage. In some embodiments, the stereochemical assignment of each chiral linking phosphorus at each chiral internucleotide linkage within an oligonucleotide is controlled.

[0042] Chirality-controlled oligonucleotide composition: The terms "chirality-controlled oligonucleotide composition," "chirality-controlled nucleic acid composition," and the like, as used herein, refer to a composition comprising multiple oligonucleotides (or nucleic acids) that share a common base sequence, and the multiple oligonucleotides (or nucleic acids) share the same linking phosphorus stereochemistry in one or more chiral internucleotide linkages (chirality-controlled or sterically-restricted internucleotide linkages, where the chiral linking phosphorus in the composition is Rp or Sp ("sterically-restricted"), rather than a random mixture of Rp and Sp as in chirality-uncontrolled internucleotide linkages). In some embodiments, a chiral controlled oligonucleotide composition comprises multiple oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, where the multiple oligonucleotides (or nucleic acids) share the same linking phosphorus stereochemistry at one or more chiral internucleotide linkages (chiral controlled or sterically restricted internucleotide linkages, the chiral linking phosphorus is Rp or Sp ("sterically restricted") in the composition, rather than the random Rp and Sp mixtures present in non-chiral controlled internucleotide linkages). The level of multiple oligonucleotides (or nucleic acids) in a chiral controlled oligonucleotide composition is predetermined / controlled or enhanced (e.g., by preparing a chiral controlled oligonucleotide that stereoselectively forms one or more chiral internucleotide linkages) compared to the random level in a non-chiral controlled oligonucleotide composition.In some embodiments, about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 20%, 30%, or 40% of all oligonucleotides in the chiral controlled oligonucleotide composition. %, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) is a plurality of oligonucleotides. In some embodiments, about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95 ... Up to 90% or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are a plurality of oligonucleotides.In some embodiments, the level is between about 1% and 100% (e.g., between about 5% and 10%) of all oligonucleotides in a composition, or of all oligonucleotides in a composition (e.g., of multiple oligonucleotides or types of oligonucleotides) that share a common base sequence, a common backbone linkage pattern, and a common pattern of backbone phosphorus modifications, or of all oligonucleotides in a composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and / or a common internucleotide linkage modification pattern. 0%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 50% to 90% or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%). In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1 to 50 (e.g., about 1 to 10, 1 to 20, 5 to 10, 5 to 20, 10 to 15, 10 to 20, 10 to 25, 10 to 30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotide linkages.In some embodiments, the plurality of oligonucleotides have between about 1% and 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, about 5%, 10%, 15%, 20% to 20% (e.g., about 5%, 10%, 15%, 20%) of chiral internucleotide linkages. %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% share the same stereochemistry. In some embodiments, multiple oligonucleotides (or nucleic acids) all share the same pattern of sugar and / or nucleobase modifications. In some embodiments, multiple oligonucleotides (or nucleic acids) are various forms of the same oligonucleotide (e.g., acid and / or various salts of the same oligonucleotide). In some embodiments, multiple oligonucleotides (or nucleic acids) are of the same composition. In some embodiments, the level of the plurality of oligonucleotides (or nucleic acids) is about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, or about 1% to 1% of all oligonucleotides (or nucleic acids) in the composition that share the same construct as the plurality of oligonucleotides (or nucleic acids). In some embodiments, the chiral internucleotide linkage is a chiral controlled internucleotide linkage, and the composition is a completely chiral oligonucleotide composition.In some embodiments, the multiple oligonucleotides (or nucleic acids) are structurally identical. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 95%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 96%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 97%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 98%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 99%. In some embodiments, the percentage of the level is (DS). nc or at least (DS) nc where DS is the diastereomeric purity as described herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater), and nc is the number of chiral bonded phosphorus as described herein (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, the percentage level is (DS) nc or at least (DS) ncwhere DS is the diastereomeric purity as described herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater), and nc is the number of chiral-controlled internucleotide linkages as described herein (e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, the percentage level is (DS) nc or at least (DS) nc and DS is 95% to 100%. For example, when DS is 99% and nc is 10, the percentage is 90% or at least 90% (99%). 10≈0.90=90%). In some embodiments, the level of multiple oligonucleotides in a composition is expressed as the product of the diastereopurities of each chiral-controlled internucleotide linkage in the oligonucleotide. In some embodiments, the diastereopurity of an internucleotide linkage linking two nucleosides in an oligonucleotide (or nucleic acid) is expressed by the diastereopurity of the internucleotide linkage of a dimer linking the same two nucleosides, the dimer being prepared using comparable conditions, in some instances identical synthesis cycle conditions (e.g., for a linkage between Nx and Ny in an oligonucleotide...NxNy..., the dimer is NxNy). In some embodiments, not all chiral internucleotide linkages are chiral-controlled internucleotide linkages, and the composition is a partially chiral-controlled oligonucleotide composition. In some embodiments, the non-chirally controlled internucleotide linkages have a diastereomeric purity of less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50%, as typically observed in stereoirregular oligonucleotide compositions (e.g., those from conventional oligonucleotide synthesis, e.g., phosphoramidite methods, as will be understood by those skilled in the art). In some embodiments, the multiple oligonucleotides (or nucleic acids) are of the same type. In some embodiments, the chiral controlled oligonucleotides The composition contains non-random or controlled levels of individual oligonucleotide or nucleic acid types. For example, in some embodiments, a chiral controlled oligonucleotide composition contains no more than one oligonucleotide type. In some embodiments, a chiral controlled oligonucleotide composition contains two or more oligonucleotide types. In some embodiments, a chiral controlled oligonucleotide composition contains multiple oligonucleotide types. In some embodiments, a chiral controlled oligonucleotide composition is a composition of oligonucleotides of a certain oligonucleotide type, and the composition contains non-random or controlled levels of multiple oligonucleotides of that oligonucleotide type.

[0043] Equivalent: The term "equivalent" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit a comparison of the results obtained or the events observed. In some embodiments, comparable sets of conditions or circumstances are characterized by multiple substantially equivalent characteristics and one or a few varying characteristics. One skilled in the art will understand that sets of conditions are comparable to one another when they are characterized by a sufficient number and variety of substantially identical characteristics to warrant a reasonable conclusion that differences in the results obtained or the events observed under the different sets of conditions or circumstances are due to or represent changes in those altered characteristics.

[0044] Alicyclic: The terms "alicyclic," "carbocycle," "carbocyclyl," "carbocyclic group," and "carbocyclic ring" are used interchangeably and, as used herein, unless otherwise specified, refer to a saturated or partially unsaturated but non-aromatic cycloaliphatic monocyclic, bicyclic, or polycyclic ring system as described herein, having 3 to 30 ring members. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, an alicyclic group has 3 to 6 carbons. In some embodiments, an alicyclic group is saturated and is cycloalkyl. The term "alicyclic" can also include an alicyclic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, an alicyclic group is bicyclic. In some embodiments, an alicyclic group is tricyclic. In some embodiments, an alicyclic group is polycyclic. In some embodiments, "alicyclic" refers to a C3-C6 monocyclic hydrocarbon or C8-C6 alkylene group that has a single point of attachment to the rest of the molecule, which is fully saturated or contains one or more units of unsaturation, but is not aromatic. 10 Bicyclic or polycyclic hydrocarbons or C9-C6 rings that are fully saturated or contain one or more units of unsaturation but are not aromatic, with a single point of attachment to the rest of the molecule 16 Refers to polycyclic hydrocarbons.

[0045] Heteroaliphatic: The term "heteroaliphatic," as used herein, has its ordinary meaning in the art and refers to an aliphatic group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, one or more units selected from C, CH, CH, and CH are independently replaced with one or more heteroatoms (including oxidized and / or substituted forms thereof). In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0046] Heteroalkyl: The term "heteroalkyl," as used herein, has its ordinary meaning in the art and refers to an alkyl group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0047] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy," as used herein, refer to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, in which at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, heteroaryl groups are groups having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), in some embodiments, 5, 6, 9, or 10 ring atoms. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, heteroaryl groups have 6, 10, or 14 pi electrons shared in the cyclic arrangement, and have 1 to 5 heteroatoms in addition to the carbon atoms. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl. The terms "heteroaryl" and "heteroar-," as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic.The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl group, where the alkyl and heteroaryl portions independently are optionally substituted.

[0048] Heteroatom: As used herein, the term "heteroatom" refers to an atom that is not carbon or hydrogen. In some embodiments, the heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized forms of nitrogen, sulfur, phosphorus, or silicon; nitrogen (e.g., quaternized forms, forms such as iminium groups, etc.), phosphorus, sulfur, charged forms of oxygen, etc.). In some embodiments, the heteroatom is silicon, phosphorus, oxygen, sulfur, or nitrogen. In some embodiments, the heteroatom is silicon, oxygen, sulfur, or nitrogen. In some embodiments, the heteroatom is oxygen, sulfur, or nitrogen.

[0049] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic group," and "heterocyclic ring" are used interchangeably herein and refer to a monocyclic, bicyclic, or polycyclic ring moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +It can be NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic group" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. Heterocyclyl groups can be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions independently are optionally substituted.

[0050] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Next, nucleotides at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons performed with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0051] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to the bond connecting the nucleoside units of an oligonucleotide or nucleic acid. In some embodiments, the internucleotide linkage is a phosphodiester bond (a natural phosphate bond (-OP(=O)(OH)O-), which, as will be understood by those of skill in the art, may exist as a salt form) that is widely found in naturally occurring DNA and RNA molecules. In some embodiments, the internucleotide linkage is a modified internucleotide linkage (not a natural phosphate bond). In some embodiments, the internucleotide linkage is a "modified internucleotide linkage," in which at least one oxygen atom or -OH of the phosphodiester bond is replaced with a different organic or inorganic moiety. In some embodiments, such organic or inorganic moiety is selected from =S, =Se, =NR', -SR', -SeR', -N(R')2, B(R')3, -S-, -Se-, and -N(R')-, where each R' is independently as defined and described in this disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate linkage (or a phosphorothioate diester linkage, -OP(=O)(SH)O-, which, as will be understood by those of skill in the art, may exist as a salt form), or a phosphorothioate triester linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the internucleotide linkage is, for example, one of a PNA (peptide nucleic acid) or PMO (phosphorodiamidate morpholino oligomer) linkage. In some embodiments, the modified internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage (e.g., n001 in certain provided oligonucleotides). It will be understood by those of skill in the art that an internucleotide linkage can exist as an anion or cation at a given pH due to the presence of an acid or base moiety in the linkage.In some embodiments, the modified internucleotide linkages are those designated s, s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, and s18, as described in WO 2017 / 210647.

[0052] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment (e.g., in a test tube or reaction vessel, in cell culture, etc.) rather than within an organism (e.g., an animal, plant, and / or microorganism).

[0053] In vivo: As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, a plant, and / or a microorganism).

[0054] Bound phosphorus: As defined herein, the phrase "bound phosphorus" is used to indicate that the particular phosphorus atom being referenced is a phosphorus atom present in an internucleotide linkage, where the phosphorus atom corresponds to the phosphorus atom of a phosphodiester internucleotide linkage as it occurs in naturally occurring DNA and RNA. In some embodiments, the bound phosphorus atom is present in a modified internucleotide linkage, where each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the bound phosphorus atom is chiral (e.g., in the case of a phosphorothioate internucleotide linkage). In some embodiments, the bound phosphorus atom is achiral (e.g., in the case of a natural phosphate linkage).

[0055] Modified nucleobase: The terms "modified nucleobase," "modified base," and the like refer to a chemical moiety that is chemically different from a nucleobase but can perform at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase that includes a modification. In some embodiments, a modified nucleobase is capable of performing at least one function of a nucleobase, for example, forming a moiety in a polymer capable of base pairing to a nucleic acid comprising at least a complementary sequence of bases. In some embodiments, a modified nucleobase is a substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobase in the context of an oligonucleotide refers to a nucleobase that is not A, T, C, G, or U.

[0056] Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but containing a chemical modification that distinguishes it from a natural nucleoside. Non-limiting examples of modified nucleosides include those containing modifications at the base and / or sugar. Non-limiting examples of modified nucleosides include those having a 2' modification at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (lacking a nucleobase). In some embodiments, modified nucleosides are capable of forming moieties in polymers that retain at least one function of a nucleoside, e.g., capable of base pairing to a nucleic acid containing a complementary sequence of at least bases.

[0057] Modified Nucleotide: The term "modified nucleotide" includes any chemical moiety that is structurally different from a naturally occurring nucleotide but that can perform at least one function of a naturally occurring nucleotide. In some embodiments, a modified nucleotide comprises a modification at the sugar, base, and / or internucleotide linkage. In some embodiments, a modified nucleotide comprises a modified sugar, a modified nucleobase, and / or a modified internucleotide linkage. In some embodiments, a modified nucleotide is capable of forming a subunit in a polymer that is capable of at least one function of a nucleotide, e.g., capable of base pairing to a nucleic acid comprising a complementary sequence of at least bases.

[0058] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of the sugar. In some embodiments, as described in this disclosure, the modified sugar is a substituted ribose or deoxyribose. In some embodiments, the modified sugar comprises a 2'-modification. Examples of useful 2'-modifications are widely available in the art and described herein. In some embodiments, the 2'-modification is 2'-F. In some embodiments, the 2'-modification is 2'-OR (where R is an optionally substituted C 1~10 In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.). In some embodiments, in the context of an oligonucleotide, the modified sugar is a sugar that is not a ribose or deoxyribose as typically found in natural RNA or DNA.

[0059] Nucleic Acid: As used herein, the term "nucleic acid" includes any nucleotide and polymers thereof. The term "polynucleotide," as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), or a combination thereof. These terms refer to the primary structure of the molecule and thus include double- and single-stranded DNA and double- and single-stranded RNA. These terms include, as equivalents, any analog of RNA or DNA, including modified nucleotides and / or modified polynucleotides, such as, but not limited to, through methylated, protected, and / or capped nucleotides or polynucleotides. These terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from nucleobases and / or modified nucleobase N-glycosides or C-glycosides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotide linkages. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified internucleotide linkages. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to nucleic acids containing from 2 to about 10,000 nucleotide monomer units, and the prefix oligo- refers to nucleic acids containing from 2 to about 200 nucleotide monomer units.

[0060] Nucleobase: The term "nucleobase" refers to the portion of a nucleic acid that participates in hydrogen bonding to link one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, naturally occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, naturally occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase comprises a heteroaryl ring, the ring atom of which is nitrogen, and in the case of a nucleoside, the nitrogen is attached to the sugar moiety. In some embodiments, a nucleobase comprises a heterocyclic ring, the ring atom of which is nitrogen, and in the case of a nucleoside, the nitrogen is attached to the sugar moiety. In some embodiments, the nucleobase is a "modified nucleobase," a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a substituted A, T, C, G, or U. In some embodiments, the modified nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the hydrogen bonding properties that bind one nucleic acid strand to another in a sequence-specific manner. In some embodiments, the modified nucleobase can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior or recognition by intracellular enzymes or activities of the oligonucleotide duplex. As used herein, the term "nucleobase" also encompasses modified nucleobases and structural analogs that are used in place of natural or naturally occurring nucleotides, such as nucleobase analogs. In some embodiments, the nucleobase is an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U.In some embodiments, "nucleobase" refers to a nucleobase unit in an oligonucleotide or nucleic acid (e.g., A, T, C, G, or U as in an oligonucleotide or nucleic acid).

[0061] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar. In some embodiments, the nucleoside is a naturally occurring nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted naturally occurring nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted tautomer of a naturally occurring nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid.

[0062] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a nucleobase, a sugar, and one or more internucleotide linkages (e.g., phosphate linkages in natural DNA and RNA). Naturally occurring bases [guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)] are derivatives of purines or pyrimidines, although it should be understood that naturally occurring and non-naturally occurring base analogs are also included. Naturally occurring sugars are the pentose (five-carbon sugar) deoxyribose (forming DNA) or ribose (forming RNA), although it should be understood that naturally occurring and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotide linkages to form nucleic acids or polynucleotides. Many internucleotide linkages are known in the art (such as, but not limited to, via phosphate, phosphorothioate, boranophosphate, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriester, phosphorothioate, H-phosphonate, phosphoramidate, boranophosphate, methylphosphonate, phosphonoacetate, thiophosphonoacetate, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, natural nucleotides contain naturally occurring bases, sugars, and internucleotide linkages. As used herein, the term "nucleotide" also encompasses structural analogs that are used in place of natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, "nucleotide" refers to a nucleotide unit in an oligonucleotide or nucleic acid.

