Oligonucleotide compositions and methods thereof

Abstract

To provide designed DMD oligonucleotides, compositions, and methods of use thereof.SOLUTION: In some embodiments, the present disclosure provides a technique useful for repairing a mutant DMD transcript by skipping exon 51 such that a transcript can be translated into internally truncated but at least partially functional dystrophin protein variant. In some embodiments, the present disclosure provides a technique useful for adjusting DMD transcript splicing. In some embodiments, provided technique can change splicing of dystrophin (DMD) DMD transcript. In some embodiments, the present disclosure provides a method for treating diseases, such as muscular dystrophy including, but not limited to Duchenne muscular dystrophy, Becker's muscular dystrophy or the like.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos. 62 / 776,432, filed December 6, 2018, 62 / 916,192, filed October 16, 2019, and 62 / 916,194, filed October 16, 2019, and PCT application PCT / U.S. Patent Application Publication No. 2019 / 027109, filed April 11, 2019, published on October 17, 2019 as WO 2019 / 200185, and PCT application PCT / U.S. Patent Application Publication No. 2019 / 031672, filed May 10, 2019, published on November 14, 2019 as WO 2019 / 217784, each of which is incorporated by reference in its entirety. [Background technology]

[0002] background Oligonucleotides are useful in therapeutic, diagnostic, research and nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) as therapeutic agents can be limited due to, for example, their instability to extracellular and intracellular nucleases and / or their lack of cellular penetration and distribution. There is a need for new and improved oligonucleotides and oligonucleotide compositions, including novel oligonucleotides and oligonucleotide compositions suitable for the treatment of various diseases. Summary of the Invention [Means for solving the problem]

[0003] overview In particular, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications and patterns of sugars, bases and / or internucleotide linkages), and / or stereochemistry (e.g., stereochemistry and / or patterns of backbone chiral centers (chiral internucleotide linkages)) can greatly affect oligonucleotide properties, such as exon skipping (e.g., exon 51 of DMD), toxicity, stability, protein binding properties, etc.

[0004] In some embodiments, the present disclosure provides an oligonucleotide or oligonucleotide composition that can mediate skipping of an exon, such as exon 51, of the DMD gene and is useful for treating muscular dystrophy. In some embodiments, the oligonucleotide or oligonucleotide composition is useful for treating muscular dystrophy. In some embodiments, the oligonucleotide or oligonucleotide composition is a DMD oligonucleotide or DMD oligonucleotide composition that is a DMD oligonucleotide or DMD oligonucleotide composition disclosed herein (e.g., Table A1).

[0005] In some embodiments, as demonstrated herein, the provided technologies (e.g., oligonucleotides, compositions, methods, etc.) are particularly useful for reducing levels of mutant mRNA (e.g., DMD transcripts containing a deleterious mutation) and / or the protein encoded thereby, and for increasing levels of repaired mRNA (e.g., DMD transcripts in which a deleterious mutation has been deleted, corrected, or compensated for by skipping exon 51) and / or the protein encoded thereby.

[0006] In some embodiments, the technology provided provides a method for modulating splicing of a DMD transcript. In some embodiments, the provided technology is particularly useful for reducing the levels of DMD transcripts, e.g., pre-mRNA, RNA, etc., and often for reducing the levels of products, such as mRNA, proteins, etc., that arise from or are encoded by such DMD transcripts. In some embodiments, the pre-mRNA, or mRNA, or RNA, has been transported from one subcellular compartment (e.g., nucleus, cytoplasm, etc.) to another and / or has been modified by one or more enzymes.

[0007] For example, in some embodiments, a dystrophin gene can include an exon that contains one or more mutations associated with muscular dystrophy (including, but not limited to, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD)). In some embodiments, the disease-associated exon contains a mutation in the exon (e.g., a missense mutation, a frameshift mutation, a nonsense mutation, a premature stop codon, etc.). In some embodiments, the present disclosure provides compositions and methods for effectively skipping one or more disease-associated dystrophin exons while maintaining or restoring the reading frame such that a shorter (e.g., internally truncated), yet partially functional dystrophin (e.g., mutant) can be produced.

