Oligonucleotide compositions and methods thereof

Abstract

In particular, the present disclosure provides designed oligonucleotides and compositions thereof. In some embodiments, the oligonucleotides and compositions of the present disclosure can provide high levels of adenosine editing. In some embodiments, the oligonucleotides and compositions of the present disclosure are useful for treating various conditions, disorders, or diseases, such as alpha-1 antitrypsin deficiency. In some embodiments, the present disclosure provides methods for treating various conditions, disorders, or diseases that may benefit from adenosine editing.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 248,520, filed September 26, 2021, U.S. Provisional Patent Application No. 63 / 331,756, filed April 15, 2022, and U.S. Provisional Patent Application No. 63 / 397,320, filed August 11, 2022, each of which is incorporated by reference in its entirety, and to PCT application PCT / US2021 / 058495, filed November 8, 2021, and published as WO 2022 / 099159 on May 12, 2022. [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 for treating conditions, disorders, or diseases associated with such target genes. The SERPINA1 gene encodes the serine protease inhibitor alpha-1 antitrypsin (A1AT). A1AT has been reported to protect tissues from certain inflammatory enzymes, including neutrophil elastase. A deficiency in A1AT (alpha-1 antitrypsin deficiency, A1AD) can cause excessive degradation of elastin in the lungs by neutrophil elastase. This can lead to reduced elasticity in the lungs and subsequent respiratory complications, including emphysema and chronic obstructive pulmonary disease (COPD). Mutant A1AT can also accumulate in the liver, causing cirrhosis and liver failure. Summary of the Invention [Means for solving the problem]

[0003] overview In particular, the present disclosure recognizes the need for new treatments and therapies that correct pathogenic mutations in SERPINA1, e.g., 1024 G>A (E342K in A1AT), to treat alpha 1 antitrypsin deficiency (A1AD), which can lead to liver failure and / or emphysema. In some embodiments, the present disclosure provides techniques, e.g., oligonucleotides, compounds, compositions, methods, etc., for preventing or treating conditions, disorders, or diseases associated with 1024 G>A (E342K in A1AT) in SERPINA1.

[0004] 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 present disclosure provides compounds and methods for selectively and efficiently editing the SERPINA1 gene, as well as compounds and methods for correcting pathogenic mutations in the gene to treat alpha-1 antitrypsin deficiency (A1AD). Also provided are methods useful for preventing or ameliorating at least one symptom of a condition, disorder, or disease associated with a SERPINA1 mutation. 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, as demonstrated herein, the provided techniques can significantly improve the efficiency of nucleic acid editing (e.g., modification of 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 an A residue, e.g., converting A to I). In some embodiments, the present disclosure provides techniques for editing in transcripts (e.g., mRNA) (e.g., modifying an A residue, e.g., converting A to I). In particular, the provided techniques offer the benefits of using 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 use of endogenous proteins may avoid some challenges and / or offer various advantages compared to these techniques that require the 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 results in a desired activity), nucleic acids encoding proteins, viruses, etc.).In some embodiments, the present invention provides oligonucleotides, compounds, compositions, and methods for editing SERPINA1 transcripts and / or treating or preventing conditions, disorders, or diseases associated with SERPINA1 mutations, e.g., 1024 G>A, in a subject. In some embodiments, the oligonucleotides, compounds, or compositions are capable of effecting adenosine deaminases acting on RNA (ADAR)-mediated adenosine to inosine modification in the transcript. In some embodiments, the deamination corrects the pathogenic mutation 1024 G>A in SERPINA1, reverts the E342K mutation in the A1AT polypeptide to wild-type, and / or reverses or delays the 1024 G>A-associated condition, disorder, or disease and associated symptoms experienced by the patient.

[0005] In particular, in some embodiments, the oligonucleotides of the provided technology include useful sugar modifications and / or patterns thereof (e.g., the presence and / or absence of certain modifications), nucleobase modifications and / or patterns thereof (e.g., the presence and / or absence of certain 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.), which, when combined with one or more other structural elements (e.g., additional chemical moieties) described herein, may provide enhanced activity and / or various desirable properties (e.g., enhanced efficiency of nucleic acid editing, enhanced selectivity, enhanced stability, enhanced cellular uptake, reduced immune stimulation, reduced toxicity, improved distribution, improved affinity, etc.). In some embodiments, the provided oligonucleotides provide enhanced stability, for example, compared to oligonucleotides having a high percentage of natural RNA sugars and / or 2'-F modified sugars utilized for adenosine editing. In some embodiments, the provided oligonucleotides provide enhanced activity (e.g., adenosine editing activity). In some embodiments, provided oligonucleotides provide high selectivity, e.g., 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).

[0006] 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 2, 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 oligonucleotides in a composition, or all oligonucleotides sharing a common base sequence, share the pattern of chiral centers in the backbones of multiple oligonucleotides. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or all oligonucleotides sharing a common base sequence, are multiple oligonucleotides. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or all oligonucleotides sharing a common base sequence, are multiple oligonucleotides.

[0007] 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 of the same configuration as the oligonucleotide 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.).

[0008] 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, compounds, 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 multiple oligonucleotides sharing the same base sequence with the same pattern of stereochemistry at the chiral phosphorus (e.g., each chiral 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 multiple oligonucleotides share the same pattern of stereochemistry at the chiral phosphorus, or are part of the multiple oligonucleotides. In some embodiments, provided compositions include multiple oligonucleotides sharing the same base sequence with the same pattern of stereochemistry at the chiral 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 multiple oligonucleotides share the same pattern of stereochemistry at the chiral phosphorus, or are part of the multiple oligonucleotides. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 90%. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 95%. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 97%. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 98%. In some embodiments, the diastereomeric purity is about or at least about (DS)nc where DS is about 90-100%, and nc is the number of chiral bonded phosphorus atoms. In some embodiments, DS is about 90% or greater. In some embodiments, DS is about 95% or greater. In some embodiments, DS is about 96% or greater. In some embodiments, DS is about 97% or greater. In some embodiments, DS is about 98% or greater. In some embodiments, DS is about 99% or greater.

[0009] In some embodiments, the oligonucleotide is WV-46312, WV-47606, WV-47608, WV-49085, WV-49086, WV-49087, WV-49088, WV-49089, WV-49090, or WV-49092. In some embodiments, the oligonucleotide is WV-46312. In some embodiments, the oligonucleotide is WV-47606. In some embodiments, the oligonucleotide is WV-47608. In some embodiments, the oligonucleotide is WV-49085. In some embodiments, the oligonucleotide is WV-49086. In some embodiments, the oligonucleotide is WV-49087. In some embodiments, the oligonucleotide is WV-49088. In some embodiments, the oligonucleotide is WV-49089. In some embodiments, the oligonucleotide is WV-49090. In some embodiments, the oligonucleotide is WV-49092.

[0010] In some embodiments, the oligomeric compound comprises an oligonucleotide, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide has the formula: Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn 001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn0 01RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU or Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU.

[0011] In some embodiments, the oligomeric compound comprises an oligonucleotide, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide has the formula: mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU; mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU or mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU is of this kind.

[0012] 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 -O(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)(OH)-O-)), a base form, a base form, a salt form (e.g., a sodium salt (-O(P(O)(OH)-O-)), ... - Na + )-O-), and phosphorothioate internucleotide linkages exist in salt form (e.g., sodium salt (-O(P(O)(S - Na + )-O-), etc.) As will be appreciated by those skilled in the art, oligonucleotides can exist in various salt forms, including pharmaceutically acceptable salts, and in solutions (e.g., various aqueous buffer systems), where the cation can 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.

[0013] As will be appreciated by those skilled in the art, oligonucleotides can be provided, administered, or delivered in a variety of forms, including various salt forms, such as pharmaceutically acceptable salt forms. In some embodiments, the oligonucleotide is provided, administered, or delivered in a salt form. In some embodiments, the oligonucleotide is provided, administered, or delivered in a pharmaceutically acceptable salt form. In some embodiments, the oligonucleotide is provided, administered, or delivered in multiple forms. In some embodiments, the oligonucleotide is provided, administered, or delivered in multiple salt forms. In some embodiments, the oligonucleotide is provided, administered, or delivered in multiple pharmaceutically acceptable salt forms. In some embodiments, the multiple combine to form an effective amount of oligonucleotide.

[0014] In some embodiments, the provided oligonucleotide comprises 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 some embodiments, the additional moiety is or comprises GalNAc. 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. In some embodiments, to deliver the oligonucleotide, a conjugated oligonucleotide comprising such an oligonucleotide with an additional chemical moiety is administered. In some embodiments, the oligonucleotide is delivered by administering a conjugate of such an oligonucleotide with one or more additional chemical moieties, optionally via one or more linkers.

[0015] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0016] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*Sin001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0017] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0018] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*Sin001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0019] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0020] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*Sin001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0021] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*Sin001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0022] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0023] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0024] In some embodiments, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof. In some embodiments, the present disclosure provides oligonucleotides that are conjugates of such oligonucleotides with one or more additional chemical moieties, optionally via one or more linkers. In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof.

[0025] The provided techniques can be utilized for a variety of purposes. For example, one skilled in the art will appreciate that the provided techniques are useful for many purposes, including modifying adenosines, e.g., correcting G to A mutations, regulating the levels of certain nucleic acids and / or products encoded thereby, etc.

[0026] In some embodiments, the present disclosure provides techniques for preventing or treating a condition, disorder, or disease that is susceptible to an adenosine modification (e.g., an A to I or G conversion). As will be understood by one of skill in the art, an I can perform one or more functions of a 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 a condition, disorder, or disease associated with 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 edit the mutation. In some embodiments, the present disclosure provides techniques for preventing or treating a condition, disorder, or disease 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, the A form 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 G form. In some embodiments, the A form 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 G form. In some embodiments, the product encoded by the A form is structurally different (e.g., a longer, in some embodiments, full-length protein) than that encoded by its corresponding G form. In some embodiments, the A form provides a structurally identical product (e.g., protein) compared to its corresponding G form. In some embodiments, the mutation is 1024 G>A in SERPINA1.In some embodiments, the condition, disorder, or disease is associated with 1024 G>A in SERPINA1.

[0027] This application incorporates by reference U.S. Provisional Patent Application Nos. 63 / 111,079, filed November 8, 2020, 63 / 175,036, filed April 14, 2021, 63 / 188,415, filed May 13, 2021, 63 / 196,178, filed June 2, 2021, 63 / 248,520, filed September 26, 2021, 63 / 331,756, filed April 15, 2022, and 63 / 397,320, filed August 11, 2022, as well as International Publication Nos. WO 2021 / 071858 and WO 2022 / 099159. [Brief explanation of the drawings]

[0028] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] The provided technology can provide permanent editing in vivo. Mice transgenic for hADAR and the SERPINA1-Z allele were treated with an oligonucleotide composition targeting the SERPINA1-Z allele at a dose of 10 mg / kg by subcutaneous administration on days 0, 2, and 4. Mouse serum was collected by weekly bleeding at the indicated days after treatment. (a) Human AAT protein levels were measured by ELISA. Data are presented as mean ± standard error. Statistics: matched two-way ANOVA; ns: not significant, **: P<0.01, ***: 0.001. (b) The relative proportions of wild-type (WT / M-AAT) and mutant (Z-AAT / mutant) AAT proteins were determined using mass spectrometry and ELISA. [Figure 2]The provided technology can provide editing. Primary mouse hepatocytes transgenic for hADAR p110 and the SERPINA1-Z allele were treated with an oligonucleotide composition containing the indicated GAlNAc-conjugated oligonucleotide targeting the SERPINA1-Z allele at the indicated concentrations. RNA was isolated 48 hours after treatment, and RNA editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 3] The provided technology can provide editing in vivo. Mice transgenic for hADAR and the SERPINA1-Z allele were treated with an oligonucleotide composition targeting the SERPINA1-Z allele at a dose of 5 mg / kg by subcutaneous administration on days 0, 2, and 4. Liver biopsies of the mice were taken on day 7 after treatment. RNA editing was measured by Sanger sequencing in male (left bar) and female (right bar) mice (n=3 animals per sex). [Figure 4] The provided technology can provide editing. Primary mouse hepatocytes transgenic for hADAR p110 and the SERPINA1-Z allele were treated with oligonucleotide compositions targeting the SERPINA-Z allele at the indicated concentrations. RNA was isolated 48 hours after treatment, and RNA editing was measured by Sanger sequencing (n=3 biological replicates). [Figure 5] The provided technology can provide functional edited polypeptides in vivo. Mice transgenic for hADAR and the SERPINA1-Z allele were treated with an oligonucleotide composition targeting the SERPINA1-Z allele at a dose of 10 mg / kg by subcutaneous administration on days 0, 2, and 4. Mouse serum was collected by weekly bleeding on the indicated days. Human AAT protein levels were quantified by ELISA and mass spectrometry to assess the relative proportions of wild-type AAT protein (PiM / WT, left bar) and mutant AAT protein (PiZ / mutant, right bar). [Figure 6]The provided technology can provide editing. Oligonucleotide compositions containing various modifications, such as base modifications (e.g., b008U, etc.), linkage modifications (e.g., PS (phosphorothioate), PN (e.g., phosphorylguanidine linkages such as n001), etc.), and sugar modifications (e.g., 2'-F, 2'-OMe, 2'-MOE, etc.), were prepared and evaluated. Editing of the target adenosine at the SERPINA1-Z allele was confirmed in primary mouse hepatocytes transgenic for human ADAR p110 and the SERPINA1-Z allele (N=2 biological replicates). [Figure 7] The provided technology can provide editing. Oligonucleotide compositions containing various modifications, such as base modifications (e.g., b008U, etc.), linkage modifications (e.g., PS (phosphorothioate), PN (e.g., phosphorylguanidine linkages such as n001), etc.), and sugar modifications (e.g., 2'-F, 2'-OMe, 2'-MOE, etc.), were prepared and evaluated. Editing of the target adenosine at the SERPINA1-Z allele was confirmed in primary mouse hepatocytes transgenic for human ADAR p110 and the SERPINA1-Z allele (N=2 biological replicates). [Figure 8] The provided technology can provide editing in vivo. We confirmed in vivo editing of a target adenosine at the SERPINA1-Z allele in mice transgenic for human ADAR and the SERPINA1-Z allele. Serum levels of AAT were also increased in treated mice. [Figure 9] The provided technology can provide editing in vivo. Oligonucleotides containing various nucleobases (e.g., b008U, hypoxanthine, etc.), linkages (e.g., PO, PS, PN (e.g., phosphorylguanidine linkages such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, 2'-MOE, etc.), and their patterns were prepared. Editing of the targeted adenosine and an increase in serum AAT were confirmed (N=4 animals per group). Top: SERPINA1 editing at day 10. Bottom: serum AAT fold change. [Figure 10]The provided technology can provide targeted transcript editing. Editing of the SERPINA1-Z allele was confirmed (N=2 biological replicates). Human patient iPSC-derived hepatocytes with the ZZ genotype were plated on day 0 and treated on day 2 with various concentrations of the indicated oligonucleotides (e.g., WV-46312, WV-49090, WV-49092) (5, 1.25, 0.31, and 0.08 μM for each oligonucleotide, from left to right). Medium was changed every two days (e.g., days 4, 6, and 8). RNA was harvested on day 10, and RNA editing was quantified by Sanger sequencing. Error bars represent the standard error of the mean (SEM). [Figure 11] The provided technology can provide targeted transcript editing. Editing of the SERPINA1-Z allele was confirmed (N=2 biological replicates). Human patient iPSC-derived hepatocytes with the ZZ genotype were plated on day 0 and treated on day 2 with various concentrations (e.g., 5, 1.25, 0.31, and 0.08 μM) of the indicated oligonucleotides (WV-46312 on the left, WV-44515 on the right). Medium was changed every two days (e.g., days 4, 6, and 8), and the indicated oligonucleotides were re-administered every two days (e.g., days 4, 6, and 8). RNA was harvested on day 10, and RNA editing was quantified by Sanger sequencing. Error bars represent the standard error of the mean (SEM). [Figure 12]The provided technology can provide editing in vivo. Editing of transcripts from the SERPINA1 PiZ allele was confirmed. Seven-week-old NSG-PiZ mice (JAX strain #028842; N=5 per treatment group) were subcutaneously administered 10 mg / kg of the indicated oligonucleotide composition (e.g., WV-49090) per dose. One group of mice received a loading dose during week 1 (days 0, 2, and 4), while the other group received a single dose (no loading dose) during week 1 (day 0). Thereafter, groups received additional doses every two weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). A control group of mice received PBS. Liver biopsies were taken from the mice 13 weeks after treatment. RNA was collected from the liver biopsies, and RNA editing was quantified by Sanger sequencing. Error bars represent the standard error of the mean (SEM). One-way ANOVA with adjustment for multiple comparisons (Tukey) was used to test for differences in % edit between loading and non-loading doses (ns: non-significant). [Figure 13] The provided technology can increase SERPINA1 mRNA levels in vivo. Seven-week-old NSG-PiZ mice (JAX strain #028842; N=5 per treatment group) were subcutaneously administered 10 mg / kg of the designated oligonucleotide composition (e.g., WV-49090) per dose. One group of mice received a loading dose during week 1 (days 0, 2, and 4), while the other group received a single dose (no loading dose) during week 1 (day 0). Thereafter, the groups received additional doses every two weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). Liver biopsies of the mice were taken 13 weeks after treatment. A control group of mice received PBS. RNA was collected from the liver biopsies, and relative SERPINA1 mRNA levels (SERPINA1 / HPRT) were quantified using qPCR. Baseline measurements of relative SERPINA1 mRNA levels were determined from mouse liver biopsies taken before dosing (week 0). Error bars represent the standard error of the mean (SEM). One-way ANOVA with adjustment for repeated measures and multiple comparisons (Dunnett's) was used to test for differences in relative SERPINA1 mRNA levels (****: P value < 0.001; ns: non-significant). [Figure 14]The provided technology can reduce mutant Z-AAT protein levels and increase wild-type (M) AAT protein levels in serum. Seven-week-old NSG-PiZ mice (JAX strain #028842; N=5 per treatment group) were subcutaneously administered 10 mg / kg of the designated oligonucleotide composition (e.g., WV-49090) per dose. One group of mice received a loading dose during week 1 (days 0, 2, and 4), while the other group received a single dose (no loading dose) during week 1 (day 0). Thereafter, the groups received additional doses every two weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). The control group of mice received PBS. Serum was collected from the mice 13 weeks after treatment. The relative abundance of Z (mutant) versus M (wild-type) AAT isoforms was determined by liquid chromatography-mass spectrometry (LC-MS). Error bars represent the standard error of the mean (SEM). [Figure 15] Editing with various provided oligonucleotide compositions resulted in functional wild-type AAT protein. Seven-week-old NSG-PiZ mice (JAX strain #028842; N=5 per treatment group) were subcutaneously administered 10 mg / kg of the indicated oligonucleotide composition per dose. One group of mice received a loading dose during week 1 (days 0, 2, and 4), while the other group received a single dose during week 1 (day 0). Thereafter, the groups received additional doses every two weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). A control group of mice received PBS. Serum was collected from the mice before administration and 13 weeks after treatment. Relative elastase inhibitory activity in the serum was determined in vitro using a commercially available kit. Treatment groups, from left to right for each time point, were: PBS control, WV-49090 (with loading doses on days 0, 2, and 4), and WV-49090 (without loading dose). Error bars represent the standard error of the mean (SEM). Two-way ANOVA with adjustment for multiple comparisons (Bonferroni) was used to test for differences in serum elastase inhibitory activity for treatment groups receiving the indicated oligonucleotide compositions versus the PBS control. (****: P value < 0.001; ns: non-significant). DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of Certain Embodiments The techniques of the present disclosure may be more readily understood by reference to the following detailed description of certain 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," "comprise," "including" (whether or not used in conjunction with "limited to"), and "include" (whether or not used in conjunction with "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 may 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 may 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, for example, in 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 may 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 forms (e.g., all cations, if present, may be H) may be used. + 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 chains 20 ), 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, where 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, the characteristic moiety 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 at 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 the case of non-chirality-controlled internucleotide linkages). In some embodiments, a chiral controlled oligonucleotide composition comprises a plurality of 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 plurality of 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 the plurality of 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 less) 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%) are 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 ... 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, or of all oligonucleotides in a composition 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 pattern of base modifications, a common pattern of sugar modifications, a common pattern of internucleotide linkage types, and / or a common pattern of internucleotide linkage modifications. 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 approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 9 0%, 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%, 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% of the oligonucleotides (or nucleic acids) 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 between about 1% and 100% (e.g., between about 5% and 100%, 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, 90% and 100%, 95% and 100%, or between about 1% and 100%, or between about 2% and 20%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, 90% and 100%, 95 ... %, 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, each chiral internucleotide linkage is a chiral-controlled internucleotide linkage, and the composition is a completely chiral-controlled oligonucleotide composition. In some embodiments, the multiple oligonucleotides (or nucleic acids) are structurally identical. In some embodiments, the chiral-controlled internucleotide linkage has 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 linkage has a diastereomeric purity of at least 95%. In some embodiments, the chiral-controlled internucleotide linkage has a diastereomeric purity of at least 96%. In some embodiments, the chiral-controlled internucleotide linkage has a diastereomeric purity of at least 97%. In some embodiments, the chiral controlled internucleotide linkage has a diastereomeric purity of at least 98%. In some embodiments, the chiral controlled internucleotide linkage has a diastereomeric purity of at least 99%. In some embodiments, the percentage 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 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 a plurality of oligonucleotides in a composition is expressed as the product of the diastereopurities of each chiral linkage phosphorus in the oligonucleotide. In some embodiments, the level of a plurality of 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 chiral non-controlled internucleotide linkages are those typically observed in stereoirregular oligonucleotide compositions (e.g., those resulting from conventional oligonucleotide synthesis, e.g., phosphoramidite methods, as will be appreciated by those skilled in the art). The diastereomeric purities are less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50%. In some embodiments, the multiple oligonucleotides (or nucleic acids) are of the same type. In some embodiments, the chiral controlled oligonucleotide composition contains non-random or controlled levels of individual oligonucleotide or nucleic acid types. For example, in some embodiments, the chiral controlled oligonucleotide composition contains only one oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide composition contains two or more oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide composition contains multiple oligonucleotide types. In some embodiments, the 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 alicyclic rings fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, alicyclic groups are bicyclic. In some embodiments, alicyclic groups are tricyclic. In some embodiments, alicyclic groups are polycyclic. In some embodiments, "alicyclic" refers to a C3-C6 monocyclic hydrocarbon having a single point of attachment to the rest of the molecule, or a C8-C6 monocyclic hydrocarbon having 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-C, with a single point of attachment to the rest of the molecule, that are fully saturated or contain one or more units of unsaturation but are not aromatic 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 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, the 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 Meyers and Miller algorithm (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 may exist as a salt form as will be understood by those of skill in the art), 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 the internucleotide linkage may 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 that is 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, the modified sugar is a substituted ribose or deoxyribose, as described in this disclosure. 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, analogs of either RNA or DNA, including modified nucleotides and / or modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. These terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from nucleobases and / or modified nucleobase N-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" 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, a nucleoside is a modified nucleoside, e.g., 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, the oligonucleotide is 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, 25, 26, 27, 28, 29, or 30 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 linked 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 linking phosphorus and / or a specific modification at the linking phosphorus, and / or a specific base and / or a specific sugar. In some embodiments, the oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the linking phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of modifications at the linking phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the above structural features. 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 multiple 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 if more than one position in any given structure can 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 allow for their production, detection, and, in certain embodiments, their collection, purification, and use for one or more of the purposes disclosed herein. Certain 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) 0~4N(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 -(C1~4 linear 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- (5-14 membered heteroaryl ring), a 5-20 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 5-20 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 · (In the formula, 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 is selected from 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 and unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from aliphatic, nitrogen, oxygen, and sulfur. Preferred divalent substituents attached to adjacent substitutable carbon atoms of an "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, partially unsaturated, 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 a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently 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, partially unsaturated, 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 only with one or more halogens), and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, 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 compatible with the following: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeting 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, that 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 connection with medicines, 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 using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates, where appropriate. 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 cations. 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 any, 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 will understand that the present disclosure provides techniques that allow for the selection of specific chemical and / or stereochemical features to be incorporated into oligonucleotide compositions, and further allow for 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 was accidentally generated 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 (carbamante), 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), and 2-trimethylsilylethyl carbamate (Teoc). , 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamido)ethyl, 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, carbamate Alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitribenzyl carbamate (nitobenzyl), p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylsulfinylbenzyl carbamate -methylthioethyl, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-Dimethoxy-6-nitrobenzyl, 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 carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenyl) carbamate p-(phenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- Butylphenyl, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, 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, 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-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamine (Fcm), N -2-Picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-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-diphenylborinic 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), benzene Sulfonamides, 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, and 4-methoxytetrahydrothiopyranyl. S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-furanyl Oroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-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, 4-(methyl (ethylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, 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, 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 borate, and phenyl borate.