[0063] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, which may contain any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0064] Oligonucleotides can be single-stranded or double-stranded. Single-stranded oligonucleotides can have a double-stranded region (formed by two portions of the single-stranded oligonucleotide), and double-stranded oligonucleotides containing two oligonucleotide strands can have a single-stranded region, for example, in the region where the two oligonucleotide strands are not complementary to each other. Examples of oligonucleotides include, but are not limited to, structural genes, genes including regulatory and termination regions, self-replicating systems such as viruses or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adapters, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0065] Oligonucleotides of the present disclosure can be of various lengths. In certain embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, single-stranded, double-stranded, or triple-stranded oligonucleotides can range in length from about 4 to about 10 nucleosides, about 10 to about 50 nucleosides, about 20 to about 50 nucleosides, about 15 to about 30 nucleosides, or about 20 to about 30 nucleosides in length. In some embodiments, oligonucleotides are about 9 to about 39 nucleosides in length. In some embodiments, oligonucleotides are about 25 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 26 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 27 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 28 to about 70 nucleosides in length. In some embodiments, oligonucleotides are about 29 to about 70 nucleosides in length. In some embodiments, the oligonucleotide is about 30 to about 70 nucleosides in length. In some embodiments, the oligonucleotide is about 31 to about 70 nucleosides in length. In some embodiments, the oligonucleotide is about 32 to about 70 nucleosides in length. In some embodiments, the oligonucleotide is about 25 to about 60 nucleosides in length. In some embodiments, the oligonucleotide is about 25 to about 50 nucleosides in length. In some embodiments, the oligonucleotide is about 25 to about 40 nucleosides in length. In some embodiments, the oligonucleotide is about 30 to about 40 nucleosides in length. In some embodiments, the oligonucleotide is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides in length. In some embodiments, the oligonucleotide is at least 4 nucleosides in length. In some embodiments, the oligonucleotide is at least 5 nucleosides in length. In some embodiments, the oligonucleotide is at least 6 nucleosides in length. In some embodiments, the oligonucleotide is at least 7 nucleosides in length.In some embodiments, the oligonucleotide is at least 8 nucleosides in length. In some embodiments, the oligonucleotide is at least 9 nucleosides in length. In some embodiments, the oligonucleotide is at least 10 nucleosides in length. In some embodiments, the oligonucleotide is at least 11 nucleosides in length. In some embodiments, the oligonucleotide is at least 12 nucleosides in length. In some embodiments, the oligonucleotide is at least 15 nucleosides in length. In some embodiments, the oligonucleotide is at least 15 nucleosides in length. In some embodiments, the oligonucleotide is at least 16 nucleosides in length. In some embodiments, the oligonucleotide is at least 17 nucleosides in length. In some embodiments, the oligonucleotide is at least 18 nucleosides in length. In some embodiments, the oligonucleotide is at least 19 nucleosides in length. In some embodiments, the oligonucleotide is at least 20 nucleosides in length. In some embodiments, the oligonucleotide is at least 25 nucleosides in length. In some embodiments, the oligonucleotide is at least 26 nucleosides in length. In some embodiments, the oligonucleotide is at least 27 nucleosides in length. In some embodiments, the oligonucleotide is at least 28 nucleosides in length. In some embodiments, the oligonucleotide is at least 29 nucleosides in length. In some embodiments, the oligonucleotide is at least 30 nucleosides in length. In some embodiments, the oligonucleotide is at least 31 nucleosides in length. In some embodiments, the oligonucleotide is at least 32 nucleosides in length. In some embodiments, the oligonucleotide is at least 33 nucleosides in length. In some embodiments, the oligonucleotide is at least 34 nucleosides in length. In some embodiments, the oligonucleotide is at least 35 nucleosides in length. In some embodiments, the oligonucleotide is at least 36 nucleosides in length.In some embodiments, the oligonucleotide is at least 37 nucleosides in length. In some embodiments, the oligonucleotide is at least 38 nucleosides in length. In some embodiments, the oligonucleotide is at least 39 nucleosides in length. In some embodiments, the oligonucleotide is at least 40 nucleosides in length. In some embodiments, the oligonucleotide is 25 nucleosides in length. In some embodiments, the oligonucleotide is 26 nucleosides in length. In some embodiments, the oligonucleotide is 27 nucleosides in length. In some embodiments, the oligonucleotide is 28 nucleosides in length. In some embodiments, the oligonucleotide is 29 nucleosides in length. In some embodiments, the oligonucleotide is 30 nucleosides in length. In some embodiments, the oligonucleotide is 31 nucleosides in length. In some embodiments, the oligonucleotide is 32 nucleosides in length. In some embodiments, the oligonucleotide is 33 nucleosides in length. In some embodiments, the oligonucleotide is 34 nucleosides in length. In some embodiments, the oligonucleotide is 35 nucleosides in length. In some embodiments, the oligonucleotide is 36 nucleosides in length. In some embodiments, the oligonucleotide is 37 nucleosides in length. In some embodiments, the oligonucleotide is 38 nucleosides in length. In some embodiments, the oligonucleotide is 39 nucleosides in length. In some embodiments, the oligonucleotide is 40 nucleosides in length. In some embodiments, each nucleoside counted in the oligonucleotide length independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In some embodiments, each nucleoside counted in the oligonucleotide length independently comprises A, T, C, G, or U, or an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U.

[0066] Oligonucleotide Type: As used herein, the phrase "oligonucleotide type" is used to define oligonucleotides having a particular base sequence, backbone linkage pattern (i.e., pattern of internucleotide linkage types, e.g., phosphate, phosphorothioate, phosphorothioate triester, etc.), pattern of backbone chiral centers (i.e., pattern of phosphorus stereochemistry (Rp / Sp)) and pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides of a commonly designated "type" are structurally identical to each other.

[0067] Those skilled in the art will appreciate that the synthetic methods of the present disclosure provide a degree of control during the synthesis of an oligonucleotide chain, such that each nucleotide unit of the oligonucleotide chain can be designed and / or selected in advance to have a specific stereochemistry at the binding phosphorus and / or a specific modification at the binding phosphorus and / or a specific base and / or a specific sugar. In some embodiments, the oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the structural features described above. In some embodiments, the present disclosure provides compositions (e.g., chiral controlled oligonucleotide compositions) comprising or consisting of a plurality of oligonucleotide molecules. In some embodiments, all such molecules are of the same type (i.e., structurally identical to one another). However, in some embodiments, the provided compositions typically comprise a plurality of oligonucleotides of different types in predetermined relative amounts.

[0068] Optionally substituted: As described herein, compounds (e.g., oligonucleotides) of the present disclosure may contain optionally substituted and / or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety have been replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. In some embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by the present disclosure preferably result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that remain substantially unchanged when subjected to conditions that permit their production, detection, and, in certain embodiments, their collection, purification, and use for one or more of the purposes disclosed herein. Specific substituents are described below.

[0069] Suitable monovalent substituents on substitutable atoms, for example suitable carbon atoms, are independently halogen; -(CH) 0~4 R°;-(CH2) 0~4 OR°;-O(CH2) 0~4 R°, -O-(CH2) 0~4 C(O)OR°;-(CH2) 0~4 CH(OR°)2; R° can be substituted with -(CH2) 0~4 Ph; R° may be substituted with -(CH2) 0~4 O(CH2) 0~1 Ph; -CH=CHPh, which may be substituted with R°; -(CH2), which may be substituted with R° 0~4 O(CH2) 0~1 -pyridyl; -NO2; -CN; -N3; ​​(CH2) 0~4 N(R°)2;-(CH2) 0~4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0~4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2)0~4 N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2) 0~4 C(O)R°; -C(S)R°; -(CH2) 0~4 C(O)OR°; -(CH2) 0~4 C(O)SR°; -(CH2) 0~4 C(O)OSiR°3; -(CH2) 0~4 OC(O)R°; -OC(O)(CH2) 0~4 SR°、 -SC(S)SR°; -(CH2) 0~4 SC(O)R°; -(CH2) 0~4 C(O)NR°2; -C(S)NR°2; -C(S)SR°; (CH2) 0~4 OC(O)NR°2; C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; (CH2) 0~4 SSR°; -(CH2) 0~4 S(O)2R°; -(CH2) 0~4 S(O)2OR°; -(CH2) 0~4 OS(O)2R°; -S(O)2NR°2; (CH2) 0~4 S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -Si(R°)3; -OSi(R°)3; -B(R°)2; -OB(R°)2; -OB(OR°)2; -P(R°)2; -P(OR°)2; -P(R°)(OR°); -OP(R°)2; -OP(OR°)2; -OP(R°)(OR°); -P(O)(R°)2; -P(O)(OR°)2; -OP(O)(R°)2; -OP(O)(OR°)2; -OP(O)(OR°)(SR°); -SP(O)(R°)2; -SP(O)(OR°)2; -N(R°)P(O)(R°)2; -N(R°)P(O)(OR°)2; -P(R°)2[B(R°)3]; -P(OR°)2[B(R°)3]; -OP(R°)2[B(R°)3]; -OP(OR°)2[B(R°)3]; -(C 1~4 linear or branched alkylene)O-N(R°)2; or -(C 1~4linear or branched alkylene)C(O)ON(R°), where each R° may be substituted as defined herein and independently represents hydrogen, C 1~20 C having 1 to 5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon, and phosphorus 1~20 Heteroaliphatic, -CH2-(C 6~14 aryl), -O(CH2) 0~1 (C 6~14 aryl), -CH2- (5- to 14-membered heteroaryl ring), a 5- to 20-membered monocyclic, bicyclic, or polycyclic saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, or, notwithstanding the above definitions, two independent occurrences of R° taken together with their intervening atoms form a 5- to 20-membered monocyclic, bicyclic, or polycyclic saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, which may be substituted as defined below.

[0070] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° taken together with their intervening atoms) are independently halogen, —(CH) 0~2 R · ,-(Halo R · ), -(CH2) 0~2 OH, -(CH2) 0~2 OR · , -(CH2) 0~2 CH(OR · )2;-O(HaloR · ), -CN, -N3, -(CH2) 0~2 C(O)R · , -(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR · , -(CH2) 0~2 SR · , -(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR · , -(CH2) 0~2 NR · 2, -NO2, -SiR· 3. -OSiR · 3. -C(O)SR · , -(C 1~4 Linear or branched alkylene)C(O)OR · or -SSR · (where each R · is unsubstituted or, if preceded by "halo", substituted only with one or more halogens), and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 and a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0071] For example, suitable divalent substituents on suitable carbon atoms are, independently, the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2~3 O- or -S(C(R * 2)) 2~3 S- and R * Each independent occurrence of is hydrogen, C which may be substituted as defined below 1~6 The "optionally substituted" group is selected from an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having an aliphatic ring and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Preferred divalent substituents attached to adjacent substitutable carbon atoms of the "optionally substituted" group include -O(CR * 2) 2~3 O- and R * Each independent occurrence of is hydrogen, C which may be substituted as defined below 1~6 It is selected from aliphatic and unsubstituted 5-6 membered saturated rings, partially unsaturated rings and aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0072] R * Suitable substituents on the aliphatic group are independently halogen, —R · ,-(Halo R · ), -OH, -OR · , -O(HaloR · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2 or -NO2 (each R · is unsubstituted or, if preceded by "halo", substituted only with one or more halogens), and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph or independently is a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms selected from nitrogen, oxygen and sulfur.

[0073] In some embodiments, suitable substituents on a substitutable nitrogen are independently —R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2 or -N(R † )S(O)2R † and each R † are independently hydrogen, C which may be substituted as defined below 1~6 an aliphatic, unsubstituted -OPh or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or, notwithstanding the above definitions, R † two independent occurrences of are taken together with their intervening atoms to form an unsubstituted 3-12 membered saturated ring, partially unsaturated ring, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0074] R † Suitable substituents on the aliphatic group are independently halogen, —R · , (Halo R · ), -OH, -OR · , -O(HaloR · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2 or -NO2 (each R · is unsubstituted or, if preceded by "halo", substituted with one or more halogens only), and independently is selected from C1-C4 aliphatic, -CH2Ph, -O(CH2) 0~1 Ph or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0075] P-modification: As used herein, the term "P-modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In some embodiments, a P-modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus.

[0076] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0077] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeting buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained-release formulation; topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a suppository, cream, or foam; sublingual; ocular; transdermal; or intranasal, pulmonary, and other mucosal surfaces.

[0078] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0079] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent that encapsulates a material, which is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0080] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in a pharmaceutical context, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. provide a detailed description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, These include, but are not limited to, salts of lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.In some embodiments, provided compounds comprise one or more acidic groups (e.g., oligonucleotides), and pharmaceutically acceptable salts are alkali, alkaline earth metal, or ammonium (e.g., ammonium salts of N(R)3, where each R is independently defined and described in this disclosure) salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates. In some embodiments, provided compounds comprise multiple acidic groups; for example, oligonucleotides may comprise two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts, or salts of such compounds in general, contain two or more cations, which can be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or salt in general), all ionizable hydrogens in acidic groups (e.g., in an aqueous solution having a pKa of about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less, in some embodiments, about 7 or less; in some embodiments, about 6 or less; in some embodiments, about 5 or less; in some embodiments, about 4 or less; in some embodiments, about 3 or less) are replaced with a cation. In some embodiments, each phosphorothioate and phosphate group is independently present in its salt form (e.g., in the case of a sodium salt, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively).In some embodiments, each phosphorothioate and phosphate internucleotide linkage is independently present in its salt form (e.g., in the case of sodium salts, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). In some embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, and each acidic phosphate group and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if present, is present as a salt form (all sodium salts).

[0081] Predetermined: Predetermined (or pre-determined) means deliberately selected or non-random or controlled, as opposed to, for example, randomly occurring, irregular, or achieved without control. Those skilled in the art who read this specification will understand that the present disclosure provides techniques that enable the selection of specific chemical and / or stereochemical features to be incorporated into oligonucleotide compositions and further enable the controlled preparation of oligonucleotide compositions having such chemical and / or stereochemical features. A composition so provided is "predetermined" as described herein. A composition that may contain a particular oligonucleotide is not a "predetermined" composition because it is produced by chance through an uncontrolled process that intentionally produces specific chemical and / or stereochemical features. In some embodiments, a predetermined composition is one that can be intentionally reproduced (e.g., by repeating a controlled process). In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition means that the absolute and / or relative amounts (ratios, percentages, etc.) of the plurality of oligonucleotides in the composition are controlled. In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition is achieved by the preparation of chiral-controlled oligonucleotides.

[0082] Protecting Group: The term "protecting group" as used herein refers to a group that is well known in the art and is described in Organic Synthesis, TW Greene and PGM Wuts, 3 rdedition, John Wiley & Sons, 1999. Also included are those protecting groups specifically adapted for nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012, Chapter 2 of which is incorporated herein by reference in its entirety. Suitable amino-protecting groups include methyl carbamate, ethyl carbamate, 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), carbamic acid esters such as methyl methyl carbamate, ... 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamido)ethyl, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrocarbamate Tobenzyl, 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-dimethylthiobenzyl carbamate Phenyl (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 derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-Dimethoxycarbonylvinyl, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1- Methylcyclohexyl, 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-trimethylcarbamate benzyl, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide )propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-Tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, tetra ... Ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methoximethane N-benzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboron Phosphoric acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)carbonyl]amine, N-copper chelate compounds, N-zinc chelate compounds, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridine sulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte) ), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0083] Suitable protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

[0084] 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]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-trimethyl- methylethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyl hydroxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxamate Acetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamanoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-Trichloroethyl (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthothyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl methylthiomethoxymethyl)benzoate, 4-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). To protect 1,2- or 1,3-diols, protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,Examples of the hydroxybenzoates include 4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl boronic acid, and phenyl boronic acid.

[0085] In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4′-dimethoxytrityl, (D MTr) 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 (Pixil), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting groups are selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.In some embodiments, the phosphorus-linked protecting group is a group that is attached to a phosphorus bond (e.g., an internucleotide bond) throughout oligonucleotide synthesis. In some embodiments, the protecting group is attached to the sulfur atom of a phosphorothioate group. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide phosphorothioate bond. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide phosphate bond. In some embodiments, the protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0086] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; parasites, etc.) and plants. In some embodiments, the subject is a human. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0087] Substantially: As used herein, the term "substantially" refers to the qualitative condition of indicating the entire or nearly entire extent or degree of a desired characteristic or property. A base sequence that is substantially identical to or complementary to a second sequence is not completely identical to or complementary to the second sequence, but is largely or nearly identical to or complementary to the second sequence. In some embodiments, an oligonucleotide having a sequence substantially complementary to another oligonucleotide or nucleic acid will form a duplex with that oligonucleotide or nucleic acid in a manner similar to an oligonucleotide having a completely complementary sequence. Additionally, those skilled in the art of biology and / or chemistry will understand that biological and chemical events rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, as used herein, the term "substantially" is used to capture the potential lack of completeness inherent in many biological and / or chemical events.