[0008] In particular, the present disclosure demonstrates that chemical modifications and / or stereochemistry can be used to modulate DMD transcript splicing with DMD oligonucleotide compositions. In some embodiments, the present disclosure provides combinations of chemical modifications and stereochemistry that improve the properties of the DMD oligonucleotide, such as its ability to alter splicing of the DMD transcript. In some embodiments, the disclosure provides chirality-controlled DMD oligonucleotide compositions that, when compared to a reference condition (e.g., the absence of the composition, the presence of a reference composition (e.g., a stereorandom composition of DMD oligonucleotides having the same chemical constitution (as one of skill in the art will appreciate, unless otherwise indicated, chemical constitution generally refers to a description of the identities and manner of bonding (and corresponding bond multiplicity) of atoms in a molecular entity, but excluding any differences arising from their spatial arrangement), a different chirality-controlled DMD oligonucleotide composition, etc.), combinations thereof, etc.), result in increased skipping of DMD exon 51 resulting in modified (e.g., repaired) mRNA that can be translated to produce an internally truncated, but at least partially functional, dystrophin protein mutant.

[0009] In some embodiments, compared to reference conditions, the chiral controlled oligonucleotide compositions provided are surprisingly effective in some embodiments, enhancing splicing of DMD exon 51 by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100 fold.

[0010] In particular, the present disclosure recognizes that it is a problem to provide oligonucleotide compositions and methods of use thereof that have low toxicity. In some embodiments, the present disclosure provides DMD oligonucleotide compositions and methods that have reduced toxicity. In some embodiments, the present disclosure provides DMD oligonucleotide compositions and methods that have reduced induction of immune responses.

[0011] In some embodiments, the disclosure provides oligonucleotide compositions (e.g., DMD oligonucleotides and compositions) that have enhanced antagonism of hTLR9 activity. In some embodiments, muscular dystrophy is associated, for example, with inflammation of muscle tissue. In some embodiments, the provided technology (e.g., DMD oligonucleotides, compositions, methods, etc.) provides enhanced In some embodiments, the DMD oligonucleotides and / or compositions thereof provide both enhanced activity (e.g., exon skipping activity) and hTLR9 antagonist activity, which may be beneficial in one or more conditions and / or diseases involving inflammation. In some embodiments, the DMD oligonucleotides and / or compositions thereof provide both exon skipping capability and reduced toxicity and / or inflammation levels.

[0012] In some embodiments, the oligonucleotide comprises multiple internucleotide linkages, each independently selected from various types. The various types of internucleotide linkages have different properties. Without wishing to be bound by any theory, the present disclosure points out that natural phosphate linkages (phosphodiester internucleotide linkages) are anionic and may be unstable in vivo when used alone without other chemical modifications; phosphorothioate internucleotide linkages are anionic, generally more stable in vivo than natural phosphate linkages, and in some cases may be more hydrophobic; neutral internucleotide linkages, such as those exemplified in the present disclosure, that contain cyclic guanidine moieties, are neutral at physiological pH, may be more stable in vivo than natural phosphate linkages, and may be more hydrophobic.

[0013] In some embodiments, the oligonucleotide comprises a modified internucleotide linkage that is a non-negatively charged (neutral or cationic) internucleotide linkage at a certain pH (e.g., human physiological pH (about 7.4), the pH of the delivery site (e.g., organelle, cell, tissue, organ, organism, etc.), etc.). Without wishing to be bound by any particular theory, in at least some instances, the neutral internucleotide linkage in the oligonucleotide may result in improved properties and / or skipping of exon 51, such as improved delivery, improved exonuclease and endonuclease resistance, improved cellular uptake, improved endosomal escape, and / or improved nuclear uptake, etc., compared to a comparable nucleic acid that does not comprise a neutral internucleotide linkage.

[0014] In some embodiments, the non-negatively charged internucleotide linkage comprises a cyclic guanidine moiety. In some embodiments, the non-negatively charged internucleotide linkage comprises [ka] or a stereoisomer thereof (e.g., n001R or n001S). In some embodiments, the neutral internucleotide linkage comprising a cyclic guanidine moiety is chiral controlled. In some embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage. In some embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage, at least one natural phosphate linkage, and at least one phosphorothioate internucleotide linkage.