[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-butyldiphenyl ... Triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), and 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4"-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (Pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.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 that are 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 that are 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 a 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 demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population is a population of subjects suffering from and / or susceptible to a disease, disorder, or condition. In some embodiments, the appropriate population is a population of model organisms. In some embodiments, the appropriate 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 in 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, etc. 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 will be understood by those of skill in the art, 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 Certain 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, as demonstrated herein, provided oligonucleotides and compositions thereof are particularly potent for editing a target adenosine in a target nucleic acid to correct, in some embodiments, a G to A mutation by converting A to I. In some embodiments, the provided techniques can be used to correct the 1024 G>A mutation in SERPINA1.

[0099] In some embodiments, provided techniques are chiral controlled. In particular, the present disclosure provides techniques for preparing chiral controlled (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%. , 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.

[0100] In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides sharing a common base sequence, wherein each internucleotide linkage containing a chiral phosphorus in the oligonucleotides is independently a chiral-controlled internucleotide linkage. In some embodiments, an oligonucleotide composition comprises oligonucleotides of the same configuration, wherein one or more internucleotide linkages are chiral-controlled. In some embodiments, an oligonucleotide composition comprises oligonucleotides of the same configuration, wherein each internucleotide linkage containing a chiral 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 a plurality of oligonucleotides. In some embodiments, in the provided oligonucleotide or compound compositions, each chiral phosphorus in the oligonucleotides or compounds is chiral-controlled.

[0101] In some embodiments, the present disclosure provides techniques for preparing, evaluating, and / or utilizing the provided oligonucleotides and compositions thereof.

[0102] 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.

[0103] As used in this disclosure, in some embodiments, "at least one" means one or more.

[0104] For example, variables (e.g., R, R L, L, etc. 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.)

[0105] Oligonucleotides In particular, the present disclosure provides oligonucleotides of various designs, which may contain 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 disclosed oligonucleotides are single-stranded oligonucleotides capable of site-specific editing of adenosines (A to I conversion) in a target RNA sequence. In some embodiments, the provided technology can edit 1024 G>A in SERPINA1. In some embodiments, the disclosed oligonucleotides contain lower levels of 2'-F modified sugars and no natural RNA sugars. In some embodiments, the provided technology provides high levels of activity (e.g., editing 1024 G>A in SERPINA1) and stability.

[0106] In some embodiments, provided oligonucleotides are sufficiently short to facilitate delivery and reduce manufacturing complexity and / or cost, while maintaining desired properties and activity (e.g., adenosine editing).

[0107] In some embodiments, the provided oligonucleotides comprise an additional chemical moiety. In some embodiments, the provided oligonucleotides comprise one or more carbohydrate moieties. In some embodiments, the provided oligonucleotides comprise one or more GalNAc moieties. In some embodiments, the provided oligonucleotides comprise one or more targeting moieties.

[0108] In some embodiments, provided oligonucleotides can induce correction of a G to A mutation in a target sequence or its product. In some embodiments, 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.

[0109] 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).

[0110] In some embodiments, provided oligonucleotides contain increased 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 can be used in the compositions and methods described herein.

[0111] 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.

[0112] In some embodiments, the oligonucleotide is provided, administered, or delivered in a salt form. In some embodiments, the oligonucleotide is provided, administered, or delivered as a salt containing a negatively charged internucleotide linkage (e.g., a phosphorothioate internucleotide linkage, a natural phosphate linkage, etc.) present in salt form. In some embodiments, the oligonucleotide is provided, administered, or delivered as a pharmaceutically acceptable salt. In some embodiments, the oligonucleotide is provided, administered, or delivered as a metal salt. In some embodiments, the oligonucleotide is provided, administered, or delivered as a sodium salt. In some embodiments, the oligonucleotide is provided, administered, or delivered as an ammonium salt. In some embodiments, the oligonucleotide is provided, administered, or delivered as a metal salt (e.g., a sodium salt), and each negatively charged internucleotide linkage is independently present in salt form (e.g., -OP(O)(SNa)-O- for a phosphorothioate internucleotide linkage, -OP(O)(ONa)-O- for a natural phosphate linkage, etc. for a sodium salt).

[0113] In some embodiments, the oligonucleotide is chiral controlled and comprises one or more chiral controlled internucleotide linkages. In some embodiments, each chiral phosphorus is independently chiral controlled. In some embodiments, provided oligonucleotides or compositions thereof are substantially pure from other stereoisomers with respect to the chiral phosphorus. In some embodiments, provided oligonucleotides or compositions thereof are substantially pure from other stereoisomers. In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions.

[0114] As described herein, oligonucleotides of the present disclosure can be provided with high purity (e.g., about 50% to 100%). In some embodiments, oligonucleotides of the present disclosure are of high stereochemical purity (e.g., about 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%.

[0115] In some embodiments, the oligonucleotide is selected from the group consisting of: mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn0 01RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*S fC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*Sm Gn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STe ofUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, mCn001RmC*SmC*SfA*SfG*Sm5CeoA eofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, mCn001RmC* SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU,mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU.

[0116] In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1 of WO 2022 / 099159, wherein the oligonucleotide targets SERPINA1 and does not contain an additional chemical moiety. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1 of WO 2022 / 099159, wherein the oligonucleotide targets SERPINA1 and does not contain L001. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1 of WO 2022 / 099159, wherein the oligonucleotide does not contain any Mod. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1 of WO 2022 / 099159, wherein the oligonucleotide does not contain Mod001. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1 of WO 2022 / 099159, wherein the oligonucleotide does not contain Mod012. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1 of WO 2022 / 099159, wherein the oligonucleotide does not contain Mod001 or Mod012. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1D of WO 2022 / 099159. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1E of WO 2022 / 099159. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from Table 1F of WO 2022 / 099159. In some embodiments, the oligonucleotide has a structure or a salt thereof selected from WV-42934 to WV-44247 in Table 1F of WO 2022 / 099159. In some embodiments, the oligonucleotide has a structure selected from WV-44248 to WV-44277 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-44349 to WV-44362 in Table 1F of WO 2022 / 099159, or a salt thereof.In some embodiments, the oligonucleotide has a structure selected from WV-44363 to WV-44390 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-44482 to WV-44515 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-46406 to WV-47042 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-47339 to WV-47483 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-47495 to WV-47496 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-47610 to WV-47631 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-48455 to WV-48459 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-49094 to WV-49096 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from Table 1O of WO 2022 / 099159, or a salt thereof.

[0117] In some embodiments, the oligonucleotide comprises an additional chemical moiety as described herein. In some embodiments, the additional chemical moiety facilitates delivery. In some embodiments, the additional chemical moiety comprises a targeting moiety. In some embodiments, the additional chemical moiety comprises one or more carbohydrate moieties. In some embodiments, the additional chemical moiety is a carbohydrate moiety. In some embodiments, the additional chemical moiety comprises one or more lipid moieties. In some embodiments, the additional chemical moiety is a lipid moiety. In some embodiments, the additional chemical moiety comprises one or more protein ligand moieties. In some embodiments, the additional chemical moiety targets the liver. In some embodiments, the additional chemical moiety comprises one or more ligands of one or more receptors expressed in the liver. In some embodiments, the additional chemical moiety is a ligand for one or more receptors expressed in the liver. In some embodiments, the additional chemical moiety comprises one or more ligands for one or more asialoglycoprotein receptors. In some embodiments, the additional chemical moiety is a ligand for an asialoglycoprotein receptor. In some embodiments, the additional chemical moiety comprises multiple moieties, each of which is independently a ligand for an asialoglycoprotein receptor. In some embodiments, the ligand is GalNAc or a derivative thereof. In some embodiments, the ligand is GalNAc. In some embodiments, the ligand is [ka] or a derivative thereof. In some embodiments, the ligand is [ka] In some embodiments, the additional chemical moiety comprises GalNAc. In some embodiments, the additional chemical moiety is GalNAc. In some embodiments, the additional chemical moiety comprises multiple GalNAc. In some embodiments, the additional chemical moiety comprises three GalNAc. In some embodiments, the additional chemical moiety is [ka] In some embodiments, the additional chemical moiety comprises: [ka] In some embodiments, the additional chemical moiety comprises: [ka] In some embodiments, the additional chemical moiety is [ka] In some embodiments, the additional chemical moiety comprises three [ka] In some embodiments, the additional chemical moiety comprises: [ka] In some embodiments, the additional chemical moiety is or comprises L001. In some embodiments, the additional chemical moiety is conjugated directly to the oligonucleotide strand. In some embodiments, the additional chemical moiety is conjugated to the oligonucleotide strand via a linker. In some embodiments, two or more additional chemical moieties are conjugated to the oligonucleotide strand via a linker. In some embodiments, the linker is or comprises L001. In some embodiments, the linker is a polyvalent linker. In some embodiments, the polyvalent linker conjugates two or more additional chemical moieties. For example, in some embodiments, a trivalent linker can link three additional chemical moieties, e.g., three GalNAc, to a single point on the oligonucleotide strand. The additional chemical moieties can be independently linked to various positions on the oligonucleotide strand, optionally via linkers. In some embodiments, the additional chemical moiety is conjugated to the 5' end of the oligonucleotide strand. In some embodiments, the additional chemical moiety is conjugated to the 3' end of the oligonucleotide strand. In some embodiments, the additional chemical moiety is conjugated to a portion of the oligonucleotide strand. In some embodiments, the additional chemical moiety is conjugated to a sugar. In some embodiments, the additional chemical moiety is conjugated to a nucleobase. In some embodiments, the additional chemical moiety is conjugated to an internucleotide bond. In some embodiments, the linker is linked to the 5'-carbon at the 5' end of the oligonucleotide chain. In some embodiments, the linker is linked to the 3'-carbon at the 3' end of the oligonucleotide chain. In some embodiments, the linker, e.g., L001, is linked to the 5'-carbon at the 5' end of the oligonucleotide chain via a phosphate group, e.g., via a phosphorothioate group.

[0118] In some embodiments, the oligonucleotide is Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn0 01RmCfA*SfGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, Mod001L001mCn001RmC*Sm C*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SfGn001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmA n001RmU, Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmGn001RfUm5Ceo*SfC*SfC*SfUn001RTeoT eofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*STeofUn001RmCfA*SmG n001RfUmC*SfC*SfC*SfUn001RTeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5Ce oAeofG*Sm5CeoTeo*SmUn001Rm5CeofA*SfGn001RmUm5Ceom5Ceo*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU,Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA*SfG n001RmUm5CeomC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001R mU, Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SmCTeo*SmUn001RmCfA* SfGn001RmUmCmC*SfC*SfU*STeoTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001Rm U, Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmA*SfG*SfCmU*SfUn001RmCfA*SfGn 001RfUmC*SfC*SfC*SfU*SmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or Mod001L001mCn001RmC*SmC*SfA*SfG*SmCmAfG*SfC*SmUfUn001RmCfA*SmGn001RfUmC*SfC*SfC*SfUn001RmUmUfC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU. In some embodiments, the oligonucleotide has a structure selected from Table 1 of WO 2022 / 099159, incorporated herein by reference, or a salt thereof, wherein the oligonucleotide targets SERPINA1 and comprises an additional chemical moiety, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from Table 1 of WO 2022 / 099159, incorporated herein by reference, or a salt thereof, wherein the oligonucleotide targets SERPINA1 and comprises L001. In some embodiments, the oligonucleotide has a structure selected from Table 1 of WO 2022 / 099159 or a salt thereof, wherein the oligonucleotide targets SERPINA1 and comprises Mod001. In some embodiments, the oligonucleotide has a structure selected from Table 1D of WO 2022 / 099159 or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from Table 1E of WO 2022 / 099159 or a salt thereof. In some embodiments, the oligonucleotide isIn some embodiments, the oligonucleotide has a structure selected from WV-46312 to WV-46323 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-47597 to WV-47609 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-47641 to WV-48454 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-47643 to WV-47648 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-48453 to WV-48454 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from WV-49085 to WV-49093 in Table 1F of WO 2022 / 099159, or a salt thereof. In some embodiments, the oligonucleotide has a structure selected from Table 1O of WO 2022 / 099159, or a salt thereof.

[0119] As described herein, in some embodiments, the additional chemical moiety, e.g., Mod001, can facilitate delivery of the oligonucleotide. In some embodiments, after delivery, the additional chemical moiety is cleaved. In some embodiments, the additional moiety is released after delivery or administration, resulting in the oligonucleotide to be delivered. In some embodiments, the linker (e.g., L001) for conjugating the additional chemical moiety is cleaved. In some embodiments, the oligonucleotide has the structure of an oligonucleotide chain of an oligonucleotide comprising the additional chemical moiety and optionally a linker. In some embodiments, the oligonucleotide has the structure of an oligonucleotide released after the additional chemical moiety is cleaved from the oligonucleotide comprising the additional chemical moiety. In some embodiments, the linker, e.g., L001, is also cleaved from the oligonucleotide. In some embodiments, the oligonucleotide is formed by cleaving the additional chemical moiety from the oligonucleotide chain of an oligonucleotide comprising the additional chemical moiety. In some embodiments, the additional chemical moiety is cleaved after the oligonucleotide is delivered to a cell. In some embodiments, the additional chemical moiety is cleaved after the oligonucleotide is administered to a subject.

[0120] In some embodiments, provided are techniques for delivering oligonucleotides, comprising administering a conjugate of an oligonucleotide, wherein the conjugate comprises the oligonucleotide to be delivered and an additional chemical moiety as described herein. In some embodiments, the oligonucleotide is conjugated to one or more additional chemical moieties, independently and optionally via one or more linkers. In some embodiments, the oligonucleotide is conjugated to the additional chemical moiety via a linker.

[0121] Oligonucleotides, compounds, moieties, e.g., additional chemical moieties, linker moieties, etc., may contain groups that may be or include R' or R as described herein. In some embodiments, R' is R. In some embodiments, R' is -C(O)R. In some embodiments, R' is -C(O)OR. In some embodiments, R' is -C(O)N(R). In some embodiments, R' is -S0R.

[0122] In some embodiments, R' in the various structures is a protecting group (e.g., amino, hydroxyl, etc.), e.g., one suitable for oligonucleotide synthesis. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is 4-nitrophenyl. In some embodiments, R is -CHCH-(4-nitrophenyl). In some embodiments, R' is -C(O)NPh.

[0123] In some embodiments, each R is independently —H, or C 1~10 Aliphatic, C with 1-5 heteroatoms 1~10 Heteroaliphatic, C 6~14 Aryl, C 6~20 Arylaliphatic, C with 1-5 heteroatoms 6~20 an optionally substituted group selected from arylheteroaliphatic, 5- to 14-membered heteroaryl having 1-5 heteroatoms, and 3- to 10-membered heterocyclyl having 1-5 heteroatoms; or Two R groups optionally and independently combine to form a covalent bond, or: two or more R groups on the same atom optionally and independently combine with that atom to form an optionally substituted 3-15 membered monocyclic, bicyclic, or polycyclic ring having, in addition to that atom, 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms; or: Two or more R groups on two or more atoms optionally and independently combine with their intervening atoms to form an optionally substituted 3-15 membered monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms.