[0088] Sugar: The term "sugar" refers to closed and / or open monosaccharides or polysaccharides. In some embodiments, a sugar is a monosaccharide. In some embodiments, a sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term "sugar" also encompasses structural analogs used in place of traditional sugar molecules, such as glycols, polymers that form the backbone of nucleic acid analogs, glycol nucleic acids ("GNAs"). As used herein, the term "sugar" also encompasses structural analogs used in place of natural or naturally occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is an RNA or DNA sugar (ribose or deoxyribose). In some embodiments, a sugar is a modified ribose or deoxyribose sugar (e.g., 2'-modified, 5'-modified, etc.). As described herein, in some embodiments, modified sugars, when used in oligonucleotides and / or nucleic acids, can provide one or more desirable properties, activities, etc. In some embodiments, the sugar is an optionally substituted ribose or deoxyribose. In some embodiments, "sugar" refers to the sugar unit in an oligonucleotide or nucleic acid.

[0089] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who is at higher risk than the general population of individuals of developing the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition has a predisposition to having the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0090] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any agent that induces a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it exhibits a statistically significant effect across a relevant population. In some embodiments, the relevant population is a population of subjects suffering from and / or susceptible to a disease, disorder, or condition. In some embodiments, the relevant population is a population of model organisms. In some embodiments, the relevant population may be defined by one or more criteria, such as age group, sex, genetic background, pre-existing clinical conditions, etc., prior to receiving therapy. In some embodiments, a therapeutic agent is a substance that, when administered to a subject in an effective amount, relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition of the subject. In some embodiments, a "therapeutic agent" is an agent that has been approved, or is required to be approved, by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is a drug for which a prescription is required for administration to a human. In some embodiments, a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.

[0091] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be understood by one of skill in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0092] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who show very early signs of the disease, disorder, and / or condition, e.g., to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0093] Unsaturated: The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0094] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning, which refers to an entity that has structure and / or activity as found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or situation. Those of skill in the art will understand that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0095] As one of skill in the art will appreciate, the methods and compositions described herein relating to provided compounds (e.g., oligonucleotides) generally also apply to pharmaceutically acceptable salts of such compounds.

[0096] Description of Specific Embodiments Oligonucleotides are useful in a variety of therapeutic, diagnostic, and research applications. The use of naturally occurring nucleic acids is limited, for example, by their susceptibility to endo- and exonucleases. Therefore, various synthetic counterparts have been developed to circumvent these drawbacks and / or further improve various properties and activities. These include, in particular, synthetic oligonucleotides containing chemical modifications (e.g., base modifications, sugar modifications, backbone modifications, etc.) that make these molecules less susceptible to degradation and improve other properties and / or activities.

[0097] From a structural standpoint, modifications to the internucleotide linkages can introduce chirality, and certain properties and activities can be influenced by the configuration of the bound phosphorus atoms of the oligonucleotide, such as binding affinity, sequence-specific binding to complementary RNA, stability against nucleases, activity, delivery, pharmacokinetics, etc., among others, can be influenced by the chirality of the backbone-bound phosphorus atoms.

[0098] In particular, the present disclosure utilizes techniques for controlling various structural elements (e.g., sugar modifications and their patterns, nucleobase modifications and their patterns, modified internucleotide linkages and their patterns, stereochemistry of linked phosphorus and their patterns, additional chemical moieties (moieties not normally present in an oligonucleotide chain) and their patterns, etc.). The ability to fully control the structural elements of oligonucleotides allows the present disclosure to provide oligonucleotides with improved and / or novel properties and / or activities for various applications, e.g., as therapeutic agents, probes, etc. For example, provided oligonucleotides and compositions thereof can be particularly potent for editing target adenosines in target nucleic acids, in some embodiments correcting G to A mutations by converting A to I.

[0099] In some embodiments, the oligonucleotide is a fragment of a nucleic acid (e.g., DNA, pre-mRNA, mRNA, etc.) at 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, The nucleic acid may comprise a sequence identical to or completely or substantially complementary to 57, 58, 59, 60, typically 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more consecutive bases. In some embodiments, the nucleic acid is a target nucleic acid comprising one or more target adenosines. In some embodiments, the target nucleic acid comprises one or less target adenosines. In some embodiments, an oligonucleotide may hybridize to the target nucleic acid. In some embodiments, such hybridization promotes modification of A (eg, conversion of A to I) in a nucleic acid or product thereof, for example, by ADAR1, ADAR2, etc.

[0100] In some embodiments, the disclosure provides oligonucleotides having a base sequence that is or includes about 10-40, about 15-40, about 20-40, or at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 contiguous bases of an oligonucleotide or nucleic acid disclosed herein (e.g., those in the Tables), or a sequence complementary to a target RNA sequence gene, transcript, etc. disclosed herein, wherein each T can optionally and independently be replaced with a U, or vice versa. In some embodiments, the disclosure provides oligonucleotides or oligonucleotide compositions as disclosed herein (e.g., in the Tables).

[0101] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide for site-specific editing of a nucleoside (e.g., a targeted adenosine) in a target nucleic acid (e.g., RNA).

[0102] As described herein, an oligonucleotide may contain one or more modified internucleotide linkages (non-natural phosphate linkages). In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage in which the linked phosphorus is chiral. In some embodiments, the modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, an oligonucleotide comprises one or more negatively charged internucleotide linkages (e.g., phosphorothioate internucleotide linkages, natural phosphate linkages, etc.). In some embodiments, an oligonucleotide comprises one or more non-negatively charged internucleotide linkages. In some embodiments, an oligonucleotide comprises one or more neutral internucleotide linkages.

[0103] In some embodiments, the oligonucleotide is chirally controlled. In some embodiments, the oligonucleotide is chirally pure (or "stereically pure," "stereochemically pure"), meaning that the oligonucleotide exists in a single stereoisomeric form (often a single diastereomeric (or "diastereomeric") form, since multiple chiral centers may exist in the oligonucleotide, for example, at the bond phosphorus, sugar, carbon, etc.). As will be understood by those skilled in the art, chirally pure oligonucleotides are separated from other stereoisomeric forms (to the extent that some impurities may be present, since chemical and biological processes, selectivity and / or purification, etc., are rarely, if ever, absolutely perfect). In chirally pure oligonucleotides, each chiral center is independently defined with respect to its configuration (for chirally pure oligonucleotides, each internucleotide linkage is independently stereorestricted or chiral controlled). In contrast to chirally controlled and chirally pure oligonucleotides containing sterically restricted linking phosphorus, racemic (or "sterically irregular," "chirally uncontrolled") oligonucleotides containing chiral linking phosphorus, derived, for example, from conventional phosphoramidite oligonucleotide synthesis without stereochemical control during the coupling step in combination with conventional sulfurization (which produces a stereoirregular phosphorothioate internucleotide linkage), refer to an irregular mixture of various stereoisomers (typically diastereoisomers (or "diastereomers"), since multiple chiral centers are present in the oligonucleotide; for example, derived from conventional oligonucleotide preparations using reagents that contain no chiral elements other than those at the nucleotides and linking phosphorus). For example, in terms of A*A*A (where * is a phosphorothioate internucleotide linkage (containing a chiral linking phosphorus)), the preparation of racemic oligonucleotides can yield four diastereomers [2 2= 4, considering two chiral linking phosphorus, each of which can exist in either of two configurations (Sp or Rp): A*SA*SA, A*SA*RA, A*RA*SA, and A*RA*RA (*S represents the phosphorothioate internucleotide linkage of Sp, and *R represents the phosphorothioate internucleotide linkage of Rp). With respect to chirally pure oligonucleotides (e.g., A*SA*SA), the oligonucleotide exists in a single stereoisomeric form, and the oligonucleotide is separated from other stereoisomers (e.g., the diastereomers A*SA*RA, A*RA*SA, and A*RA*RA).

[0104] In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sterically irregular internucleotide linkages (a mixture of Rp and Sp linked phosphorus at the internucleotide linkage, e.g., resulting from conventional, non-chiral oligonucleotide synthesis). In some embodiments, the oligonucleotide comprises one or more (e.g., 1-60, 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 9 In some embodiments, the oligonucleotide comprises 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more chiral-controlled internucleotide linkages (Rp or Sp linked phosphorus at the internucleotide linkage, e.g., derived from chiral-controlled oligonucleotide synthesis). In some embodiments, the internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage is a sterically disordered phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage is a chiral-controlled phosphorothioate internucleotide linkage.

[0105] In particular, the present disclosure provides techniques for preparing chiral controlled (and in some embodiments, stereochemically pure) oligonucleotides. In some embodiments, the oligonucleotides are stereochemically pure. In some embodiments, the oligonucleotides of the present disclosure have a chirality of about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% to 40%. %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% stereochemically pure.

[0106] In some embodiments, the present disclosure provides various oligonucleotide compositions. In some embodiments, the oligonucleotide compositions are sterically disordered or chirality-uncontrolled. In some embodiments, the oligonucleotides of the provided compositions have no chirality-controlled internucleotide linkages. In some embodiments, the internucleotide linkages of the oligonucleotides in the compositions comprise one or more chirality-controlled internucleotide linkages (e.g., chirality-controlled oligonucleotide compositions).

[0107] In some embodiments, an oligonucleotide composition comprises multiple oligonucleotides sharing a common base sequence, wherein one or more internucleotide linkages in the oligonucleotides are chiral controlled and one or more internucleotide linkages are sterically irregular (not chiral controlled). In some embodiments, an oligonucleotide composition comprises multiple oligonucleotides sharing a common base sequence, wherein each internucleotide linkage containing a chiral linked phosphorus in the oligonucleotides is independently a chiral controlled internucleotide linkage. In some embodiments, multiple oligonucleotides share the same base sequence and the same base and sugar modifications. In some embodiments, multiple oligonucleotides share the same base sequence and the same base, sugar, and internucleotide linkage modifications. In some embodiments, an oligonucleotide composition comprises oligonucleotides of the same configuration, wherein one or more internucleotide linkages are chiral controlled and one or more internucleotide linkages are sterically irregular (not chiral controlled). In some embodiments, an oligonucleotide composition comprises oligonucleotides of the same configuration, wherein each internucleotide linkage containing a chiral linked phosphorus is independently a chiral controlled internucleotide linkage. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or all of the oligonucleotides of the common base sequence are multiple oligonucleotides.

[0108] In some embodiments, the present disclosure provides techniques for preparing, evaluating, and / or utilizing the provided oligonucleotides and compositions thereof.

[0109] As used in this disclosure, in some embodiments, "one or more" is 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, "one or more" is 1. In some embodiments, "one or more" is 2. In some embodiments, "one or more" is 3. In some embodiments, "one or more" is 4. In some embodiments, "one or more" is 5. In some embodiments, "one or more" is 6. In some embodiments, "one or more" is 7. In some embodiments, "one or more" is 8. In some embodiments, "one or more" is 9. In some embodiments, "one or more" is 10. In some embodiments, "one or more" is at least 1. In some embodiments, "one or more" is at least 2. In some embodiments, "one or more" is at least 3. In some embodiments, "one or more" is at least 4. In some embodiments, "one or more" is at least 5. In some embodiments, "one or more" is at least 6. In some embodiments, "one or more" is at least 7. In some embodiments, "one or more" is at least 8. In some embodiments, "one or more" is at least 9. In some embodiments, "one or more" is at least ten.

[0110] As used in this disclosure, in some embodiments, "at least one" means one or more.

[0111] For example, variables (e.g., R, R L, L, etc.) various embodiments are described in terms of a variable (e.g., R). An embodiment described in terms of a variable (e.g., R) generally includes all variables that may be such a variable (e.g., R′, R″, R L , R L1 etc.)

[0112] Oligonucleotides In particular, the present disclosure provides oligonucleotides of various designs, which may include various nucleobases and their patterns, sugars and their patterns, internucleotide linkages and their patterns, and / or additional chemical moieties and their patterns, as described herein. In some embodiments, the provided oligonucleotides can induce A to I editing in a target nucleic acid. In some embodiments, the oligonucleotides of the present disclosure are single-stranded oligonucleotides capable of site-specific editing (A to I conversion) of adenosines in a target RNA sequence.

[0113] In some embodiments, the oligonucleotide is of suitable length and sequence complementary to the target nucleic acid to specifically hybridize with it. In some embodiments, the oligonucleotide is of sufficient length and sufficiently complementary to the target nucleic acid to distinguish it from other nucleic acids to reduce off-target effects. In some embodiments, the oligonucleotide is sufficiently short to facilitate delivery and reduce manufacturing complexity and / or cost, while maintaining the desired properties and activity (e.g., adenosine editing).

[0114] In some embodiments, the oligonucleotides are about 10 to 200 (e.g., about 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 120, 10 to 150, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, 20 to 100, 20 to 120, 20 to 150, 20 to 200, 25 to 30, 25 to 40, 25 to 50, The oligonucleotide has a length of nucleobases of 25 to 60, 25 to 70, 25 to 80, 25 to 90, 25 to 100, 25 to 120, 25 to 150, 25 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 70, 30 to 80, 30 to 90, 30 to 100, 30 to 120, 30 to 150, 30 to 200, 10, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, etc. In some embodiments, the oligonucleotide base sequence is about 10 to 60 nucleobases in length. In some embodiments, the base sequence is about 15 to 50 nucleobases in length. In some embodiments, the base sequence is about 15 to about 35 nucleobases in length. In some embodiments, the base sequence is about 25 to about 34 nucleobases in length. In some embodiments, the base sequence is about 26 to about 35 nucleobases in length. In some embodiments, the base sequence is about 27 to about 32 nucleobases in length. In some embodiments, the base sequence is about 29 to about 35 nucleobases in length. In some embodiments, the base sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleobases in length. In some embodiments, the base sequence is 35 nucleobases in length or at least 35 nucleobases in length. In some embodiments, the base sequence is 34 nucleobases in length or at least 34 nucleobases in length. In some embodiments, the base sequence is 33 nucleobases in length or at least 33 nucleobases in length.In some embodiments, the base sequence is 32 nucleobases in length or at least 32 nucleobases in length. In some embodiments, the base sequence is 31 nucleobases in length or at least 31 nucleobases in length. In some embodiments, the base sequence is 30 nucleobases in length or at least 30 nucleobases in length. In some embodiments, the base sequence is 29 nucleobases in length or at least 29 nucleobases in length. In some embodiments, the base sequence is 28 nucleobases in length or at least 28 nucleobases in length. In some embodiments, the base sequence is 27 nucleobases in length or at least 27 nucleobases in length. In some embodiments, the base sequence is 26 nucleobases in length or at least 26 nucleobases in length. In some embodiments, the base sequence of the complementary portions in the duplex is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 16, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more nucleobases in length. In some embodiments, it is at least 18 nucleobases in length. In some embodiments, it is at least 19 nucleobases in length. In some embodiments, it is at least 20 nucleobases in length. In some embodiments, it is at least 21 nucleobases in length. In some embodiments, it is at least 22 nucleobases in length. In some embodiments, it is at least 23 nucleobases in length. In some embodiments, it is at least 24 nucleobases in length. In some embodiments, it is at least 25 nucleobases in length. In particular, the present disclosure provides oligonucleotides of shorter length with comparable or better properties and / or comparable or higher activity compared to previously reported adenosine-editing oligonucleotides.