[0015] In particular, the present disclosure encompasses the recognition that stereorandom DMD oligonucleotide formulations include multiple individual chemical entities that differ from one another, for example, in the stereochemical configuration of individual backbone chiral centers within the DMD oligonucleotide strand. In the absence of control of the stereochemistry of the backbone chiral centers, stereorandom DMD oligonucleotide formulations will result in uncontrolled (or stereorandom) oligonucleotides that contain indeterminate levels of DMD oligonucleotide stereoisomers. The present disclosure provides compositions that are chiral controlled compositions that are or contain a specific stereoisomer of a DMD oligonucleotide of interest; in contrast to compositions that are not chiral controlled, a chiral controlled composition contains a controlled level of a specific stereoisomer of a DMD oligonucleotide. In some embodiments, a specific stereoisomer can be defined, for example, by its base sequence, its pattern of backbone bonds, its pattern of backbone chiral centers, and the pattern of backbone phosphorus modifications, etc., even though these stereoisomers may have the same base sequence and / or chemical modifications, at least due to their different backbone stereochemistry. As understood in the art, in some embodiments, a base sequence may simply refer to a sequence of bases, and / or to the identity and / or modification state of a nucleoside residue in a DMD oligonucleotide (e.g., of the sugar and / or base moiety compared to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil), and / or to the hybridization properties of such residues (i.e., the ability to hybridize with a particular complementary residue). In some embodiments, the present disclosure demonstrates that the improved properties (e.g., improved skipping of exon 51, reduced toxicity, etc.) achieved by the inclusion and / or location of a particular chiral structure within a DMD oligonucleotide can be equivalent to or even better than those achieved by using chemical modifications, such as particular backbone linkages, residue modifications, etc. (e.g., by using certain modified phosphates [e.g., phosphorothioates, substituted phosphorothioates, etc.], sugar modifications [e.g., 2'-modifications, etc.], and / or base modifications [e.g., methylation, etc.]). In some embodiments, the present disclosure demonstrates that chiral controlled DMD oligonucleotide compositions of DMD oligonucleotides containing certain chemical modifications (e.g., 2'-F, 2'-OMe, phosphorothioate internucleotide linkages, etc.) demonstrate unexpectedly high exon skipping efficiency.

[0016] In some embodiments, the present disclosure provides: 1) having a common base sequence complementary to a target sequence in the DMD transcript; and 2) containing one or more modified sugar moieties and modified internucleotide linkages; DMD oligonucleotide compositions are provided that include a plurality of DMD oligonucleotides, where the DMD oligonucleotides are DMD oligonucleotides described herein (eg, Table A1).

[0017] In some embodiments, the provided DMD oligonucleotide composition is characterized in that when it is contacted with a DMD transcript in a DMD transcript splicing system, splicing of the DMD transcript is altered (e.g., skipping of exon 51 is increased) relative to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0018] In some embodiments, the reference condition is the absence of the composition. In some embodiments, the reference condition is the presence of a reference composition. Exemplary reference compositions comprising a reference plurality of DMD oligonucleotides are described in detail in the present disclosure. In some embodiments, the reference plurality of DMD oligonucleotides have different structural elements (chemical modifications, stereochemistry, etc.) compared to the plurality of DMD oligonucleotides in the provided composition. In some embodiments, the reference composition is a stereorandom preparation of DMD oligonucleotides having the same chemical modifications. In some embodiments, the reference composition is a mixture of stereoisomers, while the provided composition is a chiral-controlled DMD oligonucleotide composition of one stereoisomer. In some embodiments, the reference plurality of DMD oligonucleotides have the same base sequence, same sugar modification, same base modification, same internucleotide bond modification, and same nucleotide sequence as the plurality of DMD oligonucleotides in the provided composition. and / or have the same stereochemistry but different chemical modifications, e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.

[0019] Exemplary splicing systems are widely known in the art. In some embodiments, the splicing system is an in vivo or in vitro system that includes sufficient components to achieve splicing of the relevant target DMD transcript. In some embodiments, the splicing system is or includes a spliceosome (e.g., protein and / or RNA components thereof). In some embodiments, the splicing system is or includes an organelle membrane (e.g., nuclear membrane) and / or an organelle (e.g., nucleus). In some embodiments, the splicing system is or includes a cell or population thereof. In some embodiments, the splicing system is or includes a tissue. In some embodiments, the splicing system is or includes an organism, e.g., an animal, e.g., a mammal, such as a mouse, rat, monkey, dog, human, etc.