[0124] In some embodiments, each R is independently —H, or C 1~20 Aliphatic, C with 1-10 heteroatoms 1~20 Heteroaliphatic, C 6~30 Aryl, C 6~30 Arylaliphatic, C with 1-10 heteroatoms 6~30 In some embodiments, each R is independently -H, or C. 1~10 Aliphatic, C with 1-5 heteroatoms 1~10 Heteroaliphatic, C 6~14 Aryl, C 6~20 Arylaliphatic, C with 1-5 heteroatoms 6~20and optionally substituted groups selected from arylheteroaliphatic, 5-14 membered heteroaryl having 1-5 heteroatoms, and 3-10 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, two R groups optionally and independently combine to form a covalent bond. In some embodiments, two or more R groups on the same atom optionally and independently combine with that atom to form an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to that atom. In some embodiments, two groups on the same atom optionally and independently combine with that atom to form an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to that atom. In some embodiments, two or more R groups on two or more atoms optionally and independently combine with their intervening atoms to form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the intervening atoms. In some embodiments, two groups on two or more atoms optionally and independently combine with their intervening atoms to form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the intervening atoms. In some embodiments, the ring formed is monocyclic. In some embodiments, the ring formed is bicyclic. In some embodiments, the ring formed is polycyclic. In some embodiments, each monocyclic ring unit independently has 3 to 10 (e.g., 3 to 8, 3 to 7, 3 to 6, 5 to 10, 5 to 8, 5 to 7, 5 to 6, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) members, is independently saturated, partially saturated, or aromatic, and independently has 0 to 5 heteroatoms. In some embodiments, the ring is saturated. In some embodiments, the ring is partially saturated. In some embodiments, the ring is aromatic. In some embodiments, the ring formed has 1 to 5 heteroatoms. In some embodiments, the ring formed has 1 heteroatom. In some embodiments, the ring formed has 2 heteroatoms. In some embodiments, the heteroatom is nitrogen.In some embodiments, the heteroatom is oxygen.

[0125] In some embodiments, R is —H.

[0126] In some embodiments, R is optionally substituted C 1~20 , C 1~15 , C 1~10 , C 1~8 , C 1~6 , C 1~5 , C 1~4 , C 1~3 , or C 1~2 In some embodiments, R is an optionally substituted alkyl. In some embodiments, R is an optionally substituted C 1~6 In some embodiments, R is an optionally substituted alkyl. In some embodiments, R is an optionally substituted methyl. In some embodiments, R is an optionally substituted alicyclic. In some embodiments, R is an optionally substituted cycloalkyl.

[0127] In some embodiments, R is an optionally substituted C 1~20 It is heteroaliphatic.

[0128] In some embodiments, R is optionally substituted C 6~20 In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl.

[0129] In some embodiments, R is optionally substituted C 6~20 In some embodiments, R is an optionally substituted C 6~20 In some embodiments, R is arylalkyl. In some embodiments, R is benzyl. In some embodiments, R is an optionally substituted C alkyl group having 1-10 heteroatoms. 6~20 Arylheteroaliphatic.

[0130] In some embodiments, R is an optionally substituted 5-20 membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 3-20 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 3-10 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 5-6 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, the heterocyclyl is saturated. In some embodiments, the heterocyclyl is partially saturated.

[0131] In some embodiments, heteroatoms are selected from boron, nitrogen, oxygen, sulfur, silicon, and phosphorus. In some embodiments, heteroatoms are selected from nitrogen, oxygen, sulfur, and silicon. In some embodiments, heteroatoms are selected from nitrogen, oxygen, and sulfur. In some embodiments, heteroatoms are nitrogen. In some embodiments, heteroatoms are oxygen. In some embodiments, heteroatoms are sulfur.

[0132] Certain oligonucleotides and / or compositions referenced in this disclosure are described in WO 2021 / 071858 or WO 2022 / 099159 or priority applications, for example, in Table 1 of WO 2021 / 071858 or WO 2022 / 099159 or priority applications. All oligonucleotides and / or compositions of WO 2021 / 071858 and WO 2022 / 099159 are incorporated herein by reference.

[0133] Certain specific oligonucleotides and / or compositions are described in Table 1 below.

[0134] [Table 1]

[0135] [Table 2]

[0136] Note: Descriptions, base sequences, and stereochemistry / linkages can be categorized into multiple series in Table 1 depending on their length. Unless otherwise specified, all oligonucleotides in Table 1 are single-stranded. As will be understood by those skilled in the art, nucleoside units, unless otherwise specified (e.g., r, m, m5, eo, etc.), are unmodified and contain unmodified nucleobases and 2'-deoxy sugars; linkages are natural phosphate linkages unless otherwise specified; and acidic / basic groups may independently be present in their salt form. If the sugar is not specified, the sugar is the natural DNA sugar; and if the internucleotide linkage is not specified, the internucleotide linkage is the natural phosphate linkage. Moieties and Modifications: m:2'-OMe; I: The nucleobase is hypoxanthine; f:2′-F; eo:2'-MOE(2'-OCH2CH2OCH3); m5Ceo:5-methyl 2'-O-methoxyethyl C; O, PO: phosphodiester (phosphate). This can be a bond or an end group (or a component thereof), e.g., the bond between the linker and the oligonucleotide chain, the internucleotide bond (natural phosphate bond), etc. A phosphodiester is usually indicated with an "O" in the stereochemistry / bonding column and is usually not labeled in the description column (if it is an end group, e.g., the 5' end group, it is indicated in the description and usually not in the stereochemistry / bonding); if a bond is not indicated in the description column, it is usually a phosphodiester unless otherwise indicated. Note that the phosphate bond between the linker (e.g., L001) and the oligonucleotide chain may not be labeled in the description column, but may be indicated with an "O" in the stereochemistry / bonding column; *, PS: phosphorothioate. This can be a terminal group (if it is a terminal group, e.g., the 5' terminal group, this is indicated in the description and usually not in the stereochemistry / bonding), or a bond, e.g., a bond between a linker (e.g., L001) and an oligonucleotide chain, an internucleotide bond (phosphorothioate internucleotide bond), etc.; S, phosphorothioate in Sp:Sp configuration. Note that *S in the description indicates a single phosphorothioate bond in the Sp configuration; n001: [ka] nR (when used in conjunction with n001) or n001R; n001 in the Rp configuration; nS (when used in relation to n001) or n001S; n001 in the Sp configuration; Mod001: [ka] L001: Mod (e.g., Mod001) via -NH- (e.g., forming an amide group -C(O)-NH-), and in various cases, -NH-(CH2)6- linker (C6 linker, C6 amine linker, or C6 amino linker) linked to the 5' end of the oligonucleotide chain via a phosphate bond (O or PO). For example, in WV-39306, L001 is linked to Mod001 via -NH- (forming an amide group -C(O)-NH-), and linked to the oligonucleotide chain via a phosphate bond (O). When Mod is absent, -NH- is bonded to -H; b008U: Base [ka] A nucleoside.

[0137] In some embodiments, the present disclosure provides a compound having the structure of Formula A-1 or a salt thereof. In some embodiments, WV-46312 is provided, administered, or delivered as one or more compounds, each independently having the structure of Formula A-1 or a salt thereof. In some embodiments, a composition of WV-46312 comprises one or more compounds, each independently having the structure of Formula A-1 or a salt thereof.

[0138] In some embodiments, the disclosure provides a compound having the structure of Formula A-2 or a salt thereof. In some embodiments, WV-49090 is provided, administered, or delivered as one or more compounds each independently having the structure of Formula A-2 or a salt thereof. In some embodiments, a composition of WV-49090 comprises one or more compounds each independently having the structure of Formula A-2 or a salt thereof.

[0139] In some embodiments, the disclosure provides a compound having the structure of Formula A-3 or a salt thereof. In some embodiments, WV-49092 is provided, administered, or delivered as one or more compounds each independently having the structure of Formula A-3 or a salt thereof. In some embodiments, a WV-49092 composition comprises one or more compounds each independently having the structure of Formula A-3 or a salt thereof.

[0140] In some embodiments, the disclosure provides a compound having the structure of Formula B-1 or a salt thereof. In some embodiments, WV-44515 is provided, administered, or delivered as one or more compounds, each independently having the structure of Formula B-1 or a salt thereof. In some embodiments, a composition of WV-44515 comprises one or more compounds, each independently having the structure of Formula B-1 or a salt thereof.

[0141] In some embodiments, the present disclosure provides a compound having the structure of Formula B-2 or a salt thereof. In some embodiments, WV-50497 is provided, administered, or delivered as one or more compounds each independently having the structure of Formula B-2 or a salt thereof. In some embodiments, a composition of WV-50497 comprises one or more compounds each independently having the structure of Formula B-2 or a salt thereof.

[0142] In some embodiments, the present disclosure provides compounds having the structure of Formula B-3 or a salt thereof. In some embodiments, WV-50498 is provided, administered, or delivered as one or more compounds each independently having the structure of Formula B-3 or a salt thereof. In some embodiments, a composition of WV-50498 comprises one or more compounds each independently having the structure of Formula B-3 or a salt thereof.

[0143] In some embodiments, the salts are pharmaceutically acceptable salts. In some embodiments, each salt is independently a pharmaceutically acceptable salt. [ka] [ka] [ka] [ka] The zigzag shape represents the bond between the 3' oxygen and phosphorus in the internucleotide linkage.

[0144] Compounds and oligonucleotides can be prepared in high purity according to the present disclosure, for example, through chiral controlled forms of chiral internucleotide linkages, such as phosphorothioate internucleotide linkages, n001 linkages, etc. In some embodiments, the diastereomeric purity of the compound or oligonucleotide is about or at least about (DS) ncand DS is about 85% to 100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more), and nc is the number of chiral bonded phosphorus atoms. In some embodiments, DS is about or at least about 90%. In some embodiments, DS is about or at least about 91%. In some embodiments, DS is about or at least about 92%. In some embodiments, DS is about or at least about 93%. In some embodiments, DS is about or at least about 94%. In some embodiments, DS is about or at least about 95%. In some embodiments, DS is about or at least about 96%. In some embodiments, DS is about or at least about 97%. In some embodiments, DS is about or at least about 98%. In some embodiments, the DS is about or at least about 99%. In some embodiments, the diastereopurity is determined as the product of the diastereopurities of each chiral internucleotide linkage in the oligonucleotide. In some embodiments, the diastereopurity of an internucleotide linkage linking two nucleosides in an oligonucleotide (or nucleic acid) is represented 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).

[0145] In some embodiments, the diastereomeric excess of one or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) chiral coupled phosphorus centers is independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric excess of one or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) chiral coupled phosphorus centers is independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 95%. In some embodiments, the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 96%. In some embodiments, the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 97%. In some embodiments, the diastereomeric excess of each phosphorothioate-linked phosphorus is independently about or at least about 98%. In some embodiments, the diastereomeric excess of each chiral linked phosphorus center is independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric excess of each chiral linked phosphorus center is independently about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.In some embodiments, the diastereomeric excess of each chiral bonded phosphorus center is independently about or at least about 95%. In some embodiments, the diastereomeric excess of each chiral bonded phosphorus center is independently about or at least about 96%. In some embodiments, the diastereomeric excess of each chiral bonded phosphorus center is independently about or at least about 97%. In some embodiments, the diastereomeric excess of each chiral bonded phosphorus center is independently about or at least about 98%.

[0146] In some embodiments, the oligonucleotide or compound is about 10% to 100% (e.g., about 10% to 95%, 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 10 ... 90%-95%, 90%-100%, or about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the oligonucleotides are 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%, 70% to 90%, 70% to 95%, 70% to 95%, 70% to 95%, 70% to 10 ... The purity may be between 0% 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%, or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, purity is expressed as the area % of the UV trace at 260 nM of a separation technique, e.g., HPLC, UPLC, etc.

[0147] Oligonucleotide Composition In particular, the present disclosure provides various oligonucleotide compositions. In some embodiments, the present disclosure provides oligonucleotide compositions of the oligonucleotides described herein. In some embodiments, the oligonucleotide composition comprises a plurality of oligonucleotides described in the present disclosure. In some embodiments, the oligonucleotide composition is chirally controlled. In some embodiments, the oligonucleotide composition is not chirally controlled (sterically irregular).

[0148] The linking phosphorus of natural phosphate bond is achiral. The linking phosphorus of many modified internucleotide linkages (for example, phosphorothioate internucleotide linkages) is chiral. In some embodiments, during the preparation of oligonucleotide compositions (for example, in traditional phosphoramidite oligonucleotide synthesis), the arrangement of chiral linking phosphorus is not intentionally designed or controlled, resulting in chiral uncontrolled (sterically irregular) oligonucleotide compositions (substantially racemic preparations) that are complex and random mixtures of various stereoisomers (diastereoisomers). For an oligonucleotide with n chiral internucleotide linkages (where the linking phosphorus is chiral), there are usually 2n stereoisomers (for example, when n is 10, 210 = 1,032; when n is 20, 220 = 1,048,576). These stereoisomers have the same constitution but differ in the stereochemical pattern of these linking phosphorus.

[0149] Stereoisomers within a stereoirregular composition may have different properties, activities, and / or toxicities, particularly when compared to certain chiral controlled oligonucleotide compositions of the same configuration, resulting in variable therapeutic effects and / or unintended side effects from the stereoirregular composition.

[0150] In some embodiments, the present disclosure encompasses techniques for designing and preparing chiral controlled oligonucleotide compositions. In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions, such as those in Table 1. In some embodiments, the chiral controlled oligonucleotide compositions include multiple oligonucleotides with a controlled / predetermined (not disordered as in stereoirregular compositions) level, where the oligonucleotides share the same bond phosphorus stereochemistry at one or more chiral internucleotide linkages (chiral controlled internucleotide linkages). In some embodiments, the oligonucleotides share the same pattern of backbone chiral centers (bond phosphorus stereochemistry). In some embodiments, the pattern of backbone chiral centers is as described in the present disclosure. In some embodiments, the multiple oligonucleotides are structurally identical.

[0151] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions comprising a plurality of oligonucleotides, the oligonucleotides comprising: 1) share a common configuration, and 2) one or more (e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 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) chiral internucleotide linkages (chiral controlled internucleotide linkages) sharing the same stereochemistry of the linked phosphorus; The composition is enriched with respect to these multiple oligonucleotides as compared to a substantially racemic preparation of oligonucleotides of a common constitution.

[0152] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides, the plurality of oligonucleotides comprising: 1) a common base sequence, and 2) independently, the stereochemistry of the same bond phosphorus at 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, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages"). Share; The stereochemical purity of the linking phosphorus of each chiral controlled internucleotide bond is independently 80% to 100% (e.g., 85 to 100%, 90 to 100%, about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).

[0153] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions comprising a plurality of oligonucleotides, the oligonucleotides comprising: 1) share a common configuration, and 2) one or more (e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 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) chiral internucleotide linkages (chiral controlled internucleotide linkages) sharing the same stereochemistry of the linked phosphorus; The stereochemical purity of the linking phosphorus of each chiral controlled internucleotide bond is independently 80% to 100% (e.g., 85 to 100%, 90 to 100%, about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).

[0154] In some embodiments, multiple oligonucleotides share the same nucleobase and / or sugar modifications. In some embodiments, multiple oligonucleotides share the same internucleotide linkage modifications (internucleotide linkages can exist in various acid, base, and / or salt forms). In some embodiments, multiple oligonucleotides share the same nucleobase modifications, sugar modifications, and internucleotide linkage modifications, if any. In some embodiments, multiple oligonucleotides are of the same form (e.g., acid form, base form, or particularly salt form (e.g., pharmaceutically acceptable salt form, e.g., salt form)). In some embodiments, oligonucleotides in a composition can exist in one or more forms (e.g., acid form, base form, and / or one or more salt forms). In some embodiments, in aqueous solution (e.g., when dissolved in a buffer such as PBS), anions and cations can dissociate. In some embodiments, multiple oligonucleotides are of the same constitution. In some embodiments, multiple oligonucleotides are structurally identical. In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions comprising a plurality of oligonucleotides, the oligonucleotides being of a common composition and comprising 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more) chiral internucleotide linkages (chiral controlled internucleotide linkages) sharing the same linkage phosphorus stereochemistry, and the composition is enriched with respect to these multiple oligonucleotides as compared to a substantially racemic preparation of oligonucleotides of the common configuration.

[0155] In some embodiments, at least one chiral internucleotide linkage is chiral-controlled. In some embodiments, at least two internucleotide linkages are independently chiral-controlled. In some embodiments, the number of chiral-controlled internucleotide linkages is at least 3. In some embodiments, it is at least 4. In some embodiments, it is at least 5. In some embodiments, it is at least 6. In some embodiments, it is at least 7. In some embodiments, it is at least 8. In some embodiments, it is at least 9. In some embodiments, it is at least 10. In some embodiments, it is at least 11. In some embodiments, it is at least 12. In some embodiments, it is at least 13. In some embodiments, it is at least 14. In some embodiments, it is at least 15. In some embodiments, it is at least 20. In some embodiments, it is at least 25. In some embodiments, it is at least 30. In some embodiments, each chiral internucleotide linkage is independently a chiral-controlled internucleotide linkage.

[0156] In some embodiments, at least 5% to 100% (e.g., 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%, 70% to 95%, 70% to 10 ... The chirality (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.) is chiral controlled. In some embodiments, at least 5% to 100% (e.g., 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., is chiral controlled.In some embodiments, at least 5% to 100% (e.g., 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 8 ... In some embodiments, the chirality is at least 50%. In some embodiments, the chirality is at least 60%. In some embodiments, the chirality is at least 50%. In some embodiments, the chirality is at least 60%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 90%. In some embodiments, each chiral internucleotide linkage is chiral controlled. In some embodiments, each phosphorothioate internucleotide linkage is chiral controlled.

[0157] In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides, each of which is independently a specific oligonucleotide or a salt thereof. In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides, each of which is independently a specific oligonucleotide or a pharmaceutically acceptable salt thereof. In some embodiments, such a composition is enriched compared to a substantially racemic preparation of the specific oligonucleotide. As will be understood by those skilled in the art, the multiple oligonucleotides share a common sequence, which is the base sequence of the specific oligonucleotide. In some embodiments, at least about 5% to 100%, 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 5% to 90%, 10% to 90%, 20 to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 5% to 85%, 10% to 85%, 20 to 85%, 30% to 85%, 40% to 85%, 50% to 85%, 5% to 80%, 10% to 80%, 20 to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 5% to 75%, 10% to 75%, 20% ... ~75%, 30%~75%, 40%~75%, 50%~75%, 5%~70%, 10%~70%, 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30%~65%, 40%~65%, 50%~65%, 5%~60%, 10%~ 60%, 20-60%, 30-60%, 40-60%, 50-60%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are multiple oligonucleotides.In some embodiments, at least about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 5% to 90%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 5% to 85%, 10% to 85%, 20% to 85%, 30% to 85%, 40% to 85%, 50% to 85%, 5% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 5% to 75%, 10% to 75%, 20~75%, 30%~75%, 40%~75%, 50%~75%, 5%~70%, 10%~70%, 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30%~65%, 40%~65%, 50%~65%, 5%~60%, 10% Up to 60%, 20-60%, 30-60%, 40-60%, 50-60%, 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 percentage is at least 10%. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, it is at least 60%. In some embodiments, it is at least 70%. In some embodiments, it is at least 80%. In some embodiments, it is at least 90%. In some embodiments, this is at least 95%. In some embodiments, this is about 5-100%. In some embodiments, this is about 10-100%. In some embodiments, this is about 20-100%. In some embodiments, this is about 30-90%. In some embodiments, this is about 30-80%.In some embodiments, this is about 30-70%. In some embodiments, this is about 40-90%. In some embodiments, this is about 40-80%. In some embodiments, this is about 40-70%. In some embodiments, the particular oligonucleotide is an oligonucleotide exemplified herein (e.g., an oligonucleotide in Table 1 or another table).

[0158] In some embodiments, enrichment relative to a substantially racemic preparation refers to at least about 5% to 100%, 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 5% to 90%, 10% to 90%, 20 to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 5% to 85%, 10% to 85%, 20 to 85%, 30% to 85%, 40% to 85%, 50% to 85%, 5% to 80%, 10% to 80%, 20 to 80%, 30% to 80 ...0%, 5% to 80%, 10% to 80%, 20 to 80%, 30% to 80%, 40% to 85%, 50% to 85%, 5% to 80%, 10% to 80%, 20 to 80%, 30% to 80%, 40% to 85%, 50% to 85%, 5% to 80%, 10% to 80%, 2 ~80%, 50%~80%, 5%~75%, 10%~75%, 20~75%, 30%~75%, 40%~75%, 50%~75%, 5%~70%, 10%~70%, 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30%~65%, 40%~65%, 50%~65%, 5% to 60%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, 50% to 60%, 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 percentage is at least 10%. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, it is at least 60%. In some embodiments, it is at least 70%. In some embodiments, it is at least 80%. In some embodiments, this is at least 90%. In some embodiments, this is at least 95%. In some embodiments, this is about 5-100%. In some embodiments, this is about 10-100%. In some embodiments, this is about 20-100%.In some embodiments, this is about 30-90%. In some embodiments, this is about 30-80%. In some embodiments, this is about 30-70%. In some embodiments, this is about 40-90%. In some embodiments, this is about 40-80%. In some embodiments, this is about 40-70%.