[0115] In some embodiments, the base sequence of the oligonucleotide is complementary to the base sequence of the target nucleic acid (e.g., complementary to a portion of the target nucleic acid containing the target adenosine) with 0 to 10 mismatches that are not Watson-Crick base pairs (AT, AU, and CG) (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some embodiments, there are no mismatches. In some embodiments, there is one mismatch. In some embodiments, there are two mismatches. In some embodiments, there are three mismatches. In some embodiments, there are four mismatches. In some embodiments, there are five mismatches. In some embodiments, there are six mismatches. In some embodiments, there are seven mismatches. In some embodiments, there are eight mismatches. In some embodiments, there are nine mismatches. In some embodiments, there are ten mismatches. In some embodiments, the oligonucleotide may contain portions not designed for complementarity (e.g., loops, protein binding sequences, etc., for recruitment of proteins, e.g., ADARs). As one of skill in the art will understand, such portions may be appropriately excluded when calculating mismatches and / or complementarity.In some embodiments, the complementarity (e.g., between the oligonucleotide and the target nucleic acid) is about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, Complementarity is typically between 70% and 90%, 70% and 95%, 70% and 100%, 75% and 80%, 75% and 85%, 75% and 90%, 75% and 95%, 75% and 100%, 80% and 85%, 80% and 90%, 80% and 95%, 80% and 100%, 85% and 90%, 85% and 95%, 85% and 100%, 90% and 95%, 90% and 100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, complementarity is at least about 60%. In some embodiments, complementarity is at least about 65%. In some embodiments, complementarity is at least about 70%. In some embodiments, complementarity is at least about 75%. In some embodiments, the complementarity is at least about 80%. In some embodiments, the complementarity is at least about 85%. In some embodiments, the complementarity is at least about 90%. In some embodiments, the complementarity is at least about 95%. In some embodiments, the complementarity is 100% throughout the length of the oligonucleotide. In some embodiments, the complementarity is 100% throughout the length of the oligonucleotide, excluding the nucleoside opposite the target nucleoside (e.g., adenosine). Typically, complementarity is based on Watson-Crick base pairs AT, AU, and CG. Those skilled in the art will understand that when assessing the complementarity of two sequences of different lengths (e.g., a provided oligonucleotide and a target nucleic acid), the complementarity can be appropriately based on the length and / or maximum complementarity of the shorter sequence between the two sequences. In many embodiments, the oligonucleotide and the target nucleic acid are sufficiently complementary so that modification is selectively directed to the target adenosine site. In some embodiments, the oligonucleotide may hybridize to a target nucleic acid or a portion thereof that includes the target adenosine.In some embodiments, the oligonucleotides can hybridize to a target nucleic acid or a portion thereof that can hybridize to an oligonucleotide listed in the table.

[0116] In some embodiments, one or more mismatches are independently perturbations. In some embodiments, each mismatch is a perturbation. In some embodiments, there are 0 to 10 perturbations (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some embodiments, this number is 0. In some embodiments, this number is 1. In some embodiments, this number is 2. In some embodiments, this number is 3. In some embodiments, this number is 4. In some embodiments, this number is 5. In some embodiments, the wobble is GU, IA, GA, IU, IC, IT, AA, or inverted AT. In some embodiments, the wobble is GU, IA, GA, IU, or IC. In some embodiments, an IC can be considered a match when an I is the nucleoside immediately 3' to the nucleoside opposite the target nucleoside. In some embodiments, a base that forms a wobble pair (e.g., U, which can form a GU wobble) can replace a base that forms a match pair (e.g., C, which matches G) and can provide an oligonucleotide with editing activity.

[0117] In some embodiments, the oligonucleotide and target nucleic acid duplexes contain one or more bulges, each independently containing one or more mismatches that are not wobble. In some embodiments, there are 0 to 10 bulges (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulges, etc.). In some embodiments, this number is 0. In some embodiments, this number is 1. In some embodiments, this number is 2. In some embodiments, this number is 3. In some embodiments, this number is 4. In some embodiments, this number is 5.

[0118] In some embodiments, the distance between two mismatches, the mismatch and one or both ends of the oligonucleotide (or portions thereof, e.g., the first domain, the second domain, the first subdomain, the second subdomain, the third subdomain) and / or the nucleoside opposite the mismatch and the target adenosine is independently 0 to 50, 0 to 40, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10 (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5 ... 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases (not including mismatches, terminal nucleosides, and nucleosides opposite the target adenosine). In some embodiments, this number is In some embodiments, the number is 0 to 30. In some embodiments, the number is 0 to 20. In some embodiments, the number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the distance between two mismatches is 0 to 20. In some embodiments, the distance between two mismatches is 1 to 10. In some embodiments, the distance between a mismatch and the 5'-terminal nucleoside of the oligonucleotide is 0 to 20. In some embodiments, the distance between a mismatch and the 5'-terminal nucleoside of the oligonucleotide is 0 to 20. The distance between the mismatch and the 5'-terminal nucleoside of the oligonucleotide is 5 to 20. In some embodiments, the distance between the mismatch and the 3'-terminal nucleoside of the oligonucleotide is 0 to 40. In some embodiments, the distance between the mismatch and the 3'-terminal nucleoside of the oligonucleotide is 5 to 20. In some embodiments, the distance between the mismatch and the nucleoside opposite the target adenosine is 0 to 20. In some embodiments, the distance between the mismatch and the nucleoside opposite the target adenosine is 1 to 10.In some embodiments, the number of nucleobases for the distance is 0. In some embodiments, it is 1. In some embodiments, it is 2. In some embodiments, it is 3. In some embodiments, it is 4. In some embodiments, it is 5. In some embodiments, it is 6. In some embodiments, it is 7. In some embodiments, it is 8. In some embodiments, it is 9. In some embodiments, it is 10. In some embodiments, it is 11. In some embodiments, it is 12. In some embodiments, it is 13. In some embodiments, it is 14. In some embodiments, it is 15. In some embodiments, it is 16. In some embodiments, it is 17. In some embodiments, it is 18. In some embodiments, it is 19. In some embodiments, it is 20. In some embodiments, the mismatch is at an end (e.g., at the 5'-end or 3'-end) of the first domain, second domain, first subdomain, second subdomain, or third subdomain. In some embodiments, the mismatch is at the nucleoside opposite the target adenosine.

[0119] In some embodiments, provided oligonucleotides are capable of inducing adenosine editing (e.g., A to I conversion) in a target nucleic acid, and consist of, comprise, or have a base sequence that includes a portion (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more consecutive bases) of the base sequence of an oligonucleotide disclosed herein, wherein each T can independently be replaced with a U, or vice versa, and the oligonucleotide includes at least one non-naturally occurring modification of the base, sugar, and / or internucleotide linkage.

[0120] In some embodiments, provided oligonucleotides comprise one or more carbohydrate moieties. In some embodiments, provided oligonucleotides comprise one or more GalNAc moieties. In some embodiments, provided oligonucleotides comprise one or more targeting moieties. Non-limiting examples of such additional chemical moieties that can be conjugated to oligonucleotide chains are described herein.

[0121] In some embodiments, provided oligonucleotides can induce correction of a G to A mutation in a target sequence or its product. In some embodiments, the correction of a G to A mutation is or includes conversion of A to I, which can be read as G during translation or other biological processes. In some embodiments, provided oligonucleotides can induce correction of a G to A mutation in a target sequence or its product via ADAR-mediated deamination. In some embodiments, provided oligonucleotides can induce correction of a G to A mutation in a target sequence or its product via ADAR-mediated deamination by recruiting endogenous ADARs (e.g., in target cells) and promoting ADAR-mediated deamination. Nevertheless, the present disclosure is not limited to any particular mechanism. In some embodiments, the present disclosure provides oligonucleotides, compositions, methods, etc. that can operate via double-stranded RNA interference, single-stranded RNA interference, RNase H-mediated knockdown, steric hindrance of translation, ADAR-mediated deamination, or a combination of two or more such mechanisms.

[0122] In some embodiments, the oligonucleotide comprises a structural element or portion thereof described herein (e.g., in a Table). In some embodiments, the oligonucleotide has a base sequence comprising a base sequence (or portion thereof), wherein each T can be independently replaced with a U, a pattern of chemical modifications (or portions thereof) disclosed herein (e.g., in a Table or Figure) or otherwise disclosed herein, and / or the format of the oligonucleotide. In some embodiments, such an oligonucleotide can induce correction of a G to A mutation in a target sequence or its product.

[0123] In particular, provided oligonucleotides can hybridize to their target nucleic acids (e.g., pre-mRNA, mature mRNA, etc.). In some embodiments, oligonucleotides can hybridize to nucleic acids of target RNA sequences at any stage of RNA processing, including, but not limited to, pre-mRNA or mature mRNA. In some embodiments, oligonucleotides can hybridize to any element of an oligonucleotide nucleic acid, or its complement, including, but not limited to, a promoter region, an enhancer region, a transcription termination region, a translation initiation signal, a translation termination signal, a coding region, a non-coding region, an exon, an intron, an intron / exon or exon / intron junction, a 5' UTR, or a 3' UTR.

[0124] In some embodiments, the oligonucleotide hybridizes to two or more variants of a transcript derived from the sense strand of a target site (eg, a target sequence).

[0125] In some embodiments, provided oligonucleotides contain enhanced levels of one or more isotopes. In some embodiments, provided oligonucleotides are labeled, for example, with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, provided oligonucleotides in a provided composition (e.g., multiple oligonucleotides of a composition) comprise base modifications, sugar modifications, and / or internucleotide linkage modifications, and the oligonucleotides contain enriched levels of deuterium. In some embodiments, provided oligonucleotides are enriched with deuterium (-) at one or more positions. 1 H- 2 In some embodiments, one or more of the oligonucleotide strands or any moieties conjugated to the oligonucleotide strands (e.g., targeting moieties, etc.) are labeled. 1 H, 2 substituted with H. Such oligonucleotides may be used in the compositions and methods described herein.

[0126] In some embodiments, the oligonucleotide comprises one or more modified nucleobases, one or more modified sugars, and / or one or more modified internucleotide linkages, as described herein. In some embodiments, the oligonucleotide comprises, for example, about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 85%, 80% to 95%, 90% to 95%, 90% to 10 ... and / or 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0127] In some embodiments, the oligonucleotide comprises one or more modified sugars, in some embodiments, the oligonucleotide comprises between about 1 and 50 (e.g., between about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, the oligonucleotide comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars with 2'-F modifications.In some embodiments, the oligonucleotide comprises about 2 to 50 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc., 2 to 40, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 3 to 40, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 4 to 40, 4 to 50, etc.) 2'-F modifications. 30, 4-25, 4-20, 4-15, 4-10, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 6-40, 6-30, 6-25, 6-20, 6-15, 6-10, 7-40, 7-30, 7-25, 7-20, 7-15, 7-10, 8-40, 8-30, 8-25, 8-20, 8- In some embodiments, the oligonucleotide comprises about 15, 8-10, 9-40, 9-30, 9-25, 9-20, 9-15, 9-10, 10-40, 10-30, 10-25, 10-20, 10-15, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises two consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises three consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises four consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises five consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises six consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises seven consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 8 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 9 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 10 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises two or more 2'-F modified sugar blocks, wherein each sugar in the 2'-F modified sugar block is independently a 2'-F modified sugar.In some embodiments, each 2'-F modified sugar block independently comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous 2'-F modified sugars as described herein. In some embodiments, two contiguous 2'-F modified sugar blocks are independently separated by a separation block, which independently comprises one or more sugars that are not 2'-F modified sugars. In some embodiments, the oligonucleotide comprises one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) 2'-F blocks and one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) separation blocks. In some embodiments, the first domain is one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) and one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) separation blocks. In some embodiments, each first domain block attached to a first domain 2'-F block is a separation block. In some embodiments, each first domain block attached to a first domain separation block is a first domain 2'-F block. In some embodiments, each sugar in a separation block is independently unmodified. In some embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) or all sugars in a separation block are independently not 2'-F modified.In some embodiments, the separation block comprises one or more bicyclic sugars (e.g., LNA sugars, cEt sugars, etc.) and / or one or more 2'-OR modified sugars (where R is optionally substituted C. 1~6 In some embodiments, the separation block comprises one or more 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, two or more 2'-F unmodified sugars are contiguous. In some embodiments, two or more 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the separation block is comprised of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the separation block comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-ORF modified sugars (wherein R is an optionally substituted C 1~6 In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe sugar or a 2'-MOE sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE sugar. In some embodiments, a separation block comprises one or more 2'-F modified sugars. In some embodiments, the 2'-F modified sugars in a separation block are not adjacent to one another. In some embodiments, a separation block does not comprise a 2'-F modified sugar. In some embodiments, each sugar in a separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6In some embodiments, each sugar in each separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each sugar in the separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each sugar in each separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each sugar in a separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in each separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in a separation block is independently a 2'-OMe modified sugar. In some embodiments, each sugar in a separation block is independently a 2'-MOE modified sugar. In some embodiments, a separation block comprises a 2'-OMe sugar and a 2'-MOE modified sugar. In some embodiments, each 2'-F block and each separation block independently comprises 1, 2, 3, 4, or 5 nucleosides. In some embodiments, each 2'-F block and each separation block independently comprises 1, 2, or 3 nucleosides.

[0128] As described herein, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) can include one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) blocks. or can consist of, each of which independently comprises one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.) sugars, wherein each sugar in the block shares the same structure or structural feature. In some embodiments, an oligonucleotide or portion thereof (e.g., first domain, second domain, etc.) can include one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) blocks. or consisting of, each of which independently contains one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.) sugars. In some embodiments, each block independently contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars. In some embodiments, each block independently contains 1 to 5 sugars. In some embodiments, each block independently contains 1, 2, or 3 sugars.In some embodiments, one or more (e.g., 1-15, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) blocks independently contain two or more sugars. In some embodiments, one or more (e.g., 1-15, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) blocks independently contain two or three sugars. In some embodiments, about or at least about 30%, 40%, or 50% of the blocks in an oligonucleotide or portion thereof independently contain two or more (e.g., two or three) sugars. In some embodiments, about 50% of the blocks in the oligonucleotide or first domain independently contain two or more (e.g., two or three) sugars. In some embodiments, the blocks are 2'-F blocks, and each sugar in the block is a 2'-F modified block. In some embodiments, the blocks are 2'-OR. sa block, each sugar in the block being independently 2'-OR sa In some embodiments, the block is a 2'-OR modified sugar, which may be the same or different. sk block, each sugar in the block being independently 2'-OR sk and the modified sugars may be the same or different. In some embodiments, the 2'-OR sa Or 2'-OR sk Each sugar in a block is the same. In some embodiments, the block is a 2'-OMe block, where each sugar is independently a 2'-OMe-modified sugar. In some embodiments, the block is a 2'-OME block, where each sugar is independently a 2'-OME-modified sugar. In some embodiments, there is at least one 2'-OR between every two 2'-F blocks in an oligonucleotide or portion thereof. sa In some embodiments, there is at least one 2'-OR block between every two 2'-F blocks in an oligonucleotide. skIn some embodiments, at least one 2'-OMe block is present between every two 2'-F blocks in the first domain. In some embodiments, at least one 2'-OME block is present between two 2'-F blocks in the first domain. In some embodiments, a 2'-MOE block and a 2'-OMe block are present between two 2'-F blocks in the first domain, and no 2'-F blocks are present. In some embodiments, each 2'-F block is independently a 2'-OR sa In some embodiments, each 2'-F block is independently linked to a 2'-OR sk In some embodiments, each block to which a 2'-F block is attached is independently a 2'-OR sa In some embodiments, each block to which a 2'-F block is attached is independently a 2'-OR sk In some embodiments, each block in the first domain to which a 2'-F block in the first domain is attached is independently a 2'-OR sa In some embodiments, each block in the first domain to which a 2'-F block in the first domain is attached is independently a 2'-OR sk In some embodiments, the 2'-OR sa Each block in the first domain to which the blocks are attached can independently be a 2'-F block or a different 2'-OR block. sa In some embodiments, the 2'-OR sk Each block in the first domain to which the blocks are attached can independently be a 2'-F block or a different 2'-OR block. skIn some embodiments, the 2'-OR block is a 2'-OMe block. In some embodiments, the 2'-OR block is a 2'-MOE block. In some embodiments, at least one block is a 2'-OMe block. In some embodiments, about or at least about 2, 3, 4, or 5 blocks are independently 2'-OMe blocks. In some embodiments, at least one block is a 2'-MOE block. In some embodiments, about or at least about 2, 3, 4, or 5 blocks are independently 2'-MOE blocks. In some embodiments, there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-OMe blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-MOE blocks in an oligonucleotide or portion thereof (e.g., first domain, second domain, etc.). In some embodiments, there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-MOE blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F blocks in the oligonucleotide or portion thereof (e.g., first domain, second domain, etc.). In some embodiments, there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-MOE blocks in the first domain. In some embodiments, there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F blocks in the first domain. In some embodiments, there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-MOE blocks in the first domain.In some embodiments, there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OMe blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-MOE blocks and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F blocks in the first domain. In some embodiments, the percentage of 2'-F modified sugars in an oligonucleotide or portion thereof (e.g., first domain, second domain, etc.) is about 20%-80%, 30-70%, 30%-60%, 30%-50%, 40%-60%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%; each of which independently represents a 2'-OR modified sugar (where R is an optionally substituted C). 1~6 In some embodiments, the percentage of 2'-F modified sugars in the first domain is about 20% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 40% to 60%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. In some embodiments, the percentage of 2'-F modified sugars in the first domain is about 20% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 40% to 60%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%; each of which independently represents a 2'-OR modified sugar (wherein R is an optionally substituted C 1~6 In some embodiments, the percentage of 2'-F modified sugars and the percentage of 2'-OR modified sugars (wherein R is an optionally substituted C 1~6The percentage of 2'-OR modified sugars that are aliphatic is less than about 50%, 40%, 30%, 20%, or 10% (calculated by subtracting the lesser of these two percentages from the greater of these two percentages). In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or a 2'-MOE modified sugar. In some embodiments, a portion of the oligonucleotide, e.g., the first domain, the second domain, the third subdomain, etc., comprises alternating 2'-OR modified sugars. sa and 2'-F blocks. sk and a 2'-F block.