[0020] In some embodiments, the present disclosure provides: 1) Nucleotide sequence; 2) pattern of skeletal bonds; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification wherein the DMD oligonucleotides are DMD oligonucleotides described herein (e.g., Table A1).

[0021] In some embodiments, the present disclosure provides: 1) Nucleotide sequence; 2) pattern of skeletal bonds; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification and providing a DMD oligonucleotide composition comprising a plurality of DMD oligonucleotides of a particular DMD oligonucleotide type defined by: The composition is chiral controlled and is enriched for DMD oligonucleotides of a particular DMD oligonucleotide type as compared to a substantially racemic preparation of the DMD oligonucleotide having the same base sequence; The DMD oligonucleotide composition is characterized in that when it is contacted with a DMD transcript in a DMD transcript splicing system, splicing of the DMD transcript is altered (e.g., skipping of exon 51 is increased) relative to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof, wherein the DMD oligonucleotide is a DMD oligonucleotide described herein (e.g., in Table A1).

[0022] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of the oligonucleotides in Table A1, wherein the oligonucleotides comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) chiral controlled internucleotide linkages (e.g., S, R, nS, or nR), wherein the oligonucleotides are optionally in a pharma- ceutically acceptable salt form. In some embodiments, the oligonucleotides are provided as sodium salts.

[0023] In some embodiments, as described herein, the oligonucleotides share the same chemical structure. In some embodiments, for the chiral controlled internucleotide linkages of the oligonucleotides in the composition, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of all the oligonucleotides in the composition that share the same chemical structure of the oligonucleotides share the same phosphorus arrangement in the chiral controlled internucleotide linkage.

[0024] In some embodiments, the DMD transcript is of the dystrophin gene or a mutant thereof.

[0025] In some embodiments, the present disclosure provides a composition comprising any DMD oligonucleotide disclosed herein.In some embodiments, the present disclosure provides a composition comprising any chiral controlled DMD oligonucleotide disclosed herein.In some embodiments, the present disclosure provides a composition comprising any chiral controlled DMD oligonucleotide disclosed herein, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51.

[0026] In some embodiments, the disclosure relates to any individual DMD oligonucleotide described herein (eg, Table A1).

[0027] In some embodiments, DMD oligonucleotides and / or compositions provided are capable of mediating skipping of exon 51. In some embodiments, non-limiting examples of such DMD oligonucleotides and compositions include those of WV-20011, WV-20052, WV-20059, WV-20072, WV-20073, WV-20074, WV-20075, WV-20076, WV-20096, WV-20097, WV-20101, and WV-20119, as well as other DMD oligonucleotides having a base sequence comprising at least 15 contiguous bases of any of these DMD oligonucleotides.

[0028] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20011 or methods of use thereof.

[0029] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20052 or methods of use thereof.

[0030] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20059 or methods of use thereof.

[0031] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20072 or methods of use thereof.

[0032] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20073 or methods of use thereof.

[0033] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20074 or methods of use thereof.

[0034] In some embodiments, the present disclosure provides a DMD oligonucleotide comprising WV-20075. The present invention relates to a method for the preparation of a compound or oligonucleotide composition and a method for using the same.

[0035] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20076 or methods of use thereof.

[0036] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20096 or methods of use thereof.

[0037] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20097 or methods of use thereof.

[0038] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20101 or methods of use thereof.

[0039] In some embodiments, the disclosure relates to DMD oligonucleotides or oligonucleotide compositions comprising WV-20119 or methods of use thereof.

[0040] In some embodiments, the disclosure relates to methods of making any of the DMD oligonucleotides disclosed herein (eg, Table A1).

[0041] In some embodiments, the disclosure relates to a medicament comprising any of the DMD oligonucleotides disclosed herein (eg, Table A1).