[0159] In some embodiments, at least about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 5% to 90%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 5% to 85%, 10% to 85%, 20% to 85%, 30% to 85%, 40% to 85%, 50% to 85%, 5% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 5% to 75%, 10% to 75%, 20% to 75%, 30% to 75%, 40% to 75%, 5 ... %, 30%~75%, 40%~75%, 50%~75%, 5%~70%, 10%~70%, 20~70%, 30%~70%, 40%~70%, 50%~70%, 5%~65%, 10%~65%, 20~65%, 30%~65%, 40%~65%, 50%~65%, 5%~60%, 10%~60 %, 20-60%, 30-60%, 40-60%, 50-60%, 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 percentage is at least 10%. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, it is at least 60%. In some embodiments, it is at least 70%. In some embodiments, it is at least 80%. In some embodiments, it is at least 90%. In some embodiments, this is at least 95%. In some embodiments, this is about 5-100%. In some embodiments, this is about 10-100%. In some embodiments, this is about 20-100%. In some embodiments, this is about 30-90%. In some embodiments, this is about 30-80%. In some embodiments, this is about 30-70%.In some embodiments, this is about 40-90%. In some embodiments, this is about 40-80%. In some embodiments, this is about 40-70%.

[0160] In chiral controlled oligonucleotide compositions, the level of multiple oligonucleotides is controlled. In contrast, in non-chiral controlled (or stereo-irregular, racemic) oligonucleotide compositions (or preparations), the level of oligonucleotides is random and uncontrolled. In some embodiments, the enrichment compared to substantially racemic preparations is at the level described herein.

[0161] In some embodiments, the level (e.g., controlled level, predetermined level, enrichment) as a percentage is expressed as (DS) nc or at least (DS) nc where DS (diastereomeric purity of individual internucleotide linkages) is 90%-100%, and nc is the number of chiral linkage phosphorus (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) as described herein. In some embodiments, the level (e.g., controlled level, predetermined level, enrichment) as a percentage is (DS) nc or at least (DS) ncand DS (diastereopurity of individual internucleotide linkages) is 90%-100%, and nc is the number of chiral-controlled internucleotide linkages as described herein (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more). In some embodiments, each chiral internucleotide linkage is chiral-controlled, and nc is the number of chiral internucleotide linkages. In some embodiments, DS is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater. In some embodiments, DS is 90% or at least 90%. In some embodiments, DS is 91% or at least 91%. In some embodiments, DS is 92% or at least 92%. In some embodiments, DS is 93% or at least 93%. In some embodiments, the DS is 94% or at least 94%. In some embodiments, the DS is 95% or at least 95%. In some embodiments, the DS is 96% or at least 96%. In some embodiments, the DS is 97% or at least 97%. In some embodiments, the DS is 98% or at least 98%. In some embodiments, the DS is 99% or at least 99%. In some embodiments, the level (e.g., controlled level, predetermined level, enrichment) is the percentage of all oligonucleotides in the composition that share the same construct, and the percentage is (DS) nc Or at least (DS) nc For example, DS is 99%, nc is 10, and the percentage is 90% or at least 90% ((99%) 10 ≈0.90=90%). As will be appreciated by those skilled in the art, in stereoirregular preparations, the percentage is usually about 1 / 2 nc (When nc is 10, the percentage is about 1 / 2 10In some embodiments, enrichment (e.g., compared to a substantially racemic preparation), level, etc., refers to at least about (DS) of all oligonucleotides in a composition, or all oligonucleotides in a composition sharing a common base sequence, or all oligonucleotides in a composition sharing a common structure. nc is a plurality of oligonucleotides. In some embodiments, this is for all oligonucleotides in a composition. In some embodiments, this is for all oligonucleotides in a composition that share a common base sequence. In some embodiments, this is for all oligonucleotides in a composition that share a common structure. In some embodiments, various forms of an oligonucleotide (e.g., various salt forms) may be properly considered to have the same structure.

[0162] In some embodiments, an oligonucleotide contains one or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) chiral-controlled chiral internucleotide linkages, where the diastereomeric excess (de) at the linked phosphorus is independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, about or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of all chiral internucleotide linkages that contain the chiral linked phosphorus are independently such chiral-controlled internucleotide linkages. In some embodiments, about or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the phosphorothioate internucleotide linkages are independently such chiral-controlled internucleotide linkages. In some embodiments, each phosphorothioate internucleotide linkage is independently such chiral-controlled internucleotide linkage. In some embodiments, each chiral internucleotide linkage comprising a chiral linking phosphorus is independently such chiral-controlled internucleotide linkage. In some embodiments, de is about or at least about 80%. In some embodiments, de is about or at least about 85%. In some embodiments, de is about or at least about 90%. In some embodiments, de is about or at least about 95%. In some embodiments, de is about or at least about 96%. In some embodiments, de is about or at least about 97%. In some embodiments, de is about or at least about 98%.

[0163] In some embodiments, the diastereopurity level of a plurality of oligonucleotides in a composition can be determined as the product of the diastereopurities of each chiral internucleotide bond in the oligonucleotide. In some embodiments, the diastereopurity level of a plurality of oligonucleotides in a composition can be determined as the product of the diastereopurities of each chiral internucleotide bond in the oligonucleotide. In some embodiments, the diastereopurity of an internucleotide bond linking two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereopurity of the internucleotide bond of a dimer linking the same two nucleosides, the dimer being prepared under comparable conditions, in some instances, under identical synthesis cycle conditions (e.g., for a bond between Nx and Ny in an oligonucleotide...NxNy..., the dimer is NxNy).

[0164] In some embodiments, a chiral controlled oligonucleotide composition comprises two or more oligonucleotides, each of which independently comprises a plurality of the oligonucleotides described herein (e.g., in various chiral controlled oligonucleotide compositions). For example, in some embodiments, each of which independently comprises a common base sequence and the same phosphorus stereochemistry at one or more chiral internucleotide linkages, and each of which independently comprises a sterically enriched or enriched level as described herein relative to a sterically unordered preparation of the plurality. In some embodiments, at least two of the or each of the plurality independently targets different adenosines. In some embodiments, at least two of the or each of the plurality independently targets different transcripts of the same or different nucleic acids. In some embodiments, at least two of the or each of the plurality independently targets transcripts of different genes. In particular, such compositions can be used to target two or more targets, in some embodiments simultaneously and in the same system.

[0165] In some embodiments, all chiral internucleotide linkages are chiral controlled and the composition is a fully chiral controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chiral controlled internucleotide linkages and the composition is a partially chiral controlled oligonucleotide composition.

[0166] In some embodiments, a chiral controlled oligonucleotide composition is a chirally pure (or stereochemically pure) oligonucleotide composition that comprises a plurality of oligonucleotides that are identical (including where each chiral element of each chiral phosphorus-containing oligonucleotide is independently defined (sterically restricted)), and the composition does not contain other stereoisomers. A chirally pure (or stereochemically pure) oligonucleotide composition of an oligonucleotide stereoisomer does not contain other stereoisomers (although, as will be understood by those skilled in the art, one or more unintended stereoisomers may be present as impurities).

[0167] Chirality-controlled oligonucleotide compositions may exhibit several advantages over stereoirregular oligonucleotide compositions. In particular, chirality-controlled oligonucleotide compositions are more uniform with respect to oligonucleotide structure than corresponding stereoirregular oligonucleotide compositions. By controlling stereochemistry, compositions of individual stereoisomers can be prepared and evaluated, resulting in the development of stereoisomeric chirality-controlled oligonucleotide compositions with desired properties and / or activity. In some embodiments, chirality-controlled oligonucleotide compositions provide, for example, better delivery, stability, clearance, activity, selectivity, and / or toxicity profiles compared to corresponding stereoirregular oligonucleotide compositions. In some embodiments, chirality-controlled oligonucleotide compositions provide better efficacy, fewer side effects, and / or more convenient and effective dosing regimens. In particular, the pattern of chiral centers in the backbone as described herein, optionally combined with other structural features described herein (e.g., modifications of nucleobases, sugars, internucleotide linkages, etc.), may be utilized to provide highly efficient, directional adenosine editing.

[0168] In some embodiments, the present disclosure provides chirality-controlled oligonucleotide compositions. In some embodiments, the provided chirality-controlled oligonucleotide compositions include multiple oligonucleotides of the same configuration and have one or more chiral internucleotide linkages. In some embodiments, the multiple oligonucleotides are, for example, multiple oligonucleotides selected from Table 1 (and / or one or more of its various salt forms), in the chirality-controlled oligonucleotide composition, where the oligonucleotides contain at least one Rp or Sp linked phosphorus in the chirality-controlled internucleotide linkage. In some embodiments, the multiple oligonucleotides are, for example, multiple oligonucleotides selected from Table 1 (and / or one or more of its various salt forms), in the chirality-controlled oligonucleotide composition, where each phosphorothioate internucleotide linkage in the oligonucleotide is independently chiral-controlled (each phosphorothioate internucleotide linkage is independently Rp or Sp). In some embodiments, the oligonucleotide composition (e.g., oligonucleotide composition) is a substantially pure preparation of a single oligonucleotide, in that oligonucleotides in the composition that are not a single oligonucleotide are, in some cases, impurities from the preparation process of the single oligonucleotide after certain purification procedures.

[0169] In some embodiments, a chirality-controlled oligonucleotide composition may have increased activity and / or stability, increased delivery, and / or reduced ability to induce adverse effects such as complement or TLR9 activation, compared to a corresponding sterically irregular oligonucleotide composition. In some embodiments, a sterically irregular (non-chirality-controlled) oligonucleotide composition differs from a chirality-controlled oligonucleotide composition in that its corresponding plurality of oligonucleotides does not contain any chirality-controlled internucleotide linkages, but the sterically irregular oligonucleotide composition is otherwise identical to the chirality-controlled oligonucleotide composition.

[0170] In some embodiments, the present disclosure relates to chiral controlled oligonucleotide compositions that can modulate the level, activity, or expression of a gene (e.g., SERPINA1) or its gene product. In some embodiments, the level, activity, or expression of the gene or its gene product is increased (e.g., A to I conversion (e.g., 1024 in SERPINA1) compared to a reference condition (e.g., in the absence of the disclosed oligonucleotides and / or compositions, and / or in the presence of a reference oligonucleotide and / or oligonucleotide composition (e.g., oligonucleotides of the same base sequence but with different modifications, sterically irregular compositions of oligonucleotides of equivalent structure (e.g., base sequence, modifications, etc.) but lacking stereochemical control, etc.)). In some embodiments, the level, activity, or expression of a gene or its gene product is reduced (e.g., by converting an A to I to create a stop codon and / or altering a codon (e.g., correcting E342K in the mutant A1AT) to reduce protein translation levels, decrease production of a particular protein isoform, modulate splicing to reduce levels of a particular splice product and the protein encoded thereby, etc.).

[0171] In some embodiments, the chiral controlled oligonucleotide composition provided is a chiral controlled oligonucleotide composition comprising a plurality of oligonucleotides. In some embodiments, the chiral controlled oligonucleotide composition is a chiral pure (or "stereochemically pure") oligonucleotide composition. In some embodiments, the present disclosure provides a chiral pure oligonucleotide composition of the oligonucleotides of Table 1, wherein each chiral internucleotide linkage of the oligonucleotide is independently chiral controlled (Rp or Sp, e.g., can be determined from R or S in "stereochemistry / linkage" but not from X). As one skilled in the art will understand, chemical selectivity is rarely, if ever, complete (absolute 100%). In some embodiments, a chirally pure oligonucleotide composition comprises a plurality of oligonucleotides, which are structurally identical and all have the same structure (the same stereoisomeric form; in the context of an oligonucleotide, typically the same diastereoisomeric form, due to the presence of multiple chiral centers in the oligonucleotide), and the chirally pure oligonucleotide composition does not contain other stereoisomers (in the context of an oligonucleotide, typically diastereomers, due to the presence of multiple chiral centers in the oligonucleotide; e.g., to the extent achievable by stereoselective preparation). As will be appreciated by those skilled in the art, a stereoirregular (or "racemic," "chiral non-controlled") oligonucleotide composition can contain many stereoisomers (e.g., 2 n where n is the number of chiral linking phosphorus for an oligonucleotide in which other chiral centers (e.g., carbon chiral centers in the sugar) are each independently present in one configuration and are chiral controlled, and the only chiral linking phosphorus center is not chiral controlled).

[0172] In some embodiments, the oligonucleotide is linked to a solid support. In some embodiments, the solid support is a support for oligonucleotide synthesis. In some embodiments, the solid support comprises glass. In some embodiments, the solid support is controlled pore glass (CPG). In some embodiments, the solid support is a polymer. In some embodiments, the solid support is polystyrene. In some embodiments, the solid support is highly cross-linked polystyrene (HCP). In some embodiments, the solid support is a hybrid support of controlled pore glass (CPG) and highly cross-linked polystyrene (HCP). In some embodiments, the solid support is a metal foam. In some embodiments, the solid support is a resin. In some embodiments, the oligonucleotide is cleaved from the solid support.

[0173] In some embodiments, the purity, particularly the stereochemical purity, and particularly the diastereomeric purity, of many oligonucleotides and compositions thereof in which all other chiral centers in the oligonucleotide other than the chiral linking phosphorus are sterically restricted (e.g., a carbon chiral center in the sugar, which is defined, for example, in a phosphoramidite for oligonucleotide synthesis) can be controlled by the stereoselectivity at the chiral linking phosphorus in the coupling step when forming the chiral internucleotide bond (diastereoselectivity, as will be understood by those skilled in the art, in many cases in oligonucleotide synthesis where the oligonucleotide contains multiple chiral centers). In some embodiments, the coupling step has 60% stereoselectivity at the linking phosphorus (or diastereoselectivity, if other chiral centers are present). After such a coupling step, the newly formed internucleotide bond can be said to have 60% stereochemical purity (for oligonucleotides, typically diastereomeric purity in light of the presence of other chiral centers). In some embodiments, each coupling step independently has a stereoselectivity of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In some embodiments, chiral-controlled internucleotide linkages are typically formed with a stereoselectivity of at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5%, or substantially 100% (in some embodiments, at least 85%; in some embodiments, at least 87%; in some embodiments, at least 90%; in some embodiments, at least 95%; in some embodiments, at least 96%; in some embodiments, at least 97%; in some embodiments, at least 98%; in some embodiments, at least 99%). In some embodiments, the stereoselectivity is at least 85%. In some embodiments, the stereoselectivity is at least 87%. In some embodiments, the stereoselectivity is at least 90%.In some embodiments, each coupling step independently has substantially 100% stereoselectivity.

[0174] In some embodiments, the stereopurity of a chiral center (e.g., chiral bond phosphorus) in the composition is at least 60%, 70%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In some embodiments, the stereopurity is at least 80%. In some embodiments, the stereopurity is at least 85%. In some embodiments, the stereopurity is at least 87%. In some embodiments, the stereopurity is at least 90%. In some embodiments, the stereopurity is substantially 100%. In some embodiments, each chiral controlled internucleotide linkage independently has at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5%, or substantially 100% stereochemical purity (typically diastereomeric purity for oligonucleotides with multiple chiral centers) at its chiral linkage phosphorus (in some embodiments, at least 85%; in some embodiments, at least 87%; in some embodiments, at least 90%; in some embodiments, at least 95%; in some embodiments, at least 96%; in some embodiments, at least 97%; in some embodiments, at least 98%; in some embodiments, at least 99%). In some embodiments, the chiral controlled internucleotide linkage has at least 90% stereochemical purity. In some embodiments, a majority of the chiral controlled internucleotide linkages independently have at least 90% stereochemical purity. In some embodiments, each chiral controlled internucleotide linkage independently has at least 90% stereochemical purity. In some embodiments, each phosphorothioate internucleotide linkage is independently chiral controlled.

[0175] Stereoselectivity and stereopurity can be assessed by various techniques. In some embodiments, the stereoselectivity and / or stereopurity is substantially 100%, in that when the composition is analyzed by analytical methods (e.g., NMR, HPLC, etc.), substantially all detectable stereoisomers have the intended stereochemistry.

[0176] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 couplings of monomers (and in many embodiments, phosphoramidites, in the context of oligonucleotide synthesis, as will be appreciated by those skilled in the art) independently have a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90% (typically diastereoselective with respect to the chiral center of the linked phosphorus that is formed, in the context of oligonucleotide synthesis).

[0177] In some embodiments, in a stereoirregular (or racemic) preparation (or stereoirregular / non-chiral oligonucleotide composition), at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chiral internucleotide linkages of the oligonucleotide independently have a stereochemical purity (typically diastereomeric purity for oligonucleotides containing multiple chiral centers) of less than about 60%, 65%, 70%, 75%, 80%, or 85% with respect to the chiral phosphorus of the internucleotide linkage. In some embodiments, the stereochemical purity is less than about 60%. In some embodiments, the stereochemical purity is less than about 65%. In some embodiments, the stereochemical purity is less than about 70%. In some embodiments, the stereochemical purity is less than about 75%. In some embodiments, the stereochemical purity (stereopurity) is less than about 80%.

[0178] In some embodiments, compounds (e.g., oligonucleotides, chiral auxiliaries, etc.) of the present disclosure contain multiple chiral elements (e.g., multiple carbon and / or phosphorus chiral centers (e.g., the linking phosphorus of a chiral internucleotide linkage)). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more chiral elements of a provided compound (e.g., an oligonucleotide) each independently have a diastereomeric purity as described herein. In some embodiments, the diastereomeric purity is at least 85%. In some embodiments, the diastereomeric purity is at least 86%. In some embodiments, the diastereomeric purity is at least 87%. In some embodiments, the diastereomeric purity is at least 88%. In some embodiments, the diastereomeric purity is at least 89%. In some embodiments, the diastereomeric purity is at least 90%. In some embodiments, the diastereomeric purity is at least 91%. In some embodiments, the diastereomeric purity is at least 92%. In some embodiments, the diastereomeric purity is at least 93%. In some embodiments, the diastereomeric purity is at least 94%. In some embodiments, the diastereomeric purity is at least 95%. In some embodiments, the diastereomeric purity is at least 96%. In some embodiments, the diastereomeric purity is at least 97%. In some embodiments, the diastereomeric purity is at least 98%. In some embodiments, the diastereomeric purity is at least 99%.

[0179] As will be appreciated by one of skill in the art, in some embodiments, the diastereoselectivity of coupling, or the diastereomeric purity of the chiral phosphorus center, can be assessed via the diastereoselectivity of dimer formation or the diastereomeric purity of dimers prepared under the same or comparable conditions, where the dimers have the same 5' and 3' nucleosides and internucleotide linkages.

[0180] A variety of techniques can be utilized to identify or confirm the stereochemistry of chiral elements (e.g., the configuration of chiral bond phosphorus) and / or the pattern of backbone chiral centers, and / or to assess the stereoselectivity (e.g., the diastereoselectivity of a coupling step in an oligonucleotide synthesis) and / or stereochemical purity (e.g., the diastereomeric purity of an internucleotide bond, a compound (e.g., an oligonucleotide), etc.). Exemplary techniques include NMR [e.g., 1D (one-dimensional) and / or 2D (two-dimensional) NMR]. 1 H- 31 These include P HETCOR (heteronuclear correlation spectroscopy), HPLC, RP-HPLC, mass spectrometry, LC-MS, and cleavage of internucleotide bonds by stereospecific nucleases, which can be used individually or in combination. Examples of useful nucleases include benzonase, micrococcal nuclease, and svPDE (snake venom phosphodiesterase), which are specific for certain internucleotide linkages with an Rp-linked phosphorus (e.g., an Rp phosphorothioate linkage); and nuclease P1, mung bean nuclease, and nuclease S1, which are specific for internucleotide linkages with an Sp-linked phosphorus (e.g., an Sp phosphorothioate linkage). While not wishing to be bound by any particular theory, the present disclosure recognizes that, at least in some cases, cleavage of oligonucleotides by certain nucleases may be influenced by structural elements, such as chemical modifications (e.g., 2'-modifications of the sugar), base sequence, or stereochemical context. For example, in some cases, benzonase and micrococcal nuclease, which are specific for the internucleotide bond with the bound phosphorus of Rp, were unable to cleave the phosphorothioate internucleotide bond of isolated Rp adjacent to the phosphorothioate internucleotide bond of Sp.