[0129] Certain modified sugars and their uses are described in International Publication No. WO 2021 / 071858, which is incorporated by reference in its entirety, and may be utilized in accordance with the present disclosure. Certain modified sugars and their uses are described in US 2021 / 058495, which is incorporated by reference in its entirety, and may be utilized in accordance with the present disclosure.

[0130] In some embodiments, the 2'-OR saModified sugars, such as 2'-OMe-modified sugars, 2'-MOE-modified sugars, and LNA sugars, can be prepared using the methods described in, for example, WO 2016 / 097212, WO 2017 / 050306, WO 2017 / 220751, WO 2018 / 041973, WO 2018 / 134301, and WO 2019 / 158 475, International Publication No. 2019 / 219581, International Publication No. 2020 / 154342, International Publication No. 2020 / 154343, International Publication No. 2020 / 154344, International Publication No. 2020 / 157008, International Publication No. 2020 / 165077, International Publication No. 2020 / 201406, International Publication No. 2020 / 2 16637, WO 2020 / 252376, WO 2021 / 130313, WO 2021 / 231673, WO 2021 / 231675, WO 2021 / 231679, WO 2021 / 231680, WO 2021 / 231685, WO 2021 / 231691, WO 2021 / 231692, WO 2021 / 231698, WO 2021 / 231830, WO 2021 / 243023, WO 2022 / 018207 or WO 2022 / 026928.

[0131] In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95% of all sugars. %, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc., is a modified sugar. In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95%, 70% and 100%, 75% and 8 ... % to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc., may refer to a 2'-F modified sugar, a 2'-OR modified sugar (wherein R is an optionally substituted C 1~6aliphatic) and bicyclic sugars (e.g., LNA sugars, cEt sugars, etc.). In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%. In some embodiments, the percentage is about or at least about 95%. -1 All sugars except those at N0 and N1 are independently modified sugars. -1 In some embodiments, all sugars except for N0 and N1 are independently 2'-modified sugars. In some embodiments, all sugars except for N0 and N1 are independently modified sugars. In some embodiments, all sugars except for N0 and N1 are independently 2'-modified sugars. In some embodiments, N0 and N -1 In some embodiments, all sugars except for N are independently modified sugars. -1 In some embodiments, all sugars except for N0 are independently 2'-modified sugars. In some embodiments, all sugars except for N0 are independently modified sugars. In some embodiments, all sugars except for N0 are independently 2'-modified sugars. In some embodiments, N -1 Each sugar of N, N, and N is independently a 2'-F modified sugar, a natural DNA sugar, or a natural RNA sugar. -1 Each sugar of N, N, and N is independently a 2'-F modified sugar or a natural DNA sugar. -1 Each sugar of N0, N1 and N2 is independently a natural DNA sugar.

[0132] In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95%, 70% and 100%, 75% and 8 ... % to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc., refer to 2'-F modified sugars and 2'-OR modified sugars (where R is an optionally substituted C 1~6In some embodiments, the modified sugars are independently selected from the group consisting of about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are modified sugars independently selected from 2'-F-modified sugars, 2'-OMe-modified sugars, and 2'-MOE-modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0133] In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95%, 70% and 100%, 75% and 80% of all sugars. Up to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are modified sugars independently selected from 2'-F-modified sugars and 2'-OMe-modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0134] In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95% of all sugars. %, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are 2'-F modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%. In some embodiments, 10 or more (e.g., about or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more, such as 10-50, 10-40, 10-30, 10-25, 15-50, 15-40, 15-30, 15-25, 20-50, 20-40, 20-30, 20-25, etc.) sugars are 2'-F modified sugars.In some embodiments, the oligonucleotide comprises two or more (e.g., 2 to 30, 2 to 25, 2 to 20, 2 to 15, 3 to 10, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises one or more 2'-F blocks, each independently comprising two or more (e.g., 2-30, 2-25, 2-20, 2-15, 3-10, 3-30, 3-25, 3-20, 3-15, 3-10, 4-30, 4-25, 4-20, 4-15, 4-10, 5-30, 5-25, 5-20, 5-15, 5-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) contiguous 2'-F modified sugars. In some embodiments, the oligonucleotide comprises two or more 2'-F blocks as described herein separated by one or more separation blocks as described herein. In some embodiments, the 2'-F block has 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, the 2'-F block has 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has 10 or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has 9 or fewer 2'-F modified sugars.In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has eight or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has seven or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has six or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has five or fewer 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has four or fewer 2'-F modified sugars. In some embodiments, each block attached to a 2'-F block is independently a block that does not contain a 2'-F modified sugar. In some embodiments, each block attached to a 2'-F block is independently a block that contains a neutral DNA or RNA sugar, a 2'-OR modified sugar (where R is an optionally substituted C). 1~6 In some embodiments, each block attached to the 2'-F block is independently a block comprising a neutral DNA or RNA sugar, a 2'-OMe-modified sugar, a 2'-MOE-modified sugar, or a bicyclic sugar. In some embodiments, each block attached to the 2'-F block is independently a block comprising a neutral DNA or RNA sugar, a 2'-OMe-modified sugar, a 2'-MOE-modified sugar. In some embodiments, each nucleoside in the first domain attached to the 2'-F block in the first domain is independently a block comprising a 2'-OR-modified sugar (where R is optionally substituted C 1~6 In some embodiments, each nucleoside in the first domain that is linked to a 2'-F block in the first domain is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6In some embodiments, each nucleoside in the first domain attached to a 2'-F block in the first domain is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each nucleoside in the second domain attached to a 2'-F block in the second domain is independently a 2'-OR modified sugar (wherein R is an optionally substituted C 1~6 In some embodiments, each nucleoside in the second domain linked to a 2'-F block in the second domain is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each nucleoside in the second domain linked to a 2'-F block in the second domain is independently a 2'-OMe or a 2'-MOE modified sugar.

[0135] In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95%, 70% and 100% %, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc., refers to a 2'-OR modified sugar (where R is an optionally substituted C 1~6In some embodiments, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% Up to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc., are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0136] In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95% of all sugars , 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are 2'-OMe modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0137] In some embodiments, between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90%, 70% and 95%, 70% and 100% %, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc., refers to a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95% of all sugars. , 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc., is a 2'-MOE modified sugar.

[0138] In some embodiments, one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, etc.) nucleosides at the first (5' end) and / or The sugars of the last (3'-terminal) one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, etc.) nucleosides are independently modified sugars. In some embodiments, the first one or several sugars are independently modified sugars. In some embodiments, the last one or several sugars are independently modified sugars. In some embodiments, both the first one or several sugars and the last one or several sugars are independently modified sugars. In some embodiments, the modified sugars are independently a 2'-F unmodified sugar (e.g., a bicyclic sugar), a 2'-OR modified sugar (where R is as described herein and is not -H (e.g., an optionally substituted C 1~6 In some embodiments, the modified sugars are independently selected from bicyclic sugars and 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the modified sugars are independently selected from 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the modified sugars are independently 2'-OMe-modified sugars and 2'-MOE-modified sugars. In some embodiments, the first several sugars are one or more 2'-OR-modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the first several sugars comprise one or more 2'-OR modified sugars (where R is an optionally substituted C 1~6In some embodiments, the first several sugars comprise one or more 2'-OMe-modified sugars. In some embodiments, the first several sugars comprise one or more 2'-MOE-modified sugars. In some embodiments, the first several sugars comprise one or more 2'-OMe-modified sugars and one or more 2'-MOE-modified sugars. In some embodiments, the last several sugars comprise one or more 2'-OR-modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the last several sugars comprise one or more 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the last several sugars comprise one or more 2'-OMe-modified sugars. In some embodiments, the last several sugars comprise one or more 2'-MOE-modified sugars. In some embodiments, the last several sugars comprise one or more 2'-OMe-modified sugars and one or more 2'-MOE-modified sugars. In some embodiments, the last several sugars are independently 2'-OMe-modified sugars. In some embodiments, the first several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive bicyclic sugars or 2'-OR-modified sugars (wherein R is an optionally substituted C 1~6 In some embodiments, the first several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OR modified sugars (where R is an optionally substituted C 1~6In some embodiments, the first several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive modified sugars, where each modified sugar is independently a 2'-OMe-modified sugar or a 2'-MOE-modified sugar. In some embodiments, the first several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OMe-modified sugars. In some embodiments, the first several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-MOE-modified sugars. In some embodiments, the last several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OR-modified sugars (wherein R is an optionally substituted C 1~6 In some embodiments, the last several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive modified sugars, where each modified sugar is independently a 2'-OMe-modified sugar or a 2'-MOE-modified sugar. In some embodiments, the last several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OMe-modified sugars. In some embodiments, the last several sugars comprise three or more consecutive 2'-OMe-modified sugars. In some embodiments, the last several sugars comprise four or more consecutive 2'-OMe-modified sugars. In some embodiments, the last several sugars comprise five or more consecutive 2'-OMe-modified sugars. In some embodiments, the last several sugars comprise six or more consecutive 2'-OMe-modified sugars. In some embodiments, the last few sugars include two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-MOE modified sugars.

[0139] In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the first several (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars. In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the first several (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, two or more of the first several sugars are modified sugars independently selected from 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, three or more of the first several sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, four or more of the first several sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the modified sugars are each independently selected from aliphatic sugars and bicyclic sugars. In some embodiments, one or more sugars are contiguous. In some embodiments, the first 1, 2, 3, or 4 sugars are modified sugars. In some embodiments, the first two sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the first three sugars are modified sugars independently selected from 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the first four sugars are modified sugars independently selected from 2'-OR modified sugars (where R is an optionally substituted C 1~6In some embodiments, each 2'-OR modified sugar is independently selected from a 2'-OMe or a 2'-MOE modified sugar. In some embodiments, each bicyclic sugar is independently an LNA sugar or a cEt sugar. In some embodiments, one or more (1, 2, 3, 4, or 5) of the first several sugars, or each of the first several (e.g., 1, 2, 3, 4, or 5) sugars, is independently a 2'-OR modified sugar (wherein R is an optionally substituted C 1~6aliphatic). In some embodiments, one or more (1, 2, 3, 4, or 5) sugars of the first several sugars, or each of the first several (e.g., 1, 2, 3, 4, or 5) sugars, are independently 2'-OMe or 2'-MOE modified sugars. In some embodiments, one or more (1, 2, 3, 4, or 5) sugars of the first several sugars, or each of the first several (e.g., 1, 2, 3, 4, or 5) sugars, are independently 2'-OMe modified sugars. In some embodiments, one or more (1, 2, 3, 4, or 5) sugars of the first several sugars, or each of the first several (e.g., 1, 2, 3, 4, or 5) sugars, are independently 2'-MOE modified sugars. In some embodiments, the first 1, 2, 3, 4, or more sugars are independently 2'-OMe modified sugars. In some embodiments, the first sugar is a 2'-OMe modified sugar. In some embodiments, the first two sugars are independently 2'-OMe-modified sugars. In some embodiments, the first three sugars are independently 2'-OMe-modified sugars. In some embodiments, the first four sugars are independently 2'-OMe-modified sugars. In some embodiments, the first 1, 2, 3, 4, or more sugars are independently 2'-MOE-modified sugars. In some embodiments, the first sugar is a 2'-MOE-modified sugar. In some embodiments, the first two sugars are independently 2'-MOE-modified sugars. In some embodiments, the first three sugars are independently 2'-MOE-modified sugars. In some embodiments, the first four sugars are independently 2'-MOE-modified sugars. In some embodiments, each such modified sugar is independently the sugar of a nucleoside whose nucleobase is optionally substituted or protected A, T, C, G, or U, or a tautomer of optionally substituted or protected A, T, C, G, or U. In some embodiments, one or more such sugars are independently linked to a PN linkage. In some embodiments, one or more such sugars are each independently linked to a non-negatively charged internucleotide linkage. In some embodiments, one or more such sugars are independently linked to a neutral internucleotide linkage, such as n001.In some embodiments, a non-negatively charged internucleotide linkage or a neutral internucleotide linkage, such as n001, is chiral-controlled. In some embodiments, it is Rp. In some embodiments, one or more such sugars are each independently linked to a PS linkage, e.g., a phosphorothioate internucleotide linkage. In some embodiments, a PS linkage, e.g., a phosphorothioate internucleotide linkage, is chiral-controlled. In some embodiments, it is Sp. In some embodiments, the internucleotide linkage between the first nucleoside and the second nucleoside is a non-negatively charged internucleotide linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a PN linkage. In some embodiments, it is a phosphorylguanidine internucleotide linkage. In some embodiments, it is n001. In some embodiments, it is chiral-controlled. In some embodiments, it is Rp. In some embodiments, each internucleotide linkage attached to one or more of the first several or the first several nucleosides containing modified sugars, excluding the internucleotide linkage between the first and second nucleosides, is independently a PS linkage, e.g., a phosphorothioate internucleotide linkage. In some embodiments, each is chiral controlled. In some embodiments, each is Sp. In some embodiments, the first nucleoside is linked to an additional moiety (e.g., Mod001) through its 5'-terminal carbon (in some embodiments, through a phosphate group), optionally via a linker (e.g., L001). In some embodiments, the first several are the first 3, 4, 5, 6, etc.

[0140] In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the last several (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars. In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the last several (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 aliphatic) and bicyclic sugars (e.g., sugars containing 2'-O-CH2-4' (where -CH2- is optionally substituted) (e.g., LNA sugars, cET sugars (e.g., (S)-cET)). In some embodiments, two or more of the last several sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, three or more of the last several sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, four or more of the last several sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the modified sugars are each independently selected from aliphatic sugars and bicyclic sugars. In some embodiments, one or more sugars are contiguous. In some embodiments, the last 1, 2, 3, or 4 sugars are modified sugars. In some embodiments, the last two sugars are 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the last three sugars are modified sugars independently selected from 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the last four sugars are modified sugars independently selected from 2'-OR modified sugars (where R is an optionally substituted C 1~6In some embodiments, each 2'-OR modified sugar is independently selected from a 2'-OMe or a 2'-MOE modified sugar. In some embodiments, each bicyclic sugar is independently an LNA sugar or a cEt sugar. In some embodiments, one or more (1, 2, 3, 4, or 5) of the last several sugars, or each of the last several (e.g., 1, 2, 3, 4, or 5) sugars, is independently a 2'-OR modified sugar (wherein R is an optionally substituted C 1~6aliphatic). In some embodiments, one or more (1, 2, 3, 4, or 5) of the last several sugars, or each of the last several (e.g., 1, 2, 3, 4, or 5) sugars, are independently 2'-OMe or 2'-MOE modified sugars. ... modified sugars. In some embodiments, one or more (1, 2, 3, 4, or 5) of the last several sugars, or each of the last several (e.g., 1, 2, 3, 4, or 5) sugars, are independently 2'-MOE modified sugars. In some embodiments, the last 1, 2, 3, 4, or more sugars are independently 2'-OMe modified sugars. In some embodiments, the last sugar is a 2'-OMe modified sugar. In some embodiments, the last two sugars are independently 2'-OMe-modified sugars. In some embodiments, the last three sugars are independently 2'-OMe-modified sugars. In some embodiments, the last four sugars are independently 2'-OMe-modified sugars. In some embodiments, the last 1, 2, 3, 4, or more sugars are independently 2'-MOE-modified sugars. In some embodiments, the last sugar is a 2'-MOE-modified sugar. In some embodiments, the last two sugars are independently 2'-MOE-modified sugars. In some embodiments, the last three sugars are independently 2'-MOE-modified sugars. In some embodiments, the last four sugars are independently 2'-MOE-modified sugars. In some embodiments, each such modified sugar is independently the sugar of a nucleoside whose nucleobase is optionally substituted or protected A, T, C, G, or U, or a tautomer of optionally substituted or protected A, T, C, G, or U. In some embodiments, one or more such sugars are each independently linked to a non-negatively charged internucleotide linkage, hi some embodiments, one or more such sugars are each independently linked to a PN linkage.In some embodiments, one or more such sugars are each independently linked to a neutral internucleotide linkage, such as n001. In some embodiments, a non-negatively charged internucleotide linkage or a neutral internucleotide linkage, such as n001, is chiralally controlled. In some embodiments, it is Rp. In some embodiments, one or more such sugars are independently linked to a PS linkage, e.g., a phosphorothioate internucleotide linkage. In some embodiments, a PS linkage, e.g., a phosphorothioate internucleotide linkage, is chiralally controlled. In some embodiments, it is Sp. In some embodiments, the internucleotide linkage between the last nucleoside and the penultimate nucleoside is a non-negatively charged internucleotide linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a PN linkage. In some embodiments, it is a phosphorylguanidine internucleotide linkage. In some embodiments, it is n001. In some embodiments, it is chiralally controlled. In some embodiments, it is Rp. In some embodiments, each internucleotide linkage attached to one or more of the last several or the last several nucleosides comprising a modified sugar, except for the internucleotide linkage between the last and penultimate nucleoside, is independently a phosphorothioate internucleotide linkage. In some embodiments, each is chiral controlled. In some embodiments, each is Sp. In some embodiments, the last several are the last 3, 4, 5, etc.