[0042] In some embodiments, in the oligonucleotide sequences herein (including but not limited to, in Table A1), if no sugar is specified, the sugar is a native DNA sugar; and if no internucleotide linkage is specified, the internucleotide linkage is a native phosphate linkage.

[0043] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition of a DMD oligonucleotide selected from any of the present table.

[0044] In some embodiments, the DMD oligonucleotides include internucleotide linkages that are natural phosphate linkages or phosphorothioate internucleotide linkages. In some embodiments, the phosphorothioate internucleotide linkages are not chiral controlled. In some embodiments, the phosphorothioate internucleotide linkages are chiral controlled internucleotide linkages (e.g., Sp or Rp).

[0045] In some embodiments, the DMD oligonucleotide comprises a non-negatively charged internucleotide linkage. In some embodiments, the DMD oligonucleotide comprises a neutral internucleotide linkage. In some embodiments, the neutral internucleotide linkage is or comprises a cyclic guanidine moiety.

[0046] In some embodiments, the internucleotide linkage comprises a guanidine moiety. In some embodiments, the internucleotide linkage comprises a cyclic guanidine moiety. In some embodiments, the internucleotide linkage comprising a cyclic guanidine moiety has the structure n001. In some embodiments, the neutral internucleotide linkage or the internucleotide linkage comprising a cyclic guanidine moiety is stereochemically controlled.

[0047] In general, the properties of DMD oligonucleotide compositions as described herein can be evaluated using any suitable assay. Typically, different compositions (e.g. The relative toxicity and / or protein binding properties of a stereocontrolled composition versus a non-controlled composition and / or another stereocontrolled composition are desirably determined in the same assay, in some embodiments substantially simultaneously, and in some embodiments with reference to previous results.

[0048] Those of skill in the art will know and / or be able to readily develop appropriate assays for a particular DMD oligonucleotide composition. The present disclosure provides a description of certain specific assays that may be useful, for example, in assessing one or more characteristics of DMD oligonucleotide composition behavior, such as complement activation, injection site inflammation, protein binding, and the like.

[0049] For example, certain assays that may be useful in assessing the toxicity and / or protein binding properties of DMD oligonucleotide compositions may include any of the assays described and / or exemplified herein.

[0050] In some embodiments, the disclosure provides DMD oligonucleotide compositions comprising a plurality of DMD oligonucleotides sharing the same base sequence, wherein the plurality of oligonucleotides comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral controlled internucleotide linkages. In some embodiments, the disclosure provides DMD oligonucleotide compositions comprising a plurality of DMD oligonucleotides sharing the same chemical structure, wherein the plurality of oligonucleotides comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral controlled internucleotide linkages. In some embodiments, when the oligonucleotide composition is contacted with a DMD transcript in a DMD transcript splicing system, splicing of the DMD transcript is altered (e.g., skipping of exon 51 is increased) relative to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof. In some embodiments, a splice product and / or protein encoded thereby in which one exon (e.g., exon 51 in some embodiments) has been skipped is provided at an increased level (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 fold or more) compared to the reference conditions.

[0051] In some embodiments, the present disclosure provides: 1) Nucleotide sequence; 2) pattern of skeletal bonds; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification and a DMD oligonucleotide composition comprising a plurality of DMD oligonucleotides of a particular DMD oligonucleotide type defined by: The plurality of oligonucleotides comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral controlled internucleotide linkages; and The DMD oligonucleotide composition is characterized in that when it is contacted with a DMD transcript in a DMD transcript splicing system, splicing of the DMD transcript is altered (e.g., skipping of exon 51 is increased) relative to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0052] In some embodiments, the disclosure provides a method of treating or preventing muscular dystrophy comprising administering to a subject a DMD oligonucleotide composition described herein.

[0053] In some embodiments, the present disclosure provides: 1) having a common base sequence complementary to a target sequence in the DMD transcript; and 2) containing one or more modified sugar moieties and modified internucleotide linkages; A method for treating or preventing muscular dystrophy is provided, comprising administering to a subject a DMD oligonucleotide composition comprising a plurality of DMD oligonucleotides, The DMD oligonucleotide composition is characterized in that when it is contacted with a DMD transcript in a DMD transcript splicing system, splicing of the DMD transcript is altered (e.g., skipping of exon 51 is increased) relative to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof, wherein the DMD oligonucleotide is a DMD oligonucleotide described herein (e.g., in Table A1).