[0181] In some embodiments, the oligonucleotide compositions are substantially pure preparations of a single oligonucleotide stereoisomer, in that the oligonucleotides in the composition that are of the same constitution but are not stereoisomers are, in some cases, impurities resulting from the process of preparation of said oligonucleotide stereoisomer after certain purification procedures.

[0182] Editing Area In some embodiments, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: -1 In some embodiments, the edited region is or includes the nucleoside opposite the target adenosine (typically when the base sequence of the oligonucleotide is aligned with the target sequence for maximum complementarity and / or when the oligonucleotide hybridizes to the target nucleic acid) and the nucleoside adjacent to this nucleoside. In some embodiments, the edited region is or includes three nucleobases, with the central nucleobase being the nucleoside opposite the target adenosine. In some embodiments, the nucleoside opposite the target adenosine is N0, as described herein.

[0183] In some embodiments, the nucleobase of the nucleoside opposite the target adenosine (which may be referred to as BA0) is b008U. In some embodiments, the sugar of N0 is a natural DNA sugar. See, for example, the various oligonucleotides in Table 1. In some embodiments, it has been observed that b008U as BA0 can provide improved adenosine editing efficiency. In some embodiments, the reference nucleobase is U. In some embodiments, the nucleobase is T. In some embodiments, the reference nucleobase is C.

[0184] In some embodiments, the nucleoside opposite the target adenosine, e.g., N0, is b008U (when used in reference to a nucleoside, unless otherwise specified, in an oligonucleotide chain). [ka] (referring to the sequence of the oligonucleotide b008U). See, for example, the various oligonucleotides in Table 1. In some embodiments, it has been observed that b008U can provide improved editing, for example, when compared to dC at the position opposite the target adenosine.

[0185] In some embodiments, substitution of hypoxanthine for guanine at position -1 (e.g., substitution of dG for dI) may provide improved editing. -1 Each sugar in is independently a natural DNA sugar. See, for example, the various oligonucleotides in Table 1.

[0186] Nucleic acid bases A variety of nucleobases can be utilized in oligonucleotides in accordance with the present disclosure. In some embodiments, the nucleobase is a natural nucleobase, the most commonly occurring of which are A, T, C, G, and U. In some embodiments, the nucleobase is a modified nucleobase in that it is not A, T, C, G, or U. In some embodiments, the nucleobase is an optionally substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, the nucleobase is an optionally substituted A, T, C, G, or U (e.g., 5mC, 5-hydroxymethylC, etc.). In some embodiments, the nucleobase is A. In some embodiments, the nucleobase is T. In some embodiments, the nucleobase is C. In some embodiments, the nucleobase is G. In some embodiments, the nucleobase is U. In some embodiments, the nucleobase is 5mC. In some embodiments, the nucleobase is a substituted A, T, C, G, or U. In some embodiments, the nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, the nucleobase is a modified base. In some embodiments, the base is U( [ka] ). In some embodiments, the base is optionally substituted b008U. In some embodiments, the base is optionally protected b008U. In some embodiments, the nucleobase is hypoxanthine. In some embodiments, the nucleobase is optionally substituted hypoxanthine or a tautomer thereof. In some embodiments, the nucleobase is optionally protected hypoxanthine or a tautomer thereof. In some embodiments, the substitution protects certain functional groups in the nucleobase to minimize undesired reactions during oligonucleotide synthesis. Suitable techniques for protecting nucleobases in oligonucleotide synthesis are widely known in the art and can be utilized in accordance with the present disclosure. In some embodiments, the modified nucleobase improves the properties and / or activity of the oligonucleotide. For example, in many cases, 5mC can be utilized in place of C to modulate certain undesired biological effects (e.g., immune response). In some embodiments, when determining sequence identity, substituted nucleobases that have the same hydrogen bonding pattern are treated as the same as unsubstituted nucleobases, e.g., 5mC may be treated as the same as C (e.g., an oligonucleotide having 5mC in place of C (e.g., AT5mCG) is considered to have the same base sequence as an oligonucleotide having a C at the corresponding position (e.g., ATCG)).

[0187] In some embodiments, the nucleobase is a modified base.

[0188] In some embodiments, the nucleoside is bU( [ka] ) or a salt thereof, where "*" indicates the linkage to the internucleotide bond, as in various oligonucleotides.

[0189] Certain useful nucleobases, nucleosides, etc. are described in WO 2021 / 071858 and WO 2022 / 099159, each of which is incorporated by reference in its entirety.

[0190] sugar A variety of sugars, including modified sugars, may be utilized in accordance with the present disclosure. In some embodiments, the present disclosure provides sugar modifications and patterns thereof, optionally combined with other structural elements (e.g., internucleotide linkage modifications and patterns thereof, patterns of chiral centers in the backbone, etc.), that, when incorporated into an oligonucleotide, may provide improved properties and / or activity.

[0191] The most common naturally occurring nucleosides comprise a ribose sugar (e.g., in RNA) or a deoxyribose sugar (e.g., in DNA) linked to the nucleobases adenosine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). In some embodiments, the sugar (e.g., the various sugars in many of the oligonucleotides in Table 1 (unless otherwise noted)) is: [ka] where the nucleobase is attached at the 1' position, and the 3' and 5' positions are linked to internucleotide linkages (as would be understood by one of skill in the art), and in the case of the 5' end of an oligonucleotide, the 5' position can be linked to the 5' terminal group (e.g., -OH), and in the case of the 3' end of an oligonucleotide, the 3' position can be linked to the 3' terminal group (e.g., -OH). In some embodiments, the sugar is [ka] where the nucleobase is attached at the 1' position, and the 3' and 5' positions are linked to internucleotide linkages (as would be understood by one of skill in the art), and in the case of the 5' end of the oligonucleotide, the 5' position can be linked to the 5' terminal group (e.g., -OH), and in the case of the 3' end of the oligonucleotide, the 3' position can be linked to the 3' terminal group (e.g., -OH). In some embodiments, the sugar is a modified sugar in that it is not a natural DNA or RNA sugar. In particular, modified sugars can provide improved stability. In some embodiments, modified sugars can be utilized to alter and / or optimize one or more hybridization properties. In some embodiments, modified sugars can be utilized to alter and / or optimize target nucleic acid recognition. In some embodiments, modified sugars can be utilized to optimize Tm. In some embodiments, modified sugars can be utilized to improve oligonucleotide activity.

[0192] In particular, the present disclosure demonstrates that a variety of non-natural RNA sugars (e.g., natural DNA sugars, various modified sugars, etc.) can be utilized in accordance with the present disclosure. For example, the oligonucleotides in Table 1 contain natural DNA sugars, 2'-F modified sugars, 2'-OMe modified sugars, and in some cases, 2'-MOE modified sugars. In particular, the present disclosure demonstrates that designed oligonucleotides such as those shown in Table 1 can be constructed with short lengths and relatively low levels of 2'-F modified sugars (e.g., about 50% or less of all sugars, 2'-OR modified sugars (where R is an optionally substituted C 1~6 These results demonstrate that high editing efficiency can be achieved with approximately or less than 2'-F modified sugars (which are aliphatic) and / or without a large number (e.g., about 5 or more) of consecutive 2'-F modified sugars.

[0193] In some embodiments, the sugar is a naturally occurring DNA or RNA sugar, optionally substituted. In some embodiments, the sugar is an optionally substituted [ka] In some embodiments, the 2' position is optionally substituted. In some embodiments, the sugar is [ka] In some embodiments, the 2'-modified sugar is: [ka] (In the formula, R 2s is a 2'-modification. In some embodiments, the sugar has the structure: [ka] (In the formula, R 2s is -H, halogen, or -OR (wherein R is an optionally substituted C 1~6 In some embodiments, R 2s is —H. In some embodiments, R 2s is -F. In some embodiments, R 2s In some embodiments, the modified nucleoside is mA, mT, mC, mC, mG, mU, etc., and R 2s is -OMe. In some embodiments, R 2s is -OCH2CH2OMe. In some embodiments, the modified sugar is Aeo, Teo, Ceo, m5Ceo, Geo, Ueo, etc., and R 2s is —OCHCHOMe. In some embodiments, R 2s is -OCH2CH2OH. In some embodiments, the oligonucleotide is [ka] (e.g., for fA, fT, fC, f5mC, fG, fU, etc.). In some embodiments, the oligonucleotide comprises a 2'-F modified sugar having the structure: [ka] (e.g., for mA, mT, mC, m5mC, mG, mU, etc.). In some embodiments, the oligonucleotide comprises a 2'-OMe modified sugar having the structure: [ka] (e.g., Aeo, Teo, Ceo, m5Ceo, Geo, Ueo, etc.)

[0194] In some embodiments, the sugar is [ka] (In the formula, R 2s and R 4s Combined, -L s - Forming L s is a covalent bond or an optionally substituted divalent C 1~6 In some embodiments, L has the structure: aliphatic or heteroaliphatic having 1 to 4 heteroatoms. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen, or sulfur. In some embodiments, L s is optionally substituted C2-O-CH2-C4. In some embodiments, L s is C2-O-CH2-C4. In some embodiments, L s is C2-O-(R)-CH(CH2CH3)-C4. In some embodiments, L s is C2-O-(S)-CH(CH2CH3)-C4.

[0195] In some embodiments, the sugar is selected from the group consisting of saccharides, saccharides, and modified sugars described in WO 2018 / 022473, WO 2018 / 098264, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2018 / 237195, WO 2018 / 237196, WO 2018 / 237197, WO 2018 / 237198, WO 2018 / 23719 ... and sugars described in Patent Nos. 2019 / 032607, 2019 / 055951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858, and / or 2022 / 099159.

[0196] internucleotide bond In particular, the present disclosure provides a variety of internucleotide linkages (e.g., various modified internucleotide linkages) that can be utilized in conjunction with other structural elements (e.g., various sugars as described herein) to provide oligonucleotides and compositions thereof.

[0197] In some embodiments, oligonucleotides include base modifications, sugar modifications, and / or internucleotide linkage modifications. A variety of internucleotide linkages can be utilized in accordance with the present disclosure to link nucleobase-containing units (e.g., nucleosides). In some embodiments, provided oligonucleotides include both one or more modified internucleotide linkages and one or more natural phosphate linkages. As is well known by those skilled in the art, natural phosphate linkages are widely found in natural DNA and RNA molecules; they have the structure -OP(O)(OH)O-, link sugars in nucleosides in DNA and RNA, and can exist in various salt forms, for example, at physiological pH (about 7.4), where natural phosphate linkages exist primarily in the salt form, with the anion being -OP(O)(O -)O-. A modified internucleotide linkage or non-natural phosphate linkage is an internucleotide linkage that is not a natural phosphate linkage or its salt form. Modified internucleotide linkages can exist in their salt forms depending on their structure. For example, as will be understood by those skilled in the art, a phosphorothioate internucleotide linkage having the structure -OP(O)(SH)O- can exist in various salt forms, for example, at physiological pH (about 7.4), and the anion is -OP(O)(S - )O-. In some embodiments, the internucleotide bond can exist in a neutral form, for example, at physiological pH (about 7.4).

[0198] In some embodiments, the linkage contains a linking phosphorus atom where the oxygen atom is bonded to an oxygen atom that is not bonded to or part of the backbone sugar (a "PO linkage," e.g., a natural phosphate linkage). In some embodiments, the linkage contains a linking phosphorus atom where the sulfur atom is bonded to a sulfur atom that is not bonded to or part of the backbone sugar (a "PS linkage," e.g., a phosphorothioate internucleotide linkage). In some embodiments, the linkage contains a linking phosphorus atom where the nitrogen atom is bonded to a nitrogen atom that is not bonded to or part of the backbone sugar (a "PN linkage," e.g., n001). In some embodiments, the oligonucleotide comprises one or more PO linkages, one or more PS linkages, and one or more PN linkages. In some embodiments, the oligonucleotide comprises one or more natural phosphate linkages, one or more phosphorothioate internucleotide linkages, and one or more n001 linkages. In some embodiments, each chiral linking phosphorus is independently chiral controlled.

[0199] In some embodiments, the internucleotide linkages are selected from the group consisting of those described in WO 2018 / 022473, WO 2018 / 098264, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, each of which is independently incorporated by reference herein. , No. 2019 / 032607, No. 2019 / 055951, No. 2019 / 075357, No. 2019 / 200185, No. 2019 / 217784, No. 2019 / 032612, No. 2020 / 191252, No. 2021 / 071858, and / or No. 2022 / 099159.

[0200] Additional chemical moieties In some embodiments, the oligonucleotide comprises one or more additional chemical moieties. A variety of additional chemical moieties (e.g., targeting moieties, carbohydrate moieties, lipid moieties, etc.) are known in the art and can be utilized in accordance with the present disclosure to adjust the properties and / or activity (e.g., stability, half-life, activity, delivery, pharmacodynamic properties, pharmacokinetic properties, etc.) of the provided oligonucleotides. In some embodiments, certain additional chemical moieties facilitate delivery of the oligonucleotide to desired cells, tissues, and / or organs, including, but not limited to, cells of the central nervous system. In some embodiments, certain additional chemical moieties facilitate internalization of the oligonucleotide. In some embodiments, certain additional chemical moieties increase oligonucleotide stability. In some embodiments, the present disclosure provides techniques for incorporating various additional chemical moieties into oligonucleotides.

[0201] In some embodiments, the additional chemical moiety is or comprises a small molecule moiety. In some embodiments, the small molecule is a ligand of a protein (e.g., a receptor). In some embodiments, the small molecule binds to a polypeptide. In some embodiments, the small molecule is an inhibitor of a polypeptide. In some embodiments, the additional chemical moiety is or comprises a peptide moiety (e.g., an antibody). In some embodiments, the additional chemical moiety is or comprises a nucleic acid moiety. In some embodiments, the nucleic acid provides a novel property and / or activity. In some embodiments, the nucleic acid moiety forms a duplex or other secondary structure with the original oligonucleotide strand (prior to conjugation) or a portion thereof. In some embodiments, the nucleic acid is or comprises an oligonucleotide that targets the same or a different target and may exert activity via the same or a different mechanism. In some embodiments, the nucleic acid is or comprises an RNAi agent. In some embodiments, the nucleic acid is or comprises an miRNA agent. In some embodiments, the nucleic acid is or comprises an RNAase H-dependent nucleic acid. In some embodiments, the nucleic acid is or comprises a gRNA. In some embodiments, the nucleic acid is or comprises an aptamer. In some embodiments, the additional chemical moiety is or includes a carbohydrate moiety as described herein. Many useful agents (e.g., small molecules, peptides, carbohydrates, nucleic acid agents, etc.) can be conjugated to the oligonucleotides herein in accordance with the present disclosure.

[0202] In some embodiments, the oligonucleotide comprises an additional chemical moiety that results in increased delivery to and / or activity in a tissue compared to a reference oligonucleotide (e.g., an otherwise identical reference oligonucleotide without the additional chemical moiety).

[0203] In some embodiments, the additional chemical moiety is a carbohydrate moiety, a targeting moiety, etc., which may improve one or more properties when incorporated into an oligonucleotide. In some embodiments, the additional chemical moiety is selected from glucose, GluNAc (N-acetylamine glucosamine), and anisamide moiety. In some embodiments, a provided oligonucleotide may include two or more additional chemical moieties, which may or may not be identical, or may or may not be of the same class (e.g., carbohydrate moiety, sugar moiety, targeting moiety, etc.).

[0204] In some embodiments, the additional chemical moiety is a targeting moiety. In some embodiments, the additional chemical moiety is or comprises a carbohydrate moiety. In some embodiments, the additional chemical moiety is or comprises a lipid moiety. In some embodiments, the additional chemical moiety is or comprises a ligand moiety for a cellular receptor, such as a sigma receptor, an asialoglycoprotein receptor, etc. In some embodiments, the ligand moiety is or comprises an anisamide moiety, which may be a ligand moiety for a sigma receptor. In some embodiments, the ligand moiety is or comprises GalNAc, which may be a ligand moiety for an asialoglycoprotein receptor. In some embodiments, the additional chemical moiety facilitates delivery to the liver.

[0205] In some embodiments, provided oligonucleotides may include one or more linkers and additional chemical moieties (e.g., targeting moieties), and / or may be chiral or non-chiral, and / or may have a base sequence and / or one or more modifications and / or formats as described herein.

[0206] A variety of linkers, carbohydrate moieties, and targeting moieties can be utilized in accordance with the present disclosure, including many known in the art. In some embodiments, the carbohydrate moiety is the targeting moiety. In some embodiments, the targeting moiety is a carbohydrate moiety.

[0207] In some embodiments, the additional chemical moiety is any of those described in the Examples, which include examples of various additional chemical moieties that are incorporated into various oligonucleotides.

[0208] In some embodiments, additional chemical moieties conjugated to the oligonucleotide may target the oligonucleotide to cells of the central nervous system.

[0209] In some embodiments, the additional chemical moiety comprises or is a cell receptor ligand. In some embodiments, the additional chemical moiety comprises or is a protein conjugate (e.g., one that binds to a cell surface protein). Such moieties may be particularly useful for targeted delivery of oligonucleotides to cells expressing the corresponding receptor or protein. In some embodiments, the additional chemical moiety of the provided oligonucleotide comprises anisamide, or a derivative or analog thereof, which may target the oligonucleotide to cells expressing a specific receptor, such as the sigma-1 receptor.

[0210] In some embodiments, provided oligonucleotides are formulated for administration to somatic cells and / or tissues that express the target. In some embodiments, additional chemical moieties conjugated to the oligonucleotide can target the oligonucleotide to the cell.

[0211] In some embodiments, the additional chemical moiety is or comprises an asialoglycoprotein receptor (ASGPR) ligand. Without wishing to be bound by any particular theory, the present disclosure recognizes that ASGPR1 has also been reported to be expressed in the hippocampal region and / or cerebellar Purkinje cell layer of mice.

[0212] Various other ASGPR ligands are known in the art and may be utilized in accordance with the present disclosure. In some embodiments, the ASGPR ligand is a carbohydrate. In some embodiments, the ASGPR ligand is GalNac or a derivative or analog thereof. In some embodiments, the ASGPR ligand is one described in Sanhueza et al. J. Am. Chem. Soc., 2017, 139 (9), pp. 3528-3536. In some embodiments, the ASGPR ligand is one described in Mamidyala et al. J. Am. Chem. Soc., 2012, 134, pp. 1978-1981. In some embodiments, the ASGPR ligand is one described in U.S. Patent Application Publication No. 20160207953. In some embodiments, the ASGPR ligand is a substituted-6,8-dioxabicyclo[3.2.1]octane-2,3-diol derivative, for example, as disclosed in U.S. Patent Application Publication No. 20160207953. In some embodiments, the ASGPR ligand is one described, for example, in U.S. Patent Application Publication No. 20150329555. In some embodiments, the ASGPR ligand is one described, for example, in U.S. Patent Application Publication No. 20150329555. In some embodiments, the ASGPR ligand is one described, for example, in U.S. Patent Application Publication No. 8,877,917, U.S. Patent Application Publication No. 20160376585, U.S. Patent No. 10,086,081, or U.S. Patent No. 8,106,022. In some embodiments, various GalNac derivatives and their uses are described in International Publication No. 2022 / 076922 and can be utilized in accordance with the present disclosure. The ASGPR ligands described in these documents are incorporated herein by reference. Those skilled in the art will appreciate that various techniques, including those described in these documents, for assessing the binding of chemical moieties to ASGPR are known in the art and can be utilized in accordance with the present disclosure. In some embodiments, the provided oligonucleotide is conjugated to an ASGPR ligand.In some embodiments, the provided oligonucleotide comprises an ASGPR ligand. In some embodiments, the additional chemical moiety is [ka] wherein each variable is independently as described in this disclosure. In some embodiments, R is -H. In some embodiments, R' is -C(O)R.