[0141] In some embodiments, the sugar at the +1 position is a 2'-F modified sugar. In some embodiments, the sugar at the +1 position is a natural DNA sugar. In some embodiments, the sugar at the 0 position is a natural DNA sugar (the nucleoside at the 0 position is opposite the target adenosine when aligned). In some embodiments, the sugar at the -1 position is a DNA sugar. In some embodiments, the sugar at the -2 position is a 2'-OR modified sugar (where R is an optionally substituted C1~6 aliphatic) or bicyclic sugars (e.g., sugars containing 2'-O-CH2-4' (where -CH2- is optionally substituted) (e.g., LNA sugars, cET sugars (e.g., (S)-cEt)). In some embodiments, this is a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, it is a 2'-OMe modified sugar. In some embodiments, it is a 2'-MOE modified sugar. In some embodiments, it is a bicyclic sugar. In some embodiments, it is an LNA sugar. In some embodiments, it is a cEt sugar. In some embodiments, the sugar at the -3 position is a 2'-F modified sugar. In some embodiments, each sugar after the -3 position (e.g., at positions -4, -5, -6, etc.) is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 aliphatic) or bicyclic sugars (e.g., sugars containing 2'-O-CH2-4' (where -CH2- is optionally substituted) (e.g., LNA sugars, cET sugars (e.g., (S)-cEt)). In some embodiments, each is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6aliphatic) or bicyclic sugars. In some embodiments, each is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each is a 2'-OMe modified sugar. In some embodiments, each is a 2'-MOE modified sugar. In some embodiments, one or more are independently a 2'-OMe modified sugar and one or more are independently a 2'-MOE modified sugar. In some embodiments, as described herein, the internucleotide linkage between the nucleoside at the -1 position and the nucleoside at the -2 position is a non-negatively charged internucleotide linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphorylguanidine internucleotide linkage. In some embodiments, it is nOOl. In some embodiments, it is chiral controlled. In some embodiments, it is Sp. In some embodiments, it is Rp. In some embodiments, the internucleotide linkage between the nucleoside at the -2 position and the nucleoside at the -3 position is a natural phosphate linkage. In some embodiments, as described herein, the internucleotide linkage between the last nucleoside and the penultimate nucleoside is a non-negatively charged internucleotide linkage. In some embodiments, it is a neutral internucleotide linkage. In some embodiments, it is a phosphorylguanidine internucleotide linkage. In some embodiments, it is n001. In some embodiments, it is chiral controlled. In some embodiments, it is Rp. In some embodiments, each internucleotide linkage between nucleosides on the 3' side of the nucleoside opposite the target adenosine is independently a phosphorothioate internucleotide linkage, except for those between the nucleoside at the -1 position and the nucleotide at the -2 position, between the nucleoside at the -2 position and the nucleoside at the -3 position, and between the last nucleoside and the penultimate nucleoside. In some embodiments, each phosphorothioate internucleotide linkage is chiral controlled. In some embodiments, each is Sp.

[0142] In some embodiments, the first and / or last one or more sugars are modified sugars, e.g., bicyclic sugars and / or 2'-OR modified sugars (where R is an optionally substituted C 1~6 aliphatic) (e.g., 2'-OMe modified sugars, 2'-MOE modified sugars, etc.). In some embodiments, such sugars may increase the stability, affinity, and / or activity of the oligonucleotide. In some embodiments, when conjugated to one or more additional chemical moieties, the sugars at the 5' and / or 3' ends of the oligonucleotide are bicyclic sugars or 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the sugar at the 5' terminal is not a bicyclic sugar or a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, such 5'-terminal sugars are not linked to an additional chemical moiety. In some embodiments, the 5'-terminal sugar is a 2'-F modified sugar. In some embodiments, the 5'-terminal sugar is a 2'-F modified sugar conjugated to an additional chemical moiety. In some embodiments, the 3'-terminal sugar is a bicyclic sugar or a 2'-OR modified sugar (wherein R is an optionally substituted C 1~6 In some embodiments, such 3'-terminal sugars are not linked to an additional chemical moiety. In some embodiments, the 3'-terminal sugar is a 2'-F modified sugar. In some embodiments, the 3'-terminal sugar is a 2'-F modified sugar conjugated to an additional chemical moiety. In some embodiments, the last several sugars are the sugar 3' to the nucleoside opposite the target adenosine (e.g., N -1 , N -2In some embodiments, the last several sugars or the 3' sugars comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F modified sugars. In some embodiments, the last several sugars or the 3' sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-F modified sugars. In some embodiments, the last several sugars or the 3' sugars comprise one or more 2'-F modified sugars or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a bicyclic sugar or a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, as described herein, the 2'-OR modified sugar is a 2'-OMe modified sugar or a 2'-MOE modified sugar, in some embodiments it is a 2'-OMe modified sugar, and in some embodiments it is a 2'-MOE modified sugar. In some embodiments, the last several sugars or the 3' sugar comprise one or more 2'-F modified sugars or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OR modified sugar (where R is an optionally substituted C 1~6aliphatic). In some embodiments, the last several sugars or the sugars on the 3' side comprise one or more 2'-F modified sugars or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OMe modified sugar or a 2'-MOE modified sugar. In some embodiments, the last several sugars or the sugars on the 3' side comprise one or more 2'-F modified sugars or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OMe modified sugar. In some embodiments, the last several sugars or the sugars on the 3' side comprise one or more 2'-F modified sugars or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-MOE modified sugar. In some embodiments, up to two nucleosides 3' to the nucleoside opposite the adenosine independently have a 2'-F modified sugar. In some embodiments, these are at the -4 and -5 positions. In some embodiments, these are the second and third penultimate nucleosides of the oligonucleotide. In some embodiments, no more than one nucleoside 3' to the nucleoside opposite the adenosine has a 2'-F modified sugar. In some embodiments, this is at the -3 position. In some embodiments, this is the fourth penultimate nucleoside of the oligonucleotide.

[0143] In some embodiments, a bicyclic sugar or a 2'-OR modified sugar (wherein R is an optionally substituted C 1~6aliphatic) are present in a region comprising one or more (e.g., 1-30, 1-25, 1-20, 1-15, 1-10, 2-30, 2-25, 2-20, 2-25, 2-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) sugars that are 2'-F modified. In some embodiments, the majority of sugars as described herein in such a region are 2'-F modified sugars. In some embodiments, two or more 2'-F modified sugars are contiguous. In some embodiments, the region is a first domain. In some embodiments, bicyclic sugars are present in such a region. In some embodiments, 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, 2'-OMe modified sugars are present in such regions. In some embodiments, 2'-MOE modified sugars are present in such regions.

[0144] In some embodiments, one or more sugars at positions -5, -4, -3, +1, +2, +4, +5, +6, +7, and +8 (position 0 is the position of the nucleoside opposite the target adenosine; "+" proceeds from the nucleoside opposite the target adenosine to the 5' end of the oligonucleotide, and "-" proceeds from the nucleoside opposite the target adenosine to the 3' end of the oligonucleotide; e.g., 5'-N1N0N -1 At -3', N0 is at the 0 position, N1 is at the +1 position, and N is at the 1 position, if it is the nucleoside opposite the target adenosine. -1 is present at the -1 position) are independently 2'-F modified sugars. In some embodiments, the sugar at the +1 position and one or more sugars at the -5, -4, -3, +2, +4, +5, +6, +7, and +8 positions are independently 2'-F modified sugars. In some embodiments, the sugar at the +1 position and one sugar at the -5, -4, -3, +2, +4, +5, +6, +7, and +8 positions are independently 2'-F modified sugars.

[0145] In some embodiments, an oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, such as 2-10, 3-10, 2-5, 2-4, 2-3, 3-5, 3-4, etc.) naturally occurring DNA sugars. In some embodiments, one or more naturally occurring DNA sugars are present in an editing region (e.g., at the +1, 0, and / or -1 positions). In some embodiments, naturally occurring DNA sugars are present within the first several nucleosides of the oligonucleotide (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides). In some embodiments, the first, second, and / or third nucleoside of the oligonucleotide independently have a naturally occurring DNA sugar. In some embodiments, the natural DNA sugar is linked to a modified internucleotide linkage (e.g., a PN linkage, a PS linkage, a non-negatively charged internucleotide linkage, a neutral internucleotide linkage, a phosphorylguanidine internucleotide linkage, an n001 or a phosphorothioate internucleotide linkage (in various embodiments, Sp)).

[0146] Oligonucleotides can contain various types of internucleotide linkages. In some embodiments, the oligonucleotide contains one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a PS linkage. In some embodiments, the modified linkage is a PN linkage. In some embodiments, the oligonucleotide contains a PO and a PS linkage. In some embodiments, the oligonucleotide contains a PO and a PN linkage. In some embodiments, the oligonucleotide contains a PN and a PS linkage. In some embodiments, the oligonucleotide contains a PO, PN, and PS linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate modified internucleotide linkage. In some embodiments, the modified internucleotide linkage, e.g., a PN linkage, is a non-negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage, e.g., a PN linkage, is a neutral internucleotide linkage. In some embodiments, the modified internucleotide linkage, e.g., a PN linkage, is a phosphorylguanidine internucleotide linkage. In some embodiments, the modified internucleotide linkage, e.g., a PN linkage, is an n001 linkage. In some embodiments, the oligonucleotide comprises one or more natural phosphate linkages. In some embodiments, the natural phosphate linkage is attached to a nucleoside containing a modified sugar that may improve stability (e.g., resistance to nucleases). In some embodiments, the natural phosphate linkage is attached to a bicyclic sugar. In some embodiments, the natural phosphate linkage is attached to a 2'-modified sugar. In some embodiments, the natural phosphate linkage is attached to a 2'-OR modified sugar (where R is an optionally substituted C 1~6aliphatic). In some embodiments, a natural phosphate linkage is linked to a 2'-OMe modified sugar. In some embodiments, a natural phosphate linkage is linked to a 2'-MOE modified sugar. In some embodiments, an oligonucleotide comprises a phosphorothioate internucleotide linkage, a non-negatively charged internucleotide linkage, and a natural phosphate linkage. In some embodiments, an oligonucleotide comprises a phosphorothioate internucleotide linkage, a neutral internucleotide linkage, and a natural phosphate linkage. In some embodiments, an oligonucleotide comprises a phosphorothioate internucleotide linkage, a phosphorylguanidine internucleotide linkage, and a natural phosphate linkage. In some embodiments, an oligonucleotide comprises a phosphorothioate internucleotide linkage, an n001, and a natural phosphate linkage. In some embodiments, each chiral internucleotide linkage is independently chiral controlled. In some embodiments, one or more chiral internucleotide linkages are not chiral controlled. In some embodiments, each PS linkage is independently chiral controlled. In some embodiments, each phosphorothioate internucleotide linkage is independently chiral controlled. In some embodiments, each chiral internucleotide linkage is independently chiral-controlled. In some embodiments, most or each phosphorothioate internucleotide linkage is Sp as described herein. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) phosphorothioate internucleotide linkages are independently Rp. In some embodiments, most or each P N or each non-negatively charged internucleotide linkage (e.g., n001) is Rp. In some embodiments, most or each non-negatively charged internucleotide linkage (e.g., n001) is Sp.

[0147] In some embodiments, the oligonucleotide comprises a phosphorothioate internucleotide linkage and a non-negatively charged internucleotide linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleotide linkage and a neutral internucleotide linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleotide linkage and a phosphorylguanidine internucleotide linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleotide linkage and an n001. In some embodiments, each chiral internucleotide linkage is independently chiral controlled. In some embodiments, one or more chiral internucleotide linkages are not chiral controlled. In some embodiments, each phosphorothioate internucleotide linkage is independently chiral controlled. In some embodiments, each chiral internucleotide linkage is independently chiral controlled. In some embodiments, most or each phosphorothioate internucleotide linkage is Sp as described herein. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) phosphorothioate internucleotide linkages are Rp. In some embodiments, most or each non-negatively charged internucleotide linkage (e.g., n001) is Rp. In some embodiments, most or each non-negatively charged internucleotide linkage (e.g., n001) is Sp. In some embodiments, the oligonucleotide does not contain a natural phosphate linkage. In some embodiments, each internucleotide linkage is independently a phosphorothioate or a non-negatively charged internucleotide linkage. In some embodiments, each internucleotide linkage is independently a phosphorothioate or a neutrally charged internucleotide linkage. In some embodiments, each internucleotide linkage is independently a phosphorothioate or a phosphorylguanidine internucleotide linkage. In some embodiments, each internucleotide linkage is independently a phosphorothioate or an n001 internucleotide linkage.In some embodiments, the final internucleotide linkage of the oligonucleotide is a non-negatively charged internucleotide linkage, a neutral internucleotide linkage, a phosphorylguanidine internucleotide linkage, or n001.

[0148] In some embodiments, the oligonucleotide of the present disclosure comprises one or more modified nucleobases.Various modifications can be introduced into the sugar and / or nucleobase according to the present disclosure.For example, in some embodiments, the modification is the modification described in U.S. Patent No. 9,006,198. In some embodiments, modifications are made to the nucleotides of interest, with each sugar, base, and internucleotide linkage independently incorporated by reference, as described in U.S. Patent Nos. 9,394,333, 9,744,183, 9,605,019, 9,982,257, U.S. Patent Application Publication Nos. 20170037399, 20180216108, 20180216107, U.S. Patent No. 9,598,458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647 ... and modifications described in Patent Publication Nos. 2018 / 098264, 2018 / 022473, 2018 / 223056, 2018 / 223073, 2018 / 223081, 2018 / 237194, 2019 / 032607, 2019 / 032612, 2019 / 055951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858 and / or 2021 / 237223.

[0149] In some embodiments, the nucleobase in the nucleoside is or comprises a ring BA or a tautomer of ring BA having the structure BA-I, BA-Ia, BA-Ib, BA-Ic, BA-Id, BA-II, BA-II-a, BA-II-b, BA-II-c, BA-II-d, BA-III, BA-III-a, BA-III-b, BA-III-c, BA-III-d, BA-III-e, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-Va, BA-Vb, or BA-VI, wherein the nucleobase is optionally substituted or protected.

[0150] In some embodiments, the sugar is a modified sugar that includes a 2'-modification, such as 2'-F, 2'-OR (where R is an optionally substituted aliphatic), or a bicyclic sugar (e.g., an LNA sugar), or an acyclic sugar (e.g., a UNA sugar).

[0151] In some embodiments, provided oligonucleotides comprise one or more domains, each independently having a particular length, modification, stereochemistry of the linked phosphorus, etc., as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising one or more modified sugars and / or one or more modified internucleotide linkages, wherein the oligonucleotide comprises a first domain and a second domain, each independently comprising one or more nucleobases. In some embodiments, the present disclosure provides oligonucleotides comprising one or more domains and / or subdomains as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a first domain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a second domain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a first subdomain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a second subdomain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a third subdomain as described herein. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more regions independently selected from a first domain, a second domain, a first subdomain, a second subdomain, and a third subdomain, each independently as described herein. In some embodiments, the present disclosure provides an oligonucleotide comprising: the first domain; and Second Domain Including, the first domain comprises one or more 2'-F modifications; The second domain provides an oligonucleotide that includes one or more sugars that do not have a 2'-F modification.

[0152] In some embodiments, an oligonucleotide or portion thereof (e.g., first domain, second domain, first subdomain, second subdomain, third subdomain, etc.) comprises a particular level of modified sugars. In some embodiments, the modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar is an acyclic sugar (e.g., by cleaving the C2-C3 bond of the corresponding cyclic sugar). In some embodiments, the modified sugar comprises a 5'-modification. Typically, oligonucleotides of the present disclosure have a free 5'-OH at their 5'-terminus and a free 3'-OH at their 3'-terminus, unless otherwise specified, e.g., by context. In some embodiments, the 5'-terminal sugar of the oligonucleotide may comprise a modified 5'-OH.

[0153] In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 80% to 85%, 80% to 85%, 80% to 85%, 80% to 85%, 80% to 9 ... 0%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

[0154] In some embodiments, a majority is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, a majority is at least about 50% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%. %, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, a majority is about or at least about 50%. In some embodiments, a majority is about or at least about 55%. In some embodiments, a majority is about or at least about 60%. In some embodiments, a majority is about or at least about 65%. In some embodiments, a majority is about or at least about 70%. In some embodiments, a majority is about or at least about 75%. In some embodiments, a majority is about or at least about 80%. In some embodiments, a majority is about or at least about 85%. In some embodiments, a majority is about or at least about 90%. In some embodiments, a majority is about or at least about 95%.

[0155] In some embodiments, the oligonucleotide or a portion thereof (e.g., the first domain, the second domain, the first subdomain, the second subdomain, the third subdomain, etc.) comprises a particular level of modified internucleotide linkages. In some embodiments, the oligonucleotide or a portion thereof (e.g., the first domain, the second domain, the first subdomain, the second subdomain, the third subdomain, etc.) comprises a particular level of chiral internucleotide linkages. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 75% to 90%, 75% to 95%, 75% to 10 ... 0% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%.In some embodiments, the percentage is about 100%.

[0156] In some embodiments, an oligonucleotide or a portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a particular level of chiral-controlled internucleotide linkages. In some embodiments, an oligonucleotide or a portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a particular level of Sp internucleotide linkages. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 75% to 90%, 75% to 95%, 75% to 10 ... 0% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

[0157] In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a particular level of Sp internucleotide linkages. In some embodiments, the level may be about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 75% to 90%, 75% to 95%, 75% to 10 ... 0% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% etc.In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 10 ... 0%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, about 1 to 50, 1 to 40, 1 to 30, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, internucleotide linkages are independently Sp chiral internucleotide linkages.In some embodiments, a higher percentage of Sp internucleotide linkages (e.g., compared to Rp internucleotide linkages and / or natural phosphate linkages) in an oligonucleotide or particular portion thereof may result in improved properties and / or activity (e.g., increased stability and / or increased adenosine editing activity).

[0158] In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a particular level of Rp internucleotide linkages. In some embodiments, the level may be about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 75% to 90%, 75% to 95%, 75% to 10 ... 0% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% etc.In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 10 ... 0%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about 5% or less than about 5%. In some embodiments, the percentage is about 10% or less than about 10%. In some embodiments, the percentage is about 15% or less than about 15%. In some embodiments, the percentage is about 20% or less than about 20%. In some embodiments, the percentage is about 25% or less than about 25%.In some embodiments, the percentage is about 30% or less. In some embodiments, the percentage is about 35% or less. In some embodiments, the percentage is about 40% or less. In some embodiments, the percentage is about 45% or less. In some embodiments, the percentage is about 50% or less. In some embodiments, about 1 to 50, 1 to 40, 1 to 30, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, internucleotide linkages are independently Rp chiral internucleotide linkages. In some embodiments, this number is about 1 or less. In some embodiments, this number is about 2 or less. In some embodiments, this number is about 3 or less. In some embodiments, this number is about 4 or less than or equal to about 4. In some embodiments, this number is about 5 or less than or equal to about 5. In some embodiments, this number is about 6 or less than or equal to about 6. In some embodiments, this number is about 7 or less than or equal to about 7. In some embodiments, this number is about 8 or less than or equal to about 8. In some embodiments, this number is about 9 or less than or equal to about 9. In some embodiments, this number is about 10 or less than or equal to about 10.

[0159] Without wishing to be bound by any particular theory, in some examples, the Rp and Sp configurations of internucleotide bonds may affect the structural changes in the helical structure of a double-stranded complex formed by an oligonucleotide and a target nucleic acid such as RNA, and ADAR proteins may recognize and interact with various targets (e.g., double-stranded complexes formed by an oligonucleotide and a target nucleic acid such as RNA) via multiple domains. In some embodiments, the provided oligonucleotides and compositions thereof facilitate and / or enhance the interaction profile of the oligonucleotide, the target nucleic acid, and / or the ADAR protein, resulting in efficient adenosine modification by the ADAR protein through the incorporation of various modifications and / or stereochemical control.

[0160] In some embodiments, an oligonucleotide may have or include a base sequence; an internucleotide linkage, a base modification, a sugar modification, an additional chemical moiety or pattern thereof; and / or any other structural element described herein (e.g., in the Tables).

[0161] In some embodiments, a provided oligonucleotide or composition is characterized in that when it is contacted with a target nucleic acid containing a target adenosine in a system (e.g., an ADAR-mediated deamination system), modification of the target adenosine (e.g., deamination of target A) is improved compared to that observed under reference conditions (e.g., selected from the group consisting of the absence of the composition, the presence of the reference oligonucleotide or composition, and combinations thereof). In some embodiments, the modification (e.g., ADAR-mediated deamination (e.g., endogenous ADAR-mediated deamination)) is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 fold or more.

[0162] In some embodiments, the oligonucleotide is provided in a salt form. In some embodiments, the oligonucleotide is provided as a salt containing a negatively charged internucleotide linkage (e.g., a phosphorothioate internucleotide linkage, a natural phosphate linkage, etc.) present in a salt form. In some embodiments, the oligonucleotide is provided as a pharmaceutically acceptable salt. In some embodiments, the oligonucleotide is provided as a metal salt. In some embodiments, the oligonucleotide is provided as a sodium salt. In some embodiments, the oligonucleotide is provided as an ammonium salt. In some embodiments, the oligonucleotide is provided as a metal salt (e.g., a sodium salt), and each negatively charged internucleotide linkage is independently in a salt form (e.g., -OP(O)(SNa)-O- for a phosphorothioate internucleotide linkage, -OP(O)(ONa)-O- for a natural phosphate linkage, etc.).

[0163] In some embodiments, the oligonucleotides are chiral controlled and contain one or more chiral controlled internucleotide linkages. In some embodiments, the provided oligonucleotides are stereochemically pure. In some embodiments, the provided oligonucleotides or compositions thereof are stereochemically pure from other stereoisomers. In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions.

[0164] In some embodiments, the internucleotide linkages at one or more of positions 1 (the first internucleotide linkage from the 5' end), 3, 26, and 29 are independently a PN internucleotide linkage. In some embodiments, the internucleotide linkages at one or more of positions 1, 3, 26, and 29 are independently a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkages at one or more of positions 1, 3, 26, and 29 are independently n001. In some embodiments, the internucleotide linkage at position 1 is a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 1 is a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at position 1 is n001. In some embodiments, the internucleotide linkage at position 3 is a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 3 is a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at position 3 is n001. In some embodiments, the internucleotide linkage at position 26 is a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 26 is a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at position 26 is n001. In some embodiments, the internucleotide linkage at position 29 is a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 29 is a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at position 29 is n001. In some embodiments, the internucleotide linkage at one or more of positions 7, 17, 27, and 28 is not a PN internucleotide linkage. In some embodiments, the internucleotide linkage at one or more of positions 7, 17, 27, and 28 is not a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at one or more of positions 7, 17, 27, and 28 is not n001. In some embodiments, the internucleotide linkage at position 7 is not a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 7 is not a phosphorylguanidine internucleotide linkage.In some embodiments, the internucleotide linkage at position 7 is not n001. In some embodiments, the internucleotide linkage at position 17 is not a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 17 is not a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at position 17 is not n001. In some embodiments, the internucleotide linkage at position 27 is not a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 27 is not a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at position 27 is not n001. In some embodiments, the internucleotide linkage at position 28 is not a PN internucleotide linkage. In some embodiments, the internucleotide linkage at position 28 is not a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkage at position 28 is not n001. In some embodiments, the internucleotide linkages at one or more of positions 7 and 17 are independently not a natural phosphate linkage. In some embodiments, the internucleotide linkage at position 7 is not a natural phosphate linkage. In some embodiments, the internucleotide linkage at position 17 is not a natural phosphate linkage. In some embodiments, the internucleotide linkages at one or more of positions 16, 18, 19, 23, and 27 are independently a natural phosphate linkage. In some embodiments, the internucleotide linkage at position 16 is a natural phosphate linkage. In some embodiments, the internucleotide linkage at position 18 is a natural phosphate linkage. In some embodiments, the internucleotide linkage at position 19 is a natural phosphate linkage. In some embodiments, the internucleotide linkage at position 23 is a natural phosphate linkage. In some embodiments, the internucleotide linkage at position 27 is a natural phosphate linkage. In some embodiments, the internucleotide linkages at one or more of positions 6, 7, 8, 9, 10, 12, 13, 15, 19, 21, 22, 23, 27, and 28 are independently not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 6 is not a phosphorothioate internucleotide linkage of Rp.In some embodiments, the internucleotide linkage at position 7 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 8 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 9 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 10 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 12 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 13 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 15 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 19 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 21 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 22 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 23 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 27 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 28 is not a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkages at one or more of positions 11, 18, 20, and 25 are independently a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 11 is a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 18 is a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 20 is a phosphorothioate internucleotide linkage of Rp. In some embodiments, the internucleotide linkage at position 25 is a phosphorothioate internucleotide linkage of Rp.In some embodiments, at a position where the internucleotide linkage is not a PN, phosphorylguanidine, n001, or natural phosphate linkage, the internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, it is an Sp phosphorothioate internucleotide linkage. In some embodiments, at a position where the internucleotide linkage is not an Rp phosphorothioate internucleotide linkage, the internucleotide linkage is an Sp phosphorothioate internucleotide linkage. In some embodiments, the sugar of one or more nucleosides at positions 1 (the first nucleoside from the 5' end), 2, 3, 5, 14, 27, 29, and 30 are independently 2'-OMe modified sugars. In some embodiments, the sugar of the nucleoside at position 1 is a 2'-OMe modified sugar. In some embodiments, the sugar of the nucleoside at position 2 is a 2'-OMe modified sugar. In some embodiments, the sugar of the nucleoside at position 3 is a 2'-OMe modified sugar. In some embodiments, the sugar of the nucleoside at position 5 is a 2'-OMe-modified sugar. In some embodiments, the sugar of the nucleoside at position 14 is a 2'-OMe-modified sugar. In some embodiments, the sugar of the nucleoside at position 18 is a 2'-OMe-modified sugar. In some embodiments, the sugar of the nucleoside at position 27 is a 2'-OMe-modified sugar. In some embodiments, the sugar of the nucleoside at position 29 is a 2'-OMe-modified sugar. In some embodiments, the sugar of the nucleoside at position 30 is a 2'-OMe-modified sugar. In some embodiments, the sugar of one or more nucleosides at positions 5, 7, 17, 22, and 23 are independently not a 2'-MOE-modified sugar. In some embodiments, the sugar of the nucleoside at position 5 is not a 2'-MOE-modified sugar. In some embodiments, the sugar of the nucleoside at position 7 is not a 2'-MOE-modified sugar. In some embodiments, the sugar of the nucleoside at position 17 is not a 2'-MOE modified sugar. In some embodiments, the sugar of the nucleoside at position 22 is not a 2'-MOE modified sugar. In some embodiments, the sugar of the nucleoside at position 23 is not a 2'-MOE modified sugar.

[0165] As described herein, oligonucleotides of the present disclosure may be provided with high purity (e.g., 50% to 100%). In some embodiments, oligonucleotides of the present disclosure are of high stereochemical purity (e.g., 50% to 100%). In some embodiments, the oligonucleotides in the provided compositions are of high stereochemical purity (e.g., a high percentage (e.g., 50% to 100%) of a stereoisomer relative to other stereoisomers of the same oligonucleotide). In some embodiments, the percentage is at least or about 50%. In some embodiments, the percentage is at least or about 60%. In some embodiments, the percentage is at least or about 70%. In some embodiments, the percentage is at least or about 75%. In some embodiments, the percentage is at least or about 80%. In some embodiments, the percentage is at least or about 85%. In some embodiments, the percentage is at least or about 90%. In some embodiments, the percentage is at least or about 95%.

[0166] First Domain As described herein, in some embodiments, the oligonucleotide comprises a first domain and a second domain. In some embodiments, the oligonucleotide consists of a first domain and a second domain. By way of example, certain embodiments are described below.

[0167] In some embodiments, the first domain has a length of about 2 to 100 nucleobases (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 nucleobases, etc.). In some embodiments, the first domain has a length of about 5 to 30 nucleobases. In some embodiments, the first domain has a length of about 10 to 30 nucleobases. In some embodiments, the first domain has a length of about 10 to 50 nucleobases. In some embodiments, the first domain has a length of about 20-50 nucleobases. In some embodiments, the first domain has a length of about 20-30 nucleobases. In some embodiments, the first domain has a length of about 10-20 nucleobases. In some embodiments, the first domain has a length of about 13-16 nucleobases. In some embodiments, the first domain has a length of 10 nucleobases. In some embodiments, the first domain has a length of 11 nucleobases. In some embodiments, the first domain has a length of 12 nucleobases. In some embodiments, the first domain has a length of 13 nucleobases. In some embodiments, the first domain has a length of 14 nucleobases. In some embodiments, the first domain has a length of 15 nucleobases. In some embodiments, the first domain has a length of 16 nucleobases. In some embodiments, the first domain has a length of 17 nucleobases. In some embodiments, the first domain has a length of 18 nucleobases. In some embodiments, the first domain has a length of 19 nucleobases. In some embodiments, the first domain has a length of 20 nucleobases. In some embodiments, the first domain has a length of 21 nucleobases. In some embodiments, the first domain has a length of 22 nucleobases.In some embodiments, the first domain has a length of 23 nucleobases. In some embodiments, the first domain has a length of 24 nucleobases. In some embodiments, the first domain has a length of 25 nucleobases. In some embodiments, the first domain has a length of about or at least about 20 nucleobases. In some embodiments, the first domain has a length of about or at least about 25 nucleobases.

[0168] In some embodiments, the first domain is about or at least about 5-95%, 10-90%, 20-80%, 30-70%, 40-70%, 40-60%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the oligonucleotide. In some embodiments, the percentage is about 30-80%. In some embodiments, the percentage is about 30-70%. In some embodiments, the percentage is about 40-60%. In some embodiments, the percentage is about 20%. In some embodiments, the percentage is about 25%. In some embodiments, the percentage is about 30%. In some embodiments, the percentage is about 35%. In some embodiments, the percentage is about 40%. In some embodiments, the percentage is about 45%. In some embodiments, the percentage is about 50%. In some embodiments, the percentage is about 55%. In some embodiments, the percentage is about 60%. In some embodiments, the percentage is about 65%. In some embodiments, the percentage is about 70%. In some embodiments, the percentage is about 75%. In some embodiments, the percentage is about 80%. In some embodiments, the percentage is about 85%. In some embodiments, the percentage is about 90%.

[0169] In some embodiments, when the oligonucleotide is aligned with a target nucleic acid in terms of complementarity, there are one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) mismatches in the first domain. In some embodiments, there is one mismatch. In some embodiments, there are two mismatches. In some embodiments, there are three mismatches. In some embodiments, there are four mismatches. In some embodiments, there are five mismatches. In some embodiments, there are six mismatches. In some embodiments, there are seven mismatches. In some embodiments, there are eight mismatches. In some embodiments, there are nine mismatches. In some embodiments, there are ten mismatches.

[0170] In some embodiments, when the oligonucleotide is aligned with a target nucleic acid in terms of complementarity, there are one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles in the first domain. In some embodiments, there is one wobble. In some embodiments, there are two wobbles. In some embodiments, there are three wobbles. In some embodiments, there are four wobbles. In some embodiments, there are five wobbles. In some embodiments, there are six wobbles. In some embodiments, there are seven wobbles. In some embodiments, there are eight wobbles. In some embodiments, there are nine wobbles. In some embodiments, there are ten wobbles.

[0171] In some embodiments, the duplex of the oligonucleotide and the target nucleic acid in the first domain region contains one or more bulges, each independently containing one or more mismatches that are not wobble. In some embodiments, there are 0 to 10 bulges (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulges, etc.). In some embodiments, this number is 0. In some embodiments, this number is 1. In some embodiments, this number is 2. In some embodiments, this number is 3. In some embodiments, this number is 4. In some embodiments, this number is 5.

[0172] In some embodiments, the first domain is perfectly complementary to the target nucleic acid.

[0173] In some embodiments, the first domain comprises one or more modified nucleobases.