[0213] In some embodiments, the additional chemical moiety is [ka] In some embodiments, the additional chemical moiety is or comprises: [ka] In some embodiments, the additional chemical moiety is or comprises: [ka] In some embodiments, the additional chemical moiety is or comprises: [ka] In some embodiments, the additional chemical moiety is or comprises optionally substituted [ka] In some embodiments, the additional chemical moiety is or comprises: [ka] In some embodiments, the additional chemical moiety is or comprises: [ka] In some embodiments, the additional chemical moiety is or comprises: [ka] In some embodiments, the additional chemical moiety is or comprises: [ka] is or contains

[0214] In some embodiments, the additional chemical moiety comprises one or more moieties that can bind, for example, to an oligonucleotide target cell. For example, in some embodiments, the additional chemical moiety comprises one or more protein ligand moieties, e.g., in some embodiments, the additional chemical moiety comprises multiple moieties, each independently an ASGPR ligand. In some embodiments, as in the case of Mod 001 and Mod083, the additional chemical moiety comprises three such ligands. Mod001: [ka] Mod083: [ka]

[0215] In some embodiments, the oligonucleotide is [ka] wherein each variable is independently as described herein. In some embodiments, each -OR' is -OAc and -N(R')2 is -NHAc. In some embodiments, the oligonucleotide comprises: [ka] In some embodiments, each R' is -H. In some embodiments, each -OR' is -OH and each -N(R')2 is -NHC(O)R. In some embodiments, each -OR' is -OH and each -N(R')2 is -NHAc. In some embodiments, the oligonucleotide is [ka] In some embodiments, a -CH2- linking moiety is utilized as the C5 linking moiety on the sugar. In some embodiments, a linking moiety on the ring is utilized as the C3 linking moiety on the sugar. Such moieties include, for example, [ka] for example, [ka] (Those skilled in the art will appreciate that one or more other protecting groups, such as protecting groups for -OH, -NH2-, -N(i-Pr)2, -OCH2CH2CN, etc., may alternatively be utilized, and that the protecting groups may optionally be removed under a variety of suitable conditions during the oligonucleotide deprotection and / or cleavage step.) In some embodiments, the oligonucleotide may comprise two, three, or more (e.g., only three) [ka] In some embodiments, the oligonucleotide comprises two, three, or more (e.g., only three) [ka] In some embodiments, copies of such portions are linked by internucleotide bonds (e.g., natural phosphate bonds) as described herein. In some embodiments, when at the 5' end, the -CH2- linkage is attached to an -OH. In some embodiments, the oligonucleotide comprises [ka] In some embodiments, the oligonucleotide comprises: [ka] In some embodiments, each -OR' is -OAc and -N(R')2 is -NHAc. In some embodiments, the oligonucleotide comprises [ka] In particular, [ka] have comparable and / or better activity and / or properties [ka] In some embodiments, this can be used to introduce the same number of [ka] This provides improved preparation efficiency and / or lower cost (eg, when compared to Mod001).

[0216] In some embodiments, the additional chemical moiety is a Mod group, as described herein (eg, Table 1).

[0217] In some embodiments, the additional chemical moiety is Mod001. In some embodiments, the additional chemical moiety is Mod083. In some embodiments, the additional chemical moiety (e.g., a Mod group) is conjugated directly to the remainder of the oligonucleotide (e.g., without a linker). In some embodiments, the additional chemical moiety is conjugated to the remainder of the oligonucleotide via a linker. In some embodiments, the additional chemical moiety (e.g., a Mod group) can be linked directly and / or via a linker to the nucleobase, sugar, and / or internucleotide linkage of the oligonucleotide. In some embodiments, the Mod group is linked to the sugar directly or via a linker. In some embodiments, the Mod group is linked to the 5'-terminal sugar directly or via a linker. In some embodiments, the Mod group is linked to the 5'-terminal sugar via the 5'-carbon directly or via a linker. See, for example, the various oligonucleotides in Table 1. In some embodiments, the Mod group is linked to the 3'-terminal sugar directly or via a linker. In some embodiments, the Mod group is linked to the 3'-terminal sugar via the 3' carbon, either directly or via a linker. In some embodiments, the Mod group is linked to a nucleobase, either directly or via a linker. In some embodiments, the Mod group is linked to an internucleotide linkage, either directly or via a linker. In some embodiments, the provided oligonucleotide comprises Mod001 linked to the 5'-end of the oligonucleotide chain via L001.

[0218] As will be appreciated by one of skill in the art, the additional chemical moiety can be linked to the oligonucleotide chain at various positions, e.g., the 5' end, the 3' end, or a mid-position (e.g., sugar, base, internucleotide linkage, etc.). In some embodiments, it is linked at the 5' end. In some embodiments, it is linked at the 3' end. In some embodiments, it is linked at a mid-nucleotide.

[0219] Certain additional chemical moieties (e.g., lipid moieties, targeting moieties, carbohydrate moieties), such as Mod012, Mod039, Mod062, Mod085, Mod086, and Mod094, as well as various linkers for linking additional chemical moieties to oligonucleotide chains, such as L001, L003, L004, L008, L009, and L010, and their uses are described in WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679 ... 7 / 210647, 2018 / 098264, 2018 / 022473, 2018 / 223056, 2018 / 223073 , 2018 / 223081, 2018 / 237194, 2019 / 032607, 2019 / 032612, 2019 / 0 55951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858, and / or 2022 / 099159 and may be utilized in accordance with the present disclosure. In some embodiments, the additional chemical moiety is digoxigenin or biotin, or a derivative thereof.

[0220] In some embodiments, the additional chemical moieties (e.g., linkers, lipids, solubilizing groups, conjugate groups, targeting groups, and / or targeting ligands) are those described in WO 2012 / 030683 or WO 2021 / 030778. In some embodiments, the provided oligonucleotides are those described in WO 2012 / 030683, WO 2021 / 030778, WO 2019112485, U.S. Patent Application Publication No. 20170362270, WO 2018156056, or WO 2018056871, WO 2021 / 030778, WO 20 and / or a chemical structure (e.g., a linker, lipid, solubilizing group, and / or targeting ligand) described in 2020 / 154344, 2020 / 154343, 2020 / 154342, 2020 / 165077, 2020 / 201406, 2020 / 216637, or 2020 / 252376.

[0221] In some embodiments, additional chemical moieties (e.g., targeting groups, conjugate groups, etc.) and / or modifications (e.g., of nucleobases, sugars, internucleotide linkages, etc.) may be prepared using methods described in U.S. Patent Nos. 5,688,941; 6,294,664; 6,320,017; 6,576,752; 5,258,506; 5,591,584; 4,958,013; 5,082,830; 5,118,802; 5,138,045; 6,783,931; 5,254,469; No. 5,414,077; No. 5,486,603; No. 5,112,963; No. 5,599,928; No. 6,900,297; No. 5,214,136; No. 5,109,124; No. 5,512,439; No. 4,667,025 ; Same No. 5,525,465; Same No. 5,514,785; Same No. 5,565,552; Same No. 5,541,313; Same No. 5,5 No. 45,730; No. 4,835,263; No. 4,876,335; No. 5,578,717; No. 5,580,731 5,451,463; 5,510,475; 4,904,582; 5,082,830; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 5,595,726 No. 5,214,136; No. 5,245,022; No. 5,317,098; No. 5,371,241; No. 5, No. 391,723; No. 4,948,882; No. 5,218,105; No. 5,112,963; No. 5,567,810 No. 5,574,142; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 5,585,481; No. 5,292,873; No. 5,552,538; No. 5,512,667; No. 5,597,696; No. 5,599,923; No. 7,037,646; No. 5,587,371; No. 5,416,203; No. 5,262,536; No. 5,272,250; or No. 8,106,022.

[0222] In some embodiments, the additional chemical moiety (e.g., Mod) is linked via a linker. A variety of linkers, such as those utilized for conjugation of various moieties to proteins (e.g., antibodies forming antibody drug conjugates), nucleic acids, etc., are available in the art and can be utilized in accordance with the present disclosure. Certain useful linkers are described in U.S. Patent No. 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 / 210 647, 2018 / 098264, 2018 / 223056, 2018 / 237194, 2019 / 032607, 2019 / 055951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858, and / or 2022 / 099159. In some embodiments, the linker is, by way of non-limiting example, L001, L004, L009, or L010. In some embodiments, the oligonucleotide comprises a linker but does not comprise any additional chemical moieties other than the linker. In some embodiments, the oligonucleotide comprises a linker but does not comprise any additional chemical moieties other than the linker, and the linker is L001, L004, L009, or L010. In some embodiments, the linker is or comprises a moiety having an internucleotide bond structure as described herein. In some embodiments, such a moiety in the linker does not link two nucleosides. In some embodiments, the linker has the structure L. In some embodiments, the linker is bivalent. In some embodiments, the linker is multivalent. In some embodiments, the linker may link two or more additional chemical moieties to the oligonucleotide as described herein.For example, in some embodiments, one, or two, or three, or more additional chemical moieties (e.g., GalNAc moieties) are linked to the oligonucleotide chain (e.g., at the 5' end) via a polyvalent linker moiety.

[0223] In some embodiments, the additional chemical moiety is cleaved from the remainder of the oligonucleotide (e.g., the oligonucleotide chain) after administration, e.g., to a system, cell, tissue, organ, subject, etc. In some embodiments, the additional chemical moiety facilitates, increases, and / or accelerates delivery to certain cells, and after delivery of the oligonucleotide to such cells, the additional chemical moiety is cleaved from the oligonucleotide. In some embodiments, the linker moiety comprises one or more cleavable moieties that can be cleaved at a desired location (e.g., in a certain type of cell, an intracellular compartment such as a lysosome, etc.) and / or timing. In some embodiments, the cleavable moiety is selectively cleaved by a polypeptide (e.g., an enzyme such as a nuclease). Many useful cleavable moieties and cleavable linkers have been described and can be utilized in accordance with the present disclosure. In some embodiments, the cleavable moiety is or comprises one or more functional groups selected from amide, ester, ether, phosphodiester, disulfide, carbamate, etc. In some embodiments, the linker is as described in WO 2012 / 030683, WO 2021 / 030778, WO 2020 / 154344, WO 2020 / 154343, WO 2020 / 154342, WO 2020 / 165077, WO 2020 / 201406, WO 2020 / 216637, or WO 2020 / 252376.

[0224] As demonstrated herein, the provided technology may, in some embodiments, provide high levels of activity and / or desired properties without utilizing particular structural elements (e.g., modifications, bonding arrangements, and / or patterns, etc.) reported to be desirable and / or necessary (e.g., those reported in WO 2019 / 219581), although certain such structural elements may be incorporated into oligonucleotides in combination with various other structural elements in accordance with the present disclosure.

[0225] ADAR In particular, the provided technology may provide for the modification / editing of a target adenosine by converting A to I. In some embodiments, the oligonucleotide and / or the duplex formed by the target nucleic acid and the oligonucleotide interacts with a protein (e.g., an ADAR protein). In some embodiments, such a protein comprises an adenosine-modifying activity and may modify the target adenosine in the target nucleic acid, e.g., converting it to inosine.

[0226] ADAR protein is a protein that is naturally expressed in various cells, tissues, organs, and / or organisms.It has been reported that some ADAR proteins (such as ADAR1 and ADAR2) can edit adenosine by deamination, converting adenosine to inosine, and can provide several functions, including being read as G or similar during translation.The mechanism of RNA editing mediated by ADAR (such as deamination) has been reported.For example, it has been reported that ADAR protein catalyzes the conversion of adenosine to inosine on mismatched double-stranded RNA substrates.As understood by those skilled in the art, inosine can be recognized as guanosine by the translation and / or splicing mechanism in cells.Therefore, ADAR can be used for functional adenosine to guanosine editing of nucleic acid (such as pre-mRNA and mRNA substrate).

[0227] In some embodiments, the present disclosure provides oligonucleotides and compositions thereof for ADAR-mediated editing of target adenosines in target nucleic acids (e.g., RNA). ADAR-mediated RNA editing may offer several advantages over DNA editing, for example, delivery is simplified because it does not require the expression of recombinant proteins such as Cas9. Because both ADAR1 and ADAR2 are endogenous enzymes, cellular delivery of oligonucleotides alone may be sufficient for editing. Off-target effects, if any, are transient and do not result in changes to genomic DNA. Furthermore, ADAR-mediated editing can be used in postmitotic cells, which does not require an HDR template for repair. Three vertebrate ADAR genes have been reported with common functional domains (Nishikura Nat Rev Mol Cell Biol. 2016 Feb; 17(2): 83-96; Nishikura Annu Rev Biochem. 2010; 79: 321-349; Thomas and Beal Bioessays. 2017 Apr; 39(4)). All three ADARs contain a dsRNA-binding domain (dsRBD) that can contact dsRNA substrates. Some ADAR1s also contain a Z-DNA-binding domain. ADAR1 has been reported to be significantly expressed in the brain, lung, kidney, liver, heart, etc., and may exist in two isoforms. In some embodiments, isoform p150 can be induced by interferon, while isoform p110 can be constitutively expressed. In some embodiments, it may be advantageous to use p110 because it has been reported to be widely and constitutively expressed. ADAR2 can be highly expressed, for example, in the brain and lung, and has been reported to be exclusively localized in the nucleus. ADAR3 has been reported to be catalytically inactive and expressed only in the brain. Potential differences in tissue expression can be taken into consideration when selecting therapeutic targets.

[0228] The use of oligonucleotides for RNA editing by ADAR has been reported.In particular, the present disclosure recognizes that previously reported techniques generally have one or more drawbacks, such as low stability (e.g., oligonucleotides with natural RNA sugars), low editing efficiency, low editing specificity (e.g., some A's are edited in the part of the target nucleic acid that is substantially complementary to the oligonucleotide), specific structures in the oligonucleotide for ADAR recognition / recruitment, foreign proteins (e.g., those engineered to recognize the oligonucleotide and / or its duplex (e.g., with the target nucleic acid) with specific structures for editing), etc.In addition, previously reported techniques usually utilize stereoirregular oligonucleotide compositions, when the oligonucleotide contains one or more chiral linking phosphorus of modified internucleotide linkage.

[0229] For example, various reported oligonucleotides contain ADAR recruitment domains. Merkle et al., Nat Biotechnol. 2019 Feb; 37(2): 133-138, disclosed an oligonucleotide containing an imperfect 20-bp hairpin ADAR recruitment domain, which is an intramolecular stem-loop that recruits endogenous human ADAR2 to transcribe endogenous transcripts. The oligonucleotide reported in Mali et al., Nat Methods. 2019 Mar; 16(3): 239-242 contains an ADAR substrate GluR2 pre-messenger RNA sequence or MS2 hairpin in addition to a specificity domain that hybridizes to the target mRNA.

[0230] Certain reported editing techniques utilize exogenous or engineered proteins (e.g., those utilizing the CRISPR / Cas9 system). For example, Komor et al., Nature 2016 volume 533, pages 420-424, disclosed a deaminase combined with CRISPR-Cas9 to create a programmable DNA base editor. This requires delivery of both the CRISPR / Cas9 system and guide RNA for binding in the exogenous editing protein.

[0231] In particular, the present disclosure provides oligonucleotides containing one or more features, such as sugar modifications, base modifications, internucleotide linkage modifications, stereochemical control, various patterns thereof, etc., to address one or more or all of the shortcomings of previous adenosine editing technologies, for example, by providing chiral-controlled oligonucleotide compositions of designed oligonucleotides as described herein. For example, as demonstrated herein, an ADAR recruitment loop is optional and not required for the provided technology.

[0232] As will be appreciated by those skilled in the art, one or more of such useful features can be utilized to improve oligonucleotides in previous techniques (e.g., those described in International Publication Nos. 2016097212, 2017220751, 2018041973, and 2018134301, each of which is independently incorporated by reference). In some embodiments, the present disclosure provides improvements over previous techniques by applying one or more useful features described herein to previously reported oligonucleotide base sequences. In some embodiments, the present disclosure provides chiral-controlled oligonucleotide compositions of previously reported oligonucleotides that may be useful for adenosine editing. In some embodiments, the present disclosure provides improvements over previously reported adenosine editing using sterically irregular oligonucleotide compositions by performing such editing using chiral-controlled oligonucleotide compositions.

[0233] As reported, ADAR proteins can have various isoforms. For example, ADAR1 has, among others, a reported p110 isoform and a reported p150 isoform. In some embodiments, certain chirality-controlled oligonucleotide compositions can provide high levels of adenosine modification (e.g., A to I conversion) in multiple isoforms, in some embodiments, in both the p110 and p150 isoforms, while stereoirregular compositions have been observed to provide low levels of adenosine modification in one or more isoforms (e.g., p110). In some embodiments, chirality-controlled oligonucleotide compositions are particularly useful for adenosine modification in systems (e.g., cells, tissues, organs, organisms, subjects, etc.) that express or contain the p110 isoform of ADAR1, particularly those that express or contain a high level of the p110 isoform of ADAR1 compared to the p150 isoform, or those that do not express or express ADAR1 p150 at all or at low levels.

[0234] In some embodiments, the present disclosure provides cis-acting (CisA) oligonucleotides that do not require a stem loop in their structure. In some embodiments, the provided oligonucleotides can form dsRNA structures with target mRNAs through base pairing. In some embodiments, the formed dsRNA structure (optionally with secondary mismatches) contains a bulge that can promote ADAR binding and thus ADAR-mediated editing (e.g., deamination of the target adenosine). In some embodiments, the oligonucleotides of the present disclosure are shorter than LSL or CSL oligonucleotides, for example, 32 nt or less, 31 nt or less, 30 nt or less, 29 nt or less, 28 nt or less, 27 nt or less, or 26 nt or less in length, which can provide high editing efficiency.

[0235] Duplexing Region and Targeting Region In some embodiments, the oligonucleotide comprises a duplexing region and a targeting region. In some embodiments, the duplexing region forms a duplex with another nucleic acid, for example, a duplexing oligonucleotide. Useful duplexing techniques, including duplexing oligonucleotides and their use with the provided oligonucleotides, are described in WO 2022 / 099159, which is incorporated herein by reference.

[0236] Preparation of Oligonucleotides and Compositions Various methods can be used to prepare oligonucleotides and compositions, and can be used in accordance with the present disclosure. For example, stereoirregular oligonucleotides and compositions can be prepared using conventional phosphoramidite chemistry (e.g., phosphoramidites containing -CH2CH2CN and -N(i-Pr)2), and certain reagents and chiral controlled techniques can be used, for example, as described in U.S. Patent No. 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 2018 / 067974, WO 2018 / 067975, WO 2018 / 067976, WO 2018 / 067977, WO 2018 / 067978, WO 2018 / 0679 ... Chiral controlled oligonucleotide compositions may be prepared as described in US Pat. Nos. 017 / 160741, 2017 / 192679, 2017 / 210647, 2018 / 098264, 2018 / 223056, 2018 / 237194, 2019 / 032607, 2019 / 055951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858, and / or 2022 / 099159.

[0237] In some embodiments, chiral-controlled / stereoselective preparation of oligonucleotides and compositions thereof involves the use of a chiral auxiliary, e.g., as part of a monomer, dimer (e.g., chirally pure dimer from separation), monomeric phosphoramidite, dimeric phosphoramidite (e.g., chirally pure dimer from separation), etc. Examples of such chiral auxiliaries, monomers, dimers, and phosphoramidites are described in U.S. Pat. No. 9,982,257, U.S. Patent Application Publication Nos. 20170037399, 20180216108, 20180216107, U.S. Pat. No. 9,598,458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741 ... / 192679, 2017 / 210647, 2018 / 098264, 2018 / 223056, 2018 / 237194, 2019 / 032607, 2019 / 055951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858, and / or 2022 / 099159. In some embodiments, the chiral auxiliary is selected from the group consisting of chiral auxiliaries described in WO 2018 / 022473, WO 2018 / 098264, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 03 2607, 2019 / 055951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858, and / or 2022 / 099159.

[0238] In some embodiments, chiral controlled preparation techniques, including oligonucleotide synthesis cycles, reagents, and conditions, are described in U.S. Patent No. 9,982,257, U.S. Patent Application Publication Nos. 20170037399, 20180216108, and 20180216107, each of which is independently incorporated by reference herein. U.S. Patent No. 9598458, International Publication Nos. 2017 / 062862, 2018 / 067973, 2017 / 160741, 2017 / 192679, 2017 / 210647, and / or 2018 / 098264, 2018 / 022473, 2018 / 223056, 2018 / 223073, and 2018 / 2230 No. 81, No. 2018 / 237194, No. 2019 / 032607, No. 2019 / 032612, No. 2019 / 055951, No. 2019 / 075357, No. 20 19 / 200185, 2019 / 217784, 2019 / 032612, 2018 / 223056, 2018 / 223073, 2018 / 22308 1, 2018 / 237194, 2019 / 032607, 2019 / 055951, 2019 / 075357, 2019 / 200185, 2019 / 217784, 2019 / 032612, 2020 / 191252, 2021 / 071858, and / or 2022 / 099159.