[0174] In some embodiments, the second domain comprises one or more sugars comprising two 2'-H (e.g., natural DNA sugars). In some embodiments, the second domain comprises one or more sugars comprising a 2'-OH (e.g., natural RNA sugars). In some embodiments, the first domain comprises one or more modified sugars. In some embodiments, the modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar (e.g., an LNA sugar). In some embodiments, the modified sugar is an acyclic sugar (e.g., by cleaving the C2-C3 bond of the corresponding cyclic sugar).

[0175] In some embodiments, the first domain comprises between about 1 and 50 (e.g., between about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars. In some embodiments, the first domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) modified sugars having a 2'-F modification.In some embodiments, the first domain comprises about 2 to 50 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc., 2 to 40, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 3 to 40, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 4 to 40, 4 to 30) nucleotides having 2'-F modifications. , 4~25, 4~20, 4~15, 4~10, 5~40, 5~30, 5~25, 5~20, 5~15, 5~10, 6~40, 6~30, 6~25, 6~20, 6~15, 6~10, 7~40, 7~30, 7~25, 7~20, 7~15, 7~10, 8~40, 8~30, 8~25, 8~20, 8~15 , 8-10, 9-40, 9-30, 9-25, 9-20, 9-15, 9-10, 10-40, 10-30, 10-25, 10-20, 10-15, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive modified sugars. In some embodiments, the first domain comprises two consecutive 2'-F modified sugars. In some embodiments, the first domain comprises three consecutive 2'-F modified sugars. In some embodiments, the first domain comprises four consecutive 2'-F modified sugars. In some embodiments, the first domain comprises five consecutive 2'-F modified sugars. In some embodiments, the first domain comprises six consecutive 2'-F modified sugars. In some embodiments, the first domain comprises seven consecutive 2'-F modified sugars. In some embodiments, the first domain comprises eight consecutive 2'-F modified sugars. In some embodiments, the first domain comprises nine consecutive 2'-F modified sugars. In some embodiments, the first domain comprises ten consecutive 2'-F modified sugars. In some embodiments, the first domain comprises two or more 2'-F modified sugar blocks, wherein each sugar in the 2'-F modified sugar block is independently a 2'-F modified sugar.In some embodiments, each 2'-F-modified sugar block independently comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive 2'-F-modified sugars as described herein. In some embodiments, two consecutive 2'-F-modified sugar blocks are independently separated by a separation block, which separation block independently comprises one or more sugars that are not 2'-F-modified sugars. In some embodiments, each sugar in a separation block is independently not 2'-F modified. In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) or all sugars in a separation block are independently not 2'-F modified. In some embodiments, a separation block comprises one or more bicyclic sugars (e.g., LNA sugars, cEt sugars, etc.) and / or one or more 2'-OR-modified sugars (wherein R is an optionally substituted C). 1~6 In some embodiments, the separation block comprises one or more 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, two or more 2'-F unmodified sugars are contiguous. In some embodiments, two or more 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the separation block is comprised of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OR modified sugars (where R is an optionally substituted C 1~6 In some embodiments, the separation block comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-ORF modified sugars (wherein R is an optionally substituted C 1~6In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe sugar or a 2'-MOE sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE sugar. In some embodiments, a separation block comprises one or more 2'-F modified sugars. In some embodiments, the 2'-F modified sugars in a separation block are not adjacent to one another. In some embodiments, a separation block does not comprise a 2'-F modified sugar. In some embodiments, each sugar in a separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each sugar in each separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each sugar in the separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6 In some embodiments, each sugar in each separation block is independently a 2'-OR modified sugar (where R is an optionally substituted C 1~6In some embodiments, each sugar in a separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in each separation block is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each sugar in a separation block is independently a 2'-OMe modified sugar. In some embodiments, each sugar in a separation block is independently a 2'-MOE modified sugar. In some embodiments, a separation block comprises a 2'-OMe sugar and a 2'-MOE modified sugar. In some embodiments, each 2'-F block and each separation block independently comprises 1, 2, 3, 4, or ...

Claims

1. An oligonucleotide comprising: a first domain; and Second Domain Including, the first domain comprises one or more 2'-F modifications; the second domain comprises one or more sugars that do not have a 2'-F modification; An oligonucleotide, the base sequence of which is complementary to a characteristic portion of the MECP2 transcript that contains the target adenosine.

2. The oligonucleotide described in claim 1, having a length of 10 to 200 nucleic acid bases.

3. 3. The oligonucleotide of claim 2, wherein between 5% and 100% of the sugars in the first domain independently comprise a 2'-F modification.

4. The first domain may comprise a 2'-OR modification (where R is a substituted or unsubstituted C １～６ 4. The oligonucleotide of claim 3, comprising one or more modified sugars, including aliphatic sugars.

5. The oligonucleotide of claim 4, wherein the second domain has a length of 2 to 50 nucleobases.

6. 6. The oligonucleotide of claim 5, wherein the second domain comprises the nucleoside opposite a target adenosine when the oligonucleotide is aligned with a target nucleic acid in terms of complementarity.

7. 7. The oligonucleotide of claim 6, wherein the opposite nucleobase is nucleobase BA, wherein BA is or comprises ring BA or a tautomer thereof, and ring BA is a substituted or unsubstituted 5-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms.

8. Ring BA is 【Chemical 1】 (wherein R' is -C(O)Ph) or hypoxanthine.

9. 7. The oligonucleotide of claim 6, wherein the second domain comprises one or more modified sugars that include a 2'-F modification.

10. The second domain may comprise a 2'-OR modification (where R is a substituted or unsubstituted C １～６ 10. The oligonucleotide of claim 9, comprising one or more modified sugars, including aliphatic sugars.

11. 11. The oligonucleotide of claim 10, wherein the second domain comprises or consists of, from 5' to 3', a first subdomain, a second subdomain, and a third subdomain.

12. 12. The oligonucleotide of claim 11, wherein the first subdomain has a length of 5 to 50 nucleobases.

13. 13. The oligonucleotide of claim 12, wherein the first subdomain comprises 1 to 50 modified sugars that independently have a modification that is not a 2'-F.

14. 12. The oligonucleotide of claim 11, wherein the second subdomain has a length of three nucleobases.

15. 15. The oligonucleotide of claim 14, wherein the second subdomain comprises the nucleoside opposite the target adenosine.

16. 16. The oligonucleotide of claim 15, wherein the second subdomain comprises one or more natural DNA sugars.

17. 17. The oligonucleotide of claim 16, wherein the second subdomain comprises 1 to 10 modified sugars.

18. The sugar of the adjacent nucleoside on the 5' side of the opposite nucleoside (5'-...N １ N ０ N at 3' １ When aligned with the target, the sugar ０ 16. The oligonucleotide of claim 15, wherein the sugar residue (opposite the target adenosine) is a natural DNA sugar, or a 2'-F modified sugar, or [thpyr] or [fana].

19. The sugar of the adjacent nucleoside on the 3' side of the opposite nucleoside (5'-...N ０ N －１ N at 3' －１ When aligned with the target, the sugar ０ 19. The oligonucleotide of claim 18, wherein the sugar residue (opposite the target adenosine) is a natural DNA sugar, or a 2'-F modified sugar, or [thpyr] or [fana].

20. 12. The oligonucleotide of claim 11, wherein the third subdomain has a length of 1 to 50 nucleobases.

21. 21. The oligonucleotide of claim 20 in a salt form.

22. 22. The oligonucleotide of claim 21, wherein the first domain comprises one or more 2'-F blocks and one or more separation blocks, wherein each sugar in each 2'-F block is independently a 2'-F modified sugar and each sugar in each separation block is independently a sugar other than a 2'-F modified sugar.

23. Each sugar in each separation block is independently a 2'-OR modified sugar or a bicyclic sugar (where R is a substituted or unsubstituted C １～６ 23. The oligonucleotide of claim 22, wherein the aryl group is aliphatic.

24. 24. The oligonucleotide of claim 23, wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or all of the phosphorothioate internucleotide linkages are Sp.

25. 1. An oligonucleotide composition comprising a plurality of oligonucleotides, said plurality of oligonucleotides comprising: 1) a common base sequence, and 2) independently, the stereochemistry of the same bond phosphorus in one or more chiral internucleotide linkages ("chiral-controlled internucleotide linkages"); Share; 25. An oligonucleotide composition, wherein each oligonucleotide of the plurality is independently an oligonucleotide according to any one of claims 1 to 24, or an acid, base or salt form thereof.

26. The composition of claim 25, wherein the level of a plurality of oligonucleotides sharing a common base sequence is (DS) nc or at least (DS) nc; 26. The composition of claim 25, wherein DS is 85% to 100% and n is the number of chiral bonded phosphorus.

27. a phosphoramidite, wherein the nucleobase of said phosphoramidite is a nucleobase described herein or a tautomer thereof, and said nucleobase or tautomer thereof is substituted or unsubstituted or protected; or A phosphoramidite, wherein the nucleobase is or comprises ring BA, wherein ring BA has the structure BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-V-a, BA-V-b, or BA-VI, or a tautomer of ring BA, and said nucleobase is substituted, unsubstituted, or protected.

28. 28. The phosphoramidite of claim 27, wherein the nucleobase of the phosphoramidite is c7In, c39z48In, or z2c3In.

29. A method for preparing an oligonucleotide or composition, comprising coupling the 5'-OH of an oligonucleotide or nucleoside with a phosphoramidite or compound according to claim 27 or 28.

30. 2. Evaluating an agent or composition thereof in a cell, tissue, or non-human animal, comprising: evaluating said cell, tissue or non-human animal is or comprises a cell, tissue or organ associated with or of a condition, disorder or disease and / or comprises a nucleotide sequence associated with a condition, disorder or disease; administering to a non-human subject susceptible to or suffering from a condition, disorder, or disease an effective amount of an agent or composition for preventing or treating said condition, disorder, or disease; or a method comprising administering to a non-human subject susceptible to or suffering from a condition, disorder or disease an effective amount of an agent or composition for preventing or treating said condition, disorder or disease, wherein said agent or composition is assessed in a cell, tissue or non-human animal which is or comprises a cell, tissue or organ associated with or of a condition, disorder or disease and / or comprises a nucleotide sequence associated with the condition, disorder or disease; or 1. A method for characterizing an oligonucleotide or composition, comprising: administering the oligonucleotide or composition to a cell or population thereof that contains or expresses an ADAR1 polypeptide or a characteristic portion thereof, or a polynucleotide encoding an ADAR1 polypeptide or a characteristic portion thereof; or administering said oligonucleotide or composition to a non-human animal or population thereof that contains or expresses an ADAR1 polypeptide or a characteristic portion thereof or a polynucleotide encoding an ADAR1 polypeptide or a characteristic portion thereof. A method comprising:

31. A method of modifying a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide according to any one of claims 1 to 24; or A method for deaminating a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide according to any one of claims 1 to 24; or 25. A method of producing, restoring or increasing the level of a specific nucleic acid or a product thereof, comprising contacting a target nucleic acid with the oligonucleotide of any one of claims 1 to 24, wherein the target nucleic acid comprises a target adenosine and the specific nucleic acid differs from the target nucleic acid in that the specific nucleic acid has an I or a G in place of the target adenosine; or A method for reducing the level of a target nucleic acid or a product thereof, comprising contacting the target nucleic acid with an oligonucleotide according to any one of claims 1 to 24, wherein the target nucleic acid comprises a target adenosine; or 25. A method comprising contacting the oligonucleotide of any one of claims 1 to 24 with a sample containing a target nucleic acid and adenosine deaminase, the base sequence of the one or more oligonucleotides in the oligonucleotide composition is substantially complementary to that of the target nucleic acid; and the target nucleic acid comprises a target adenosine; a method wherein the target adenosine is modified; or 1) obtaining a first level of modification of a target adenosine in a target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample comprising said target nucleic acid and an adenosine deaminase, said first oligonucleotide composition comprising a first plurality of oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; 2) obtaining a reference level of target adenosine modification in a target nucleic acid, said level being observed when a reference oligonucleotide composition is contacted with a sample comprising said target nucleic acid and adenosine deaminase, said reference oligonucleotide composition comprising a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; A method comprising: the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, more sugars with 2'-OR modifications (where R is not -H), and / or more chiral internucleotide linkages than the reference plurality of oligonucleotides; and a method wherein the first oligonucleotide composition provides a higher level of modification compared to the oligonucleotides of the reference oligonucleotide composition; or 1. A method comprising: obtaining a first level of modification of a target adenosine in a target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample comprising said target nucleic acid and an adenosine deaminase, said first oligonucleotide composition comprising a first plurality of oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; the first level of modification of the target adenosine is higher than a reference level of modification of the target adenosine, the reference level being observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase, the reference oligonucleotide composition comprising a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to that of the target nucleic acid; a method wherein the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, more sugars with 2'-OR modifications (where R is not -H), and / or more chiral internucleotide linkages than the reference plurality of oligonucleotides; or 1) obtaining a first level of modification of a target adenosine in a target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample comprising said target nucleic acid and an adenosine deaminase, said first oligonucleotide composition comprising a first plurality of oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; 2) obtaining a reference level of target adenosine modification in a target nucleic acid, said level being observed when a reference oligonucleotide composition is contacted with a sample comprising said target nucleic acid and adenosine deaminase, said reference oligonucleotide composition comprising a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; A method comprising: the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, more sugars with 2'-OR modifications (where R is not -H), and / or more chiral internucleotide linkages that are chiral-controlled than the reference plurality of oligonucleotides; and a method wherein the first oligonucleotide composition provides a higher level of modification compared to the oligonucleotides of the reference oligonucleotide composition; or 1. A method comprising: obtaining a first level of modification of a target adenosine in a target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample comprising said target nucleic acid and an adenosine deaminase, said first oligonucleotide composition comprising a first plurality of oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; the first level of modification of the target adenosine is higher than a reference level of modification of the target adenosine, the reference level being observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase, the reference oligonucleotide composition comprising a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to that of the target nucleic acid; a method wherein the first plurality of oligonucleotides comprises more sugars with 2'-F modifications, more sugars with 2'-OR modifications (where R is not -H), and / or more chiral internucleotide linkages that are chiral-controlled than the reference plurality of oligonucleotides; or 1) obtaining a first level of modification of a target adenosine in a target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample comprising said target nucleic acid and an adenosine deaminase, said first oligonucleotide composition comprising a first plurality of oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; 2) obtaining a reference level of target adenosine modification in a target nucleic acid, said level being observed when a reference oligonucleotide composition is contacted with a sample comprising said target nucleic acid and adenosine deaminase, said reference oligonucleotide composition comprising a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; A method comprising: the first plurality of oligonucleotides comprises one or more chiral-controlled chiral internucleotide linkages; and The reference oligonucleotides do not contain chiral internucleotide linkages that are chiral controlled (the reference oligonucleotide composition is a "sterically disordered composition"); and a method wherein the first oligonucleotide composition provides a higher level of modification compared to the oligonucleotides of the reference oligonucleotide composition; or 1. A method comprising: obtaining a first level of modification of a target adenosine in a target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample comprising said target nucleic acid and an adenosine deaminase, said first oligonucleotide composition comprising a first plurality of oligonucleotides sharing the same base sequence substantially complementary to that of said target nucleic acid; the first level of modification of the target adenosine is higher than a reference level of modification of the target adenosine, the reference level being observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and adenosine deaminase, the reference oligonucleotide composition comprising a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to that of the target nucleic acid; the first plurality of oligonucleotides comprises one or more chiral-controlled chiral internucleotide linkages; and The method wherein the reference plurality of oligonucleotides does not contain chiral internucleotide linkages that are chiral-controlled (the reference oligonucleotide composition is a "sterically disordered composition").

32. Editing a premature stop codon in an MECP2 transcript in a system, comprising administering to said system an oligonucleotide according to any one of claims 1 to 24; or Increasing or restoring the level or one or more activities of MECP2 in a system, comprising administering to said system an oligonucleotide according to any one of claims 1 to 24.

1. A method comprising: The system includes an in vitro system or a non-human organism. method.

33. 33. The method of claim 32, wherein the system comprises a premature TGA stop codon in MECP2.

34. 25. A pharmaceutical composition for preventing or treating a condition, disorder or disease in a subject, the pharmaceutical composition being administered to a subject susceptible to or suffering from the same, the pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 24.

35. 35. The pharmaceutical composition of claim 34, wherein the condition, disorder or disease is associated with an MECP2 mutation.

36. 36. The pharmaceutical composition of claim 35, wherein the condition, disorder, or disease is Rett syndrome, MECP2-associated neonatal encephalopathy, PPM-X syndrome, or Angelman syndrome.

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