[0239] Once synthesized, the provided oligonucleotides and compositions are typically further purified. Suitable purification techniques are widely known and practiced by those skilled in the art, and are described in U.S. Patent No. 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, each of which is independently incorporated herein by reference. No. 2018 / 098264, No. 2018 / 223056, No. 2018 / 237194, No. 2019 / 032607, No. 2019 / 055951, No. 2019 / 075357, No. 2019 / 200185, No. 2019 / 217784, No. 2019 / 032612, No. 2020 / 191252, No. 2021 / 071858, and / or No. 2022 / 099159.

[0240] In some embodiments, a cycle includes or consists of coupling, capping, modification, and deblocking. In some embodiments, a cycle includes or consists of coupling, capping, modification, capping, and deblocking. These steps are typically performed in the order listed, although in some embodiments, the order of certain steps (e.g., capping and modification) may be changed, as will be understood by those of skill in the art. If necessary, as those of skill in the art often do in synthesis, one or more steps may be repeated to improve conversion, yield, and / or purity. For example, in some embodiments, coupling may be repeated; in some embodiments, modification (e.g., oxidation to introduce ═O, sulfurization to introduce ═S, etc.) may be repeated; in some embodiments, coupling is repeated after a modification that may convert a P(III) bond into a P(V) bond, which may be more stable under certain conditions; coupling is routinely followed by a modification that converts the newly formed P(III) bond into a P(V) bond. In some embodiments, when steps are repeated, different conditions may be utilized (e.g., concentrations, temperatures, reagents, time, etc.).

[0241] Techniques for formulating provided oligonucleotides and / or preparing pharmaceutical compositions, for example, for administration to a subject via various routes, are readily available in the art and may be utilized in accordance with the present disclosure, e.g., as described in U.S. Pat. No. 9,982,257, U.S. Patent Application Publication Nos. 20170037399, 20180216108, 20180216107, U.S. Pat. No. 9,598,458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 223056, or WO 2018 / 237194, and the references cited therein.

[0242] Techniques for formulating provided oligonucleotides and / or preparing pharmaceutical compositions, for example, for administration to a subject via various routes, are readily available in the art and may be utilized in accordance with the present disclosure, e.g., as described in U.S. Pat. No. 9,982,257, U.S. Patent Application Publication Nos. 20170037399, 20180216108, 20180216107, U.S. Pat. No. 9,598,458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 223056, or WO 2018 / 237194, and the references cited therein.

[0243] In some embodiments, useful chiral auxiliaries are [ka] or a salt thereof (wherein, R C11 -L C1 -R C1 and L C1 is an optionally substituted —CH—, and R C1 is R, -Si(R) 3 , -SO2R, or an electron-withdrawing group, R C2 and R C3 together with their intervening atoms to form an optionally substituted 3- to 10-membered saturated ring having 0-2 heteroatoms in addition to the nitrogen atom. In some embodiments, useful chiral auxiliaries include: [ka] (In the formula, R C1 is R, -Si(R)3, or -SO2R, and R C2 and R C3 together with their intervening atoms to form an optionally substituted 3- to 7-membered saturated ring having 0-2 heteroatoms in addition to the nitrogen atom. The ring formed is an optionally substituted 5-membered ring. In some embodiments, useful chiral auxiliaries include: [ka] or a salt thereof. In some embodiments, a useful chiral auxiliary is [ka] In some embodiments, a useful chiral auxiliary is a DPSE chiral auxiliary. In some embodiments, the purity or stereochemical purity of the chiral auxiliary is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, this is at least 85%. In some embodiments, this is at least 90%. In some embodiments, this is at least 95%. In some embodiments, this is at least 96%. In some embodiments, this is at least 97%. In some embodiments, this is at least 98%. In some embodiments, this is at least 99%.

[0244] In some embodiments, L C1 is -CH-. In some embodiments, L C1 is a substituted -CH-. In some embodiments, L C1 is a monosubstituted -CH2-.

[0245] In some embodiments, R C1 is R. In some embodiments, R C1 is optionally substituted phenyl. In some embodiments, R C1 is -SiR. In some embodiments, R C1 is -SiPhMe. In some embodiments, R C1 is -SO2R. In some embodiments, R is not hydrogen. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is an optionally substituted C 1~6In some embodiments, R is C 1~6 In some embodiments, R is alkyl. In some embodiments, R is methyl. In some embodiments, R is t-butyl.

[0246] In some embodiments, R C1 is an electron-withdrawing group such as -C(O)R, -OP(O)(OR), -OP(O)(R), -P(O)(R), -S(O)R, or -S(O)R. In some embodiments, the electron-withdrawing group R C1 Chiral auxiliaries containing the group are particularly useful for preparing chiral controlled non-negatively charged internucleotide linkages and / or chiral controlled internucleotide linkages attached to natural RNA sugars.

[0247] In some embodiments, R C2 and R C3 taken together with their intervening atoms form an optionally substituted 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered saturated ring having no heteroatoms in addition to the nitrogen atom. C2 and R C3 taken together with their intervening atoms form an optionally substituted 5-membered saturated ring having no heteroatoms in addition to the nitrogen atom.

[0248] In some embodiments, methods for preparing oligonucleotides and / or compositions include, for example, using a chiral auxiliary described herein to construct one or more chiral-controlled internucleotide linkages. In some embodiments, one or more chiral-controlled internucleotide linkages are independently constructed using a DPSE chiral auxiliary. In some embodiments, each chiral-controlled phosphorothioate internucleotide linkage is independently constructed using a DPSE chiral auxiliary. In some embodiments, one or more chiral-controlled internucleotide linkages are independently constructed using [ka] or a salt thereof (wherein, RAU is as described herein). In some embodiments, each chiral, non-negatively charged internucleotide linkage (e.g., n001) is independently constructed using [ka] or a salt thereof. In some embodiments, each chiral controlled internucleotide linkage is independently [ka] or a salt thereof. In some embodiments, R AU is an optionally substituted C 1~20 , C 1~10 , C 1~6 , C 1~5 , or C 1~4 In some embodiments, R AU is an optionally substituted C 1~20 , C 1~10 , C 1~6 , C 1~5 , or C 1~4 In some embodiments, R AU is optionally substituted aryl. In some embodiments, R AU is phenyl. In some embodiments, one or more chiral controlled internucleotide linkages are constructed using a PSM asymmetric auxiliary. In some embodiments, each chiral controlled non-negatively charged internucleotide linkage (e.g., n001) is independently constructed using a PSM asymmetric auxiliary. In some embodiments, each chiral controlled internucleotide linkage is independently constructed using a PSM asymmetric auxiliary. As will be appreciated by those skilled in the art, chiral auxiliary agents can be used in conjunction with phosphoramidites (e.g., [ka] (DPSE phosphoramidite), [ka] (In the formula, R AU are independently as described herein; R AU is -Ph, it is a PSM phosphoramidite), R NS is often utilized in optionally substituted / protected nucleosides (e.g., optionally protected for oligonucleotide synthesis), or salts thereof, etc. In some embodiments, phosphoramidites are [ka] or a salt thereof, wherein each variable is independently as described herein. In some embodiments, R AU is optionally substituted phenyl. In some embodiments, R AU is phenyl. In some embodiments, R NS is an optionally substituted or protected nucleoside, including hypoxanthine. In some embodiments, R NS is an optionally substituted or protected hypoxanthine. In some embodiments, R NS is an optionally substituted or protected inosine. In some embodiments, R NS is an optionally substituted or protected deoxyinosine. In some embodiments, R NS is an optionally substituted or protected 2'-F inosine (2'-OH replaced with 2'-F). In some embodiments, R NS is an optionally substituted or protected 2'-OR modified inosine (wherein the 2'-OH is replaced with a 2'-OR modification as described herein (e.g., 2'-OMe, 2'-MOE, etc.)). In some embodiments, hypoxanthine is 6In some embodiments, hypoxanthine is an O protected with -L-Si(R)3, where L is an optionally substituted -CH2-CH2-, and each R is independently as described herein and is not -H. 6 In some embodiments, each R is independently C 1~6 In some embodiments, each R is independently an optionally substituted C 1~6 In some embodiments, -L-Si(R) is -CHCHSi(Me). In some embodiments, -L-Si(R) is -CHCHSi(Me). In some embodiments, -L-Si(R) is -CHCHSi(Me). In some embodiments, -L-Si(R) is -CHCHSi(Me). 6 The protected hypoxanthine-containing compounds were synthesized by the corresponding O 6 They have increased solubility compared to unprotected compounds and may offer various benefits and advantages when utilized in oligonucleotide synthesis in accordance with the present disclosure. [ka] or a salt thereof, R NS is (e.g., by -CH2CH2Si(Me)3) O 6 In some embodiments, R NS O 6 In some embodiments, R is a protected inosine. NS O 6 In some embodiments, R is a protected deoxyinosine. NS O 6 In some embodiments, R is a protected 2'-F inosine. NS O 6 and protected 2'-OR modified inosine, where the 2'-OR modification is as described herein (e.g., 2'-OMe, 2'-MOE, etc.). In particular, the present disclosure provides that such compounds have sufficient solubility for and can be utilized in oligonucleotide synthesis, and that O 6It encompasses the recognition that the corresponding unprotected compound may not have sufficient solubility for efficient oligonucleotide synthesis. In some embodiments, the phosphoramidite is (1S,3S,3aS)-1-(((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(2-(trimethylsilyl)ethoxy)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-3-((methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole. In some embodiments, the phosphoramidite is (1S,3S,3aS)-1-(((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(2-(trimethylsilyl)ethoxy)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-3-((phenylsulfonyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole. In some embodiments, [ka] or a salt thereof, R NS O 6 In some embodiments, R is unprotected hypoxanthine. NS is an optionally substituted or protected inosine, and hypoxanthine is unprotected. In some embodiments, R NS is an optionally substituted or protected deoxyinosine, and hypoxanthine is unprotected. In some embodiments, R NS is an optionally substituted or protected 2'-F inosine, and hypoxanthine is unprotected. NSis an optionally substituted or protected 2'-OR modified inosine, and hypoxanthine is unprotected, the 2'-OR modification being as described herein (e.g., 2'-OMe, 2'-MOE, etc.). In particular, the present disclosure provides that such compounds have sufficient solubility for oligonucleotide synthesis and that O 6 This includes the recognition that the present invention can be utilized in oligonucleotide synthesis without the use of a nucleotide sequence.

[0249] In some embodiments, the method includes providing a DPSE and / or a PSM phosphoramidite or a salt thereof. In some embodiments, the provided method includes contacting the DPSE and / or PSM phosphoramidite or a salt thereof with an -OH (e.g., the 5'-OH of a nucleoside or oligonucleotide strand). As one of skill in the art will appreciate, the contacting can be carried out under a variety of suitable conditions to form a phosphorus bond. In some embodiments, the preparation of each chirality-controlled internucleotide linkage independently includes contacting a DPSE or PSM phosphoramidite or a salt thereof with an -OH (e.g., the 5'-OH of a nucleoside or oligonucleotide strand). In some embodiments, the preparation of each chirality-controlled phosphorothioate internucleotide linkage independently includes contacting a DPSE phosphoramidite or a salt thereof with an -OH (e.g., the 5'-OH of a nucleoside or oligonucleotide strand). In some embodiments, the preparation of each chirality-controlled non-negatively charged internucleotide linkage (e.g., n001) independently comprises contacting a PSM phosphoramidite or a salt thereof with an -OH (e.g., the 5'-OH of a nucleoside or oligonucleotide chain). In some embodiments, the preparation of each chirality-controlled internucleotide linkage independently comprises contacting a PSM phosphoramidite or a salt thereof with an -OH (e.g., the 5'-OH of a nucleoside or oligonucleotide chain). In some embodiments, the contacting comprises contacting two sugars and a chiral auxiliary moiety (e.g., [ka] or a salt form thereof (e.g., derived from a DPSE phosphoramidite or a salt thereof), [ka] or a salt form thereof (wherein R AU are independently as described herein; R AU When is -Ph, a P(III) bond is formed that includes a phosphorus atom bonded to a P(V) bond, such as, for example, derived from a PSM phosphoramidite or a salt thereof. In some embodiments, the oligonucleotide includes a P(III) bond that includes a chiral auxiliary moiety, such as, for example, derived from a DPSE or a PSM phosphoramidite. In some embodiments, the P(III) bond that includes a chiral auxiliary moiety is chiral controlled. In some embodiments, the chiral auxiliary moiety can be protected, for example, before converting the P(III) bond to a P(V) bond (e.g., before sulfurization, reaction with an azide, etc.). In some embodiments, the protected chiral auxiliary is [ka] or a salt form thereof (e.g., where R' is independently as described herein; e.g., derived from a DPSE phosphoramidite or salt thereof), or [ka] or a salt form thereof, wherein each R′ and R AU are independently as described herein; R AU In some embodiments, R' is -C(O)R, where R is as described herein. In some embodiments, R is -CH. In some embodiments, the oligonucleotide comprises a protected asymmetric auxiliary. In some embodiments, each chiral controlled internucleotide linkage in the oligonucleotide is independently [ka] or a salt form thereof, or [ka] or a salt form thereof. In some embodiments, each chiral controlled internucleotide linkage in the oligonucleotide is independently: [ka] or a salt form thereof. In some embodiments, R' is -C(O)R. In some embodiments, R' is -C(O)CH. In some embodiments, R AU In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PIII-1), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PIII-2), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PIII-5), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PIII-6), wherein each variable is independently as described herein. In some embodiments, the 5'-terminal internucleotide linkage is PIII-1, PIII-2, PIII-5, or PIII-6. In some embodiments, the 5'-terminal internucleotide linkage is PIII-1 or PIII-2. In some embodiments, R' is -H. In some embodiments, R' is -C(O)R. In some embodiments, R' is -C(O)CH3. In some embodiments, R AU is -Ph. In some embodiments, the P(III) bond is converted to a P(V) bond. In some embodiments, the P(V) bond is converted to a P(V) bond. In some embodiments, the P(V) bond is converted to a P(V) bond between two sugars, a chiral auxiliary moiety (e.g., [ka] or a salt form thereof (wherein R' is as described herein; e.g., derived from a DPSE phosphoramidite or a salt thereof); [ka] or a salt form thereof (wherein R′ and R AU each is independently as described herein; R AU When is -Ph, for example, derived from a PSM phosphoramidite or a salt thereof), and S or [ka] In some embodiments, the P(V) bond comprises a phosphorus atom bonded to two sugars, [ka] or a salt form thereof, wherein each R′ and R AU are independently as described herein; R AU When is -Ph, for example, derived from a PSM phosphoramidite or a salt thereof), and S or [ka] In some embodiments, the P(V) bond comprises a phosphorus atom bonded to two sugars, [ka] or a salt form thereof, wherein each R′ and R AU are independently as described herein; R AU When is -Ph, the P(V) bond includes a phosphorus atom bonded to two sugars, e.g., from a PSM phosphoramidite or salt thereof, and S. In some embodiments, the P(V) bond is formed by bonding two sugars, [ka] or a salt form thereof, wherein each R′ and R AU are independently as described herein; R AU When is -Ph, for example, derived from a PSM phosphoramidite or a salt thereof), and [ka] Those skilled in the art will recognize that [ka] is a counterion (e.g., in some embodiments, PF6 - In some embodiments, the oligonucleotide may be present with one or more [ka] or a salt form thereof (PV-1), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PV-2), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PV-3), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PV-4), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PV-5), wherein each variable is independently as described herein. In some embodiments, the oligonucleotide comprises one or more [ka] or a salt form thereof (PV-6), wherein each variable is independently as described herein. In some embodiments, each chiral internucleotide linkage or each chiral-controlled internucleotide linkage of the oligonucleotide is independently selected from PIII-1, PIII-2, PIII-5, PIII-6, PV-1, PV-2, PV-3, PV-4, PV-5, and PV-6. In some embodiments, each chiral internucleotide linkage or each chiral-controlled internucleotide linkage of the oligonucleotide is independently selected from PIII-1, PIII-2, PV-1, PV-2, PV-3, and PV-4. In some embodiments, the PIII-1, PIII-2, PIII-5, or PIII-6 linkage is typically the 5'-terminal internucleotide linkage. In some embodiments, each chiral internucleotide linkage or each chiral-controlled internucleotide linkage of the oligonucleotide is independently selected from PV-1, PV-2, PV-3, PV-4, PV-5, and PV-6. In some embodiments, each chiral internucleotide linkage or each chiral-controlled internucleotide linkage of the oligonucleotide is independently selected from PV-1, PV-2, PV-3, or PV-4. In some embodiments, the provided oligonucleotide is an oligonucleotide as described herein (e.g., in Table 1), wherein each *S is independently replaced with PV-3 or PV-5, each *R is independently replaced with PV-4 or PV-6, each n001R is independently replaced with PV-1, and each n001S is independently replaced with PV-2. In some embodiments, the provided oligonucleotide is an oligonucleotide as described herein (e.g., in Table 1), wherein each *S is independently replaced with PV-3, each *R is independently replaced with PV-4, each n001R is independently replaced with PV-1, and each n001S is independently replaced with PV-2. In some embodiments, each native phosphate linkage is independently a precursor, e.g., [ka] In some embodiments, R' is -H. In some embodiments, R' is -C(O)R. In some embodiments, R' is -C(O)CH. In some embodiments, R AU is -Ph. In some embodiments, the method comprises removing one or more chiral auxiliary moieties such that a phosphorothioate and / or non-negatively charged internucleotide linkage (e.g., n001) is formed (e.g., from V-1, PV-2, PV-3, PV-4, PV-5, PV-6, etc.). In some embodiments, removing the chiral auxiliary (e.g., PSM) comprises contacting the oligonucleotide with a base (e.g., N(R)3, such as DEA) under anhydrous conditions.

[0250] In some embodiments, as will be understood by one of skill in the art, for the preparation of chirality-controlled internucleotide linkages, monomers or phosphoramidites (e.g., DPSE or PSM phosphoramidites) are typically utilized in chirally enriched or pure form (e.g., purity as described herein (e.g., about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or about 100%)). In some embodiments, the present disclosure provides useful reagents for the preparation of oligonucleotides and compositions thereof. In some embodiments, the monomers and phosphoramidites comprise nucleosides, nucleobases, and sugars as described herein. In some embodiments, the nucleobases and sugars are appropriately protected for oligonucleotide synthesis, as will be understood by one of skill in the art. In some embodiments, the phosphoramidites are R NS -P(OR)N(R)2 (wherein R NS is an optionally protected nucleoside moiety. In some embodiments, the phosphoramidite has the structure NSIn some embodiments, the phosphoramidite has the structure -P(OCH2CH2CN)N(i-Pr)2. In some embodiments, the phosphoramidite comprises a chiral auxiliary moiety, wherein the phosphorus is bonded to an oxygen and a nitrogen atom of the chiral auxiliary moiety. In some embodiments, the phosphoramidite has the structure [ka] or a salt thereof (wherein, R NS is a protected nucleoside moiety (e.g., a 5'-OH and / or nucleobase suitably protected for oligonucleotide synthesis), and each other variable is independently as described herein. In some embodiments, the phosphoramidite has the structure: [ka] (In the formula, R NS is a protected nucleoside moiety (e.g., a 5′-OH and / or nucleobase suitably protected for oligonucleotide synthesis), and R C1 is R, -Si(R)3 or -SO2R, and R C2 and R C3 and, taken together with their intervening atoms, form an optionally substituted 3- to 7-membered saturated ring having 0-2 heteroatoms in addition to the nitrogen atom, and the coupling forms an internucleotide bond. NS The 5'-OH of R is protected. NS In some embodiments, the 5'-OH of R is protected as -ODMTr. NS is attached to phosphorus through its 3'-O-. In some embodiments, R C2 and R C3 is an optionally substituted five-membered ring. In some embodiments, the phosphoramidite is [ka] or a salt thereof. In some embodiments, the phosphoramidite has the structure: [ka] In some embodiments, R NS comprises modified nucleobases (e.g., b001A, b002A, b003A, b008U, b001C, etc.) that are optionally protected for oligonucleotide synthesis. In some embodiments, each -OH is optionally and independently substituted or protected. In some embodiments, BA s is an optionally substituted or protected nucleobase, wherein each -OH of the nucleobase is independently protected, with at least one -OH being protected as DMTrO-. In some embodiments, the -OH intended for coupling, e.g., with another monomer or phosphoramidite, is protected as DMTrO-. In some embodiments, the -OH intended for coupling, e.g., with another monomer or phosphoramidite, is protected differently from the -OH group not intended for coupling. In some embodiments, the non-coupling -OH is protected such that it remains protected when DMTrO- is deprotected. In some embodiments, the non-coupling -OH is protected such that it remains protected during the oligonucleotide synthesis cycle. In some embodiments, BA s is an optionally protected nucleobase selected from A, T, C, G, U, u, hypoxanthine, and tautomers thereof. In some embodiments, R NS comprises an optionally substituted or protected nucleobase as described herein or a tautomer thereof and a sugar as described herein.

[0251] In some embodiments, the purity or stereochemical purity of the monomers or phosphoramidites is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, it is at least 85%. In some embodiments, it is at least 90%. In some embodiments, it is at least 95%.

[0252] In some embodiments, the present disclosure provides methods for preparing an oligonucleotide or composition, the methods comprising coupling a free -OH (e.g., a free 5'-OH) of an oligonucleotide or nucleoside with a monomer as described herein. In some embodiments, the present disclosure provides methods for preparing an oligonucleotide or composition, the methods comprising coupling a free -OH (e.g., a free 5'-OH) of an oligonucleotide or nucleoside with a phosphoramidite as described herein.

[0253] In some embodiments, the present disclosure provides an oligonucleotide comprising: -O 5 -P L (W)(R CA )-O 3 -(In the formula, P L is P or P(=W); W is O, S, or W N and; W N is =NC(-N(R 1 )2=N + (R 1 )2Q - and; Q - is an anion; R CA is or comprises an optionally capped chiral auxiliary moiety; O 5 is the oxygen attached to the 5'-carbon of the sugar, and O 3 is an oxygen attached to the 3'-carbon of the sugar).

[0254] In some embodiments, the modified internucleotide linkage is optionally chiral controlled. In some embodiments, the modified internucleotide linkage is optionally chiral controlled.

[0255] In some embodiments, provided methods comprise converting such modified internucleotide linkages into R CA In some embodiments, after the removal, R CA The bond to is replaced with -OH. In some embodiments, after removal, R CA The bond to is replaced by =O, and W N The bond to -N=C(N(R 1 )2)2 can be replaced by

[0256] In some embodiments, P L is P=S and R CA When is removed, such an internucleotide linkage is converted to a phosphorothioate internucleotide linkage.

[0257] In some embodiments, P L is P=W N and R CA is removed, such an internucleotide bond becomes [ka] In some embodiments, the internucleotide bond is converted to an internucleotide bond having the structure: [ka] The internucleotide bond has the structure [ka] In some embodiments, [ka] The internucleotide bond has the structure [ka] It has the following structure.

[0258] In some embodiments, P Lis P (e.g., at the newly formed internucleotide linkage from coupling of the phosphoramidite with the 5'-OH). In some embodiments, W is O or S. In some embodiments, W is S (e.g., after sulfurization). In some embodiments, W is O (e.g., after oxidation). In some embodiments, certain non-negatively charged or neutral internucleotide linkages are obtained by coupling a P(III) phosphite triester internucleotide linkage with an azidoimidazolium salt (e.g., [ka] In some embodiments, the azidoimidazolium salt can be prepared by reacting PF6 with a compound containing PF6 under suitable conditions. - In some embodiments, the azidoimidazolium salt is a salt of [ka] In some embodiments, the azidoimidazolium salt is 2-azido-1,3-dimethylimidazolium hexafluorophosphate.

[0259] As will be appreciated by those skilled in the art, Q - Q can be a variety of suitable anions present in a system (e.g., in oligonucleotide synthesis) and can vary during the oligonucleotide preparation process depending on the cycle, process step, reagents, solvents, etc. In some embodiments, Q - PF6 - is.

[0260] In some embodiments, R CA teeth, [ka] (In the formula, R C4 is —H or —C(O)R′, and each other variable is independently as described herein. In some embodiments, R CA teeth, [ka] (In the formula, R C1 is R, -Si(R)3 or -SO2R, and R C2 and R C3 together with their intervening atoms to form an optionally substituted 3- to 7-membered saturated ring having 0-2 heteroatoms in addition to the nitrogen atom, and R C4 is —H or —C(O)R′). In some embodiments, R C4 is —H. In some embodiments, R C4 is —C(O)CH. In some embodiments, R C2 and R C3 taken together form an optionally substituted 5-membered ring.

[0261] In some embodiments, R C4 is —H (e.g., at the newly formed internucleotide linkage from the coupling of the phosphoramidite with the 5′-OH). In some embodiments, R C4 is —C(O)R (e.g., after amine capping). In some embodiments, R is methyl.

[0262] In some embodiments, each chiral-controlled phosphorothioate internucleotide linkage is independently selected from the group consisting of -O 5 -P L (W)(R CA )-O 3 - is converted from

[0263] In some embodiments, a linker (e.g., L001) is incorporated through the ring by coupling with a suitable phosphoramidite. In some embodiments, an additional chemical moiety (e.g., Mod001) is coupled to a linker (e.g., L001). In some embodiments, an additional chemical moiety, or an additional chemical moiety and a linker, is incorporated through the ring by coupling with a phosphoramidite comprising the additional chemical moiety, or the additional chemical moiety and a linker, respectively.

[0264] Evaluation / characterization of the technology provided As will be understood by those skilled in the art, various techniques can be used to evaluate / characterize the provided techniques according to the present disclosure.Certain useful techniques are described in the examples;As demonstrated, in particular, the present disclosure describes various in vivo and in vitro techniques suitable for evaluating and characterizing the provided techniques.In some embodiments, the provided techniques are evaluated / characterized, for example, in cells, with or without exogenous ADAR polypeptide;In addition or alternatively, in some embodiments, the provided techniques are evaluated / characterized, for example, in animals (for example, non-human primates and mice).

[0265] In some embodiments, cells and non-human animals are engineered to express a human ADAR1 polypeptide or a characteristic portion thereof. In some embodiments, such cells and humans are useful for evaluating and characterizing the provided technologies. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises a human ADAR1 polypeptide or a characteristic portion thereof. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises a human ADAR1 p110 polypeptide or a characteristic portion thereof. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises a human ADAR1 p150 polypeptide or a characteristic portion thereof. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises human ADAR1. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises a human ADAR1 p110 peptide. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises a human ADAR1 p150 peptide. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises one or more or all of the following domains of human ADAR1: the Z-DNA binding domain, the dsRNA binding domain, and the deaminase domain. In some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises one or both of the human ADAR1 Z-DNA binding domains; alternatively, or in addition, in some embodiments, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises one, two, or all of the human ADAR1 dsRNA binding domains; alternatively, or in addition, the human ADAR1 polypeptide or a characteristic portion thereof is or comprises the human deaminase domain.In some embodiments, a human ADAR1 polypeptide or a characteristic portion thereof may be expressed together with a mouse ADAR1 polypeptide or a characteristic portion thereof; for example, one or more human dsRNA-binding domains may be engineered to be expressed together with a mouse deaminase domain to form a human-mouse hybrid ADAR1 polypeptide. In some embodiments, the cell and / or non-human animal has been engineered to contain and / or express a polynucleotide encoding a human ADAR1 polypeptide or a characteristic portion thereof as described herein. In some embodiments, the genome of the cell and / or non-human animal has been engineered to contain a polynucleotide encoding a human ADAR1 ...

Claims

1. an oligonucleotide having the structure Mod001L001mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof (wherein Mod001 is, 【Chemical 1】 L001: Mod001 via -NH- and -NH-(CH ２ ) ６ - represents; f represents a 2'-F modification to the nucleoside; m represents a 2'-OMe modification to the nucleoside; eo is the 2'-OCH ２ CH ２ OCH ３ represents a modification; m5Ceo represents 5-methyl 2'-O-methoxyethyl C; n001R represents an Rp n001 bond; n001S represents an Sp n001 bond; The n001 bond is 【Chemistry 2】 having the structure *S represents an Sp phosphorothioate bond; I represents a nucleobase that is hypoxanthine; and b008U is a base 【Chemistry 3】 represents a nucleoside.

2. The diastereomeric purity of the oligonucleotide is at least (DS) ｎｃ 2. The oligonucleotide of claim 1, wherein DS is 85% to 100% and n c is the number of chiral binding phosphorus.

3. 2. The oligonucleotide of claim 1, wherein the diastereomeric excess of each chiral phosphorus center is independently at least 85%.

4. The oligonucleotide of claim 2 , wherein the oligonucleotide is in a pharmaceutically acceptable salt form.

5. The oligonucleotide of claim 2 , wherein the oligonucleotide is in sodium salt form.

6. A compound having the structure of Formula A-2 or a pharmaceutically acceptable salt thereof: 【Chemistry 4】

7. The diastereomeric purity of the compound is at least (DS) ｎｃ 7. The compound according to claim 6, wherein DS is 90% to 100% and n c is the number of chiral bonded phosphorus.

8. 7. The compound of claim 6, wherein the diastereomeric excess at each chiral bonded phosphorus center is independently at least 90%.

9. 8. The compound of claim 7, wherein the compound is a sodium salt.

10. 7. The compound of claim 6, wherein the compound is a sodium salt, and each phosphorothioate group and phosphate group is independently present in sodium salt form.

11. an oligonucleotide having the structure: mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof (wherein: f represents a 2'-F modification to the nucleoside; m represents a 2'-OMe modification to the nucleoside; eo is the 2'-OCH ２ CH ２ OCH ３ represents a modification; m5Ceo represents 5-methyl 2'-O-methoxyethyl C; n001R represents an Rp n001 bond; n001S represents an Sp n001 bond; The n001 bond is 【Chemistry 5】 having the structure *S represents an Sp phosphorothioate bond; I represents a nucleobase that is hypoxanthine; and b008U is a base 【Chemistry 6】 represents a nucleoside.

12. The diastereomeric purity of the oligonucleotide is at least (DS) ｎｃ 12. The oligonucleotide of claim 11, wherein DS is 85% to 100% and n c is the number of chiral binding phosphorus.

13. 12. The oligonucleotide of claim 11, wherein the diastereomeric excess of each chiral binding phosphorus center is independently at least 85%.

14. The oligonucleotide of claim 2 , wherein the oligonucleotide is in a pharmaceutically acceptable salt form.

15. The oligonucleotide of claim 2 , wherein the oligonucleotide is in sodium salt form.

16. A compound having the structure of Formula B-2 or a pharmaceutically acceptable salt thereof: 【Chemistry 7】

17. The diastereomeric purity of the compound is at least (DS) ｎｃ 17. The compound of claim 16, wherein DS is 90% to 100% and n c is the number of chiral bonded phosphorus.

18. 17. The compound of claim 16, wherein the diastereomeric excess at each chiral bonded phosphorus center is independently at least 90%.

19. 18. The compound of claim 17, wherein the compound is a sodium salt.

20. 17. The compound of claim 16, wherein the compound is a sodium salt, and each phosphorothioate group and phosphate group is independently present in sodium salt form.

21. A conjugate of an oligonucleotide and an additional chemical moiety or a salt thereof, the oligonucleotide is mCn001RmC*SmC*SfA*SfG*Sm5CeoAeofG*SfC*SmUmUn001RmCfA*SfGn001RfUm5Ceo*SfC*SmCmUn001RmUTeofC*ST*Sb008U*SIn001SmUfC*SmG*SmAn001RmU, or a salt thereof (wherein f represents a 2'-F modification to the nucleoside; m represents a 2'-OMe modification to the nucleoside; eo is the 2'-OCH ２ CH ２ OCH ３ represents a modification; m5Ceo represents 5-methyl 2'-O-methoxyethyl C; n001R represents an Rp n001 bond; n001S represents an Sp n001 bond; The n001 bond is 【Chemistry 8】 having the structure *S represents an Sp phosphorothioate bond; I represents a nucleobase that is hypoxanthine; and b008U is a base 【Chemistry 9】 represents a nucleoside.

22. A conjugate of an oligonucleotide according to any one of claims 11 to 15 or a compound according to any one of claims 16 to 20 with an additional chemical moiety or a salt thereof.

23. 22. The conjugate of claim 21, wherein the additional chemical moiety is a targeting moiety.

24. 22. The conjugate of claim 21, wherein the additional chemical moiety is a carbohydrate moiety.

25. 22. The conjugate of claim 21, wherein the additional chemical moiety targets the liver.

26. 22. The conjugate of claim 21, wherein the additional chemical moiety comprises a ligand for the asialoglycoprotein receptor.

27. 22. The conjugate of claim 21 , wherein the additional chemical moiety comprises GalNAc.

28. said additional chemical moiety being 【Chemistry 10】 22. The conjugate of claim 21, wherein:

29. said additional chemical moiety being 【Chemistry 11】 or said additional chemical moiety being 【Chemistry 12】 22. The conjugate of claim 21, wherein:

30. 30. The conjugate of any one of claims 21 and 23 to 29, wherein the additional chemical moiety is conjugated to the oligonucleotide via a linker.

31. 31. The conjugate of claim 30, wherein the linker comprises L001.

32. The conjugate of any one of claims 21 and 23 to 29, wherein the additional chemical moiety is conjugated to the 5' end of the oligonucleotide.

33. 30. The conjugate of any one of claims 21 and 23 to 29, in the form of a pharmaceutically acceptable salt.

34. 30. The conjugate of any one of claims 21 and 23 to 29, which is in sodium salt form.

35. A pharmaceutical composition comprising or delivering an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, and a pharmaceutically acceptable carrier.

36. 36. The pharmaceutical composition of claim 35, wherein the oligonucleotide, the compound, or the conjugate is in a pharmaceutically acceptable salt form.

37. 36. The pharmaceutical composition of claim 35, wherein the oligonucleotide or the conjugate is in sodium salt form.

38. 36. The pharmaceutical composition of claim 35, comprising two or more pharmaceutically acceptable salt forms of the oligonucleotide or conjugate.

39. 36. The pharmaceutical composition of claim 35, which is a liquid.

40. 36. The pharmaceutical composition of claim 35, which is an oligonucleotide or conjugate dissolved in a buffer solution.

41. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1 to 5 and 11 to 15, the compound according to any one of claims 6 to 10 and 16 to 20, and a pharmaceutically acceptable carrier.

42. A composition comprising a plurality of oligonucleotides, each of the plurality of oligonucleotides being independently a specific oligonucleotide or a salt thereof, the specific oligonucleotide being the oligonucleotide of any one of claims 1 to 5 and 11 to 15, the compound of any one of claims 6 to 10 and 16 to 20, or the conjugate of any one of claims 21 and 23 to 29, a composition wherein 5% to 100% of all oligonucleotides in the composition share a particular oligonucleotide base sequence; The level of oligonucleotides in the composition that share a plurality of structures among the plurality of oligonucleotides is at least (DS) ｎｃ wherein DS is 95% to 100% and n is the number of chiral internucleotide linkages; a composition in which the diastereomeric excess of one or more chiral phosphorus centers is independently at least 96%; and / or A composition wherein the diastereomeric excess at each chiral bonded phosphorus center is independently at least 91%.

43. 43. The composition of claim 42, wherein the specific oligonucleotide is in a pharmaceutically acceptable salt form.

44. 43. The composition of claim 42, which is a pharmaceutical composition.

45. Either of the following methods: 1) A method for modifying a target adenosine in a target nucleic acid, comprising contacting the target nucleic acid with an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the target adenosine is 1024 G>A in human SERPINA1; or 2) A method for deaminating a target adenosine in a target nucleic acid, comprising contacting the target nucleic acid with an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the target adenosine is 1024 G>A in human SERPINA1; or 3) A method for producing a product of, or restoring or increasing the level of, a specific nucleic acid, comprising contacting a target nucleic acid with an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the target nucleic acid comprises a target adenosine, and the specific nucleic acid differs from the target nucleic acid in having an I or G instead of the target adenosine, and the target nucleic acid is a human SERPINA1 transcript with a 1024 G>A mutation, and the target adenosine is 1024 G>A in human SERPINA1; or 4) A method for reducing the level of a product of a target nucleic acid, comprising contacting the target nucleic acid with an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the target nucleic acid comprises a target adenosine, and the target nucleic acid is a human SERPINA1 transcript with a 1024 G>A mutation, and the target adenosine is 1024 G>A in human SERPINA1; or ５） contacting an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29 with a sample comprising a target nucleic acid and adenosine deaminase, wherein the target nucleic acid comprises a target adenosine; the target nucleic acid is a human SERPINA1 transcript having a 1024 G>A mutation, and the target adenosine is 1024 G>A in human SERPINA1; and a method wherein the target adenosine is modified; or 6) A method for reducing Z-AAT in the liver of a subject, comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 7) A method for reducing Z-AAT in the liver of a subject, comprising delivering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 8) A method for reducing liver inflammation in a subject, comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 9) A method for reducing liver inflammation in a subject, comprising delivering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 10) A method for inhibiting elastase in a subject, comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 11) A method for inhibiting elastase in a subject, comprising delivering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 12) A method for increasing the level and / or activity of alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject, comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 13) A method for increasing the level and / or activity of alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject, comprising delivering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1; or 14) A method for reducing the level and / or activity of a mutant alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject, comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1. 15) A method for reducing the level and / or activity of a mutant alpha-1 antitrypsin (A1AT) polypeptide in the serum or blood of a subject, comprising delivering to the subject an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1.

46. 30. A method for preventing a condition, disorder or disease, comprising administering or delivering to a subject susceptible to or suffering from the same an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1.

47. 30. A method for treating a condition, disorder or disease, comprising administering or delivering to a subject susceptible to or suffering from the same an effective amount of an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1.

48. 30. A method for treating alpha-1 antitrypsin deficiency, comprising administering or delivering to a subject an effective amount of the oligonucleotide of any one of claims 1 to 5 and 11 to 15, the compound of any one of claims 6 to 10 and 16 to 20, or the conjugate of any one of claims 21 and 23 to 29, wherein the subject comprises a 1024 G>A (E342K) mutation in human SERPINA1.

49. 49. The method of claim 48, wherein the subject is a PiZZ carrier.

50. A method for delivering an oligonucleotide according to any one of claims 11 to 15, or a compound according to any one of claims 16 to 20, to a system, the method comprising administering to said system a conjugate of said oligonucleotide or said compound with an additional chemical moiety or a salt thereof; or 19. A method for delivering an oligonucleotide according to any one of claims 11 to 15, a compound according to any one of claims 16 to 20 to a system, the method comprising administering to said system an oligonucleotide according to any one of claims 1 to 5, a compound according to any one of claims 6 to 10, or a conjugate according to any one of claims 21 and 23 to 29.

51. A method for preparing an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, or a composition thereof, the method comprising coupling a phosphoramidite comprising an asymmetric auxiliary with a hydroxyl group; or A method for preparing an oligonucleotide according to any one of claims 1 to 5 and 11 to 15, a compound according to any one of claims 6 to 10 and 16 to 20, or a conjugate according to any one of claims 21 and 23 to 29, or a composition thereof, comprising: coupling a phosphoramidite with a hydroxyl group, wherein the phosphoramidite is 【Chemistry 13】 or a salt thereof, R ＮＳ is an optionally protected nucleoside moiety; R Ｃ１ is R, -Si(R) ３ or -SO ２ R; R Ｃ２ and R Ｃ３ each is independently R; and Each R is independently —H or C １～２０ Aliphatic, C with 1-10 heteroatoms １～２０ Heteroaliphatic, C ６～２０ Aryl, C ６～２０ Arylaliphatic, C with 1-10 heteroatoms ６～２０ an optionally substituted group selected from arylheteroaliphatic, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms, or: two R groups optionally and independently combine to form a covalent bond, or two or more R groups on the same atom optionally and independently combine with said atom to form an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to said atom; or two or more R groups on two or more atoms optionally and independently combine with their intervening atoms to form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to said intervening atoms; or 30. A method for preparing an oligonucleotide according to any one of claims 1 to 5, a compound according to any one of claims 6 to 10, or a conjugate according to any one of claims 21 and 23 to 29, or a composition thereof, comprising coupling with a phosphoramidite comprising an optionally substituted additional chemical moiety.

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