[0054] In some embodiments, the present disclosure provides: 1) Nucleotide sequence; 2) pattern of skeletal bonds; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification The present invention provides a method for treating or preventing muscular dystrophy, comprising administering to a subject a chiral-controlled DMD oligonucleotide composition comprising a plurality of DMD oligonucleotides of a particular DMD oligonucleotide type defined by: The composition is chiral controlled, and is enriched for DMD oligonucleotides of a particular DMD oligonucleotide type as compared to a substantially racemic preparation of the DMD oligonucleotide having the same base sequence; The DMD oligonucleotide composition is characterized in that when it is contacted with a DMD transcript in a DMD transcript splicing system, splicing of the DMD transcript is altered (e.g., skipping of exon 51 is increased) relative to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof, wherein the DMD oligonucleotide is a DMD oligonucleotide described herein (e.g., in Table A1).

[0055] In some embodiments, the oligonucleotide provided comprises at least one, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, non-negatively charged internucleotide linkages, which are optionally and independently chiral controlled. In some embodiments, the oligonucleotide provided comprises chiral controlled non-negatively charged internucleotide linkages. In some embodiments, the non-negatively charged internucleotide linkage is n001.

[0056] In some embodiments, the present disclosure provides: 1) Nucleotide sequence; 2) pattern of skeletal bonds; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification and a DMD oligonucleotide composition comprising a plurality of DMD oligonucleotides of a particular DMD oligonucleotide type defined by: The plurality of oligonucleotides may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral controlled containing internucleotide bonds; and The plurality of oligonucleotides comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotide linkages.

[0057] In some embodiments, in muscular dystrophies, dystrophin function can be restored or at least partially restored by an internally truncated but at least partially functional dystrophin protein mutant following skipping of DMD exon 51.

[0058] In some embodiments, muscular dystrophies include, but are not limited to, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0059] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a DMD oligonucleotide or DMD oligonucleotide composition of the present disclosure and a pharma- ceutically acceptable carrier.

[0060] In some embodiments, the present disclosure provides a method of treating muscular dystrophy, Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), comprising administering to a subject susceptible to or suffering from the same a composition described in the present disclosure.

[0061] In some embodiments, the present disclosure provides a method for treating muscular dystrophy, Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), comprising administering to a subject susceptible to or suffering from a composition comprising any of the DMD oligonucleotides disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising an effective amount of an oligonucleotide, and is chiral controlled. In some embodiments, the oligonucleotide is provided as a salt form, for example, a sodium salt.

[0062] In some embodiments, the disclosure provides a method of treating muscular dystrophy, Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), comprising: (a) administering to a subject susceptible to or suffering from the same a composition comprising any of the DMD oligonucleotides disclosed herein; and (b) administering to the subject an additional treatment capable of preventing, treating, ameliorating, or slowing the progression of at least one symptom of the muscular dystrophy, Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0063] definition As used herein, the following definitions shall apply unless otherwise stated. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M. b. and March, J., John Wiley & Sons, New York: 2001.

[0064] Aliphatic: As used herein, the term “aliphatic” or “aliphatic group” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or that contains one or more units of unsaturation; It refers to a monocyclic, bicyclic, or polycyclic hydrocarbon that contains multiple units of unsaturation, but is not aromatic (also referred to herein as "carbocyclic," "alicyclic," or "cycloalkyl") or any combination thereof. In some embodiments, an aliphatic group contains 1-100 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, an aliphatic group contains 1-5 aliphatic carbon atoms, and in still other embodiments, an aliphatic group contains 1, 2, 3, or 4 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic, bicyclic or polycyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C ring that is fully saturated or contains one or more units of unsaturation, but is not aromatic. 3~ C 6 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.

[0065] Alkenyl: As used herein, the term "alkenyl," as defined herein, refers to an aliphatic group having one or more double bonds.

[0066] Alkyl: As used herein, the term "alkyl" is given 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 with 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., C for straight chain). 1~ C 20 , C for branched chains 2~ C 20 ), or alternatively having about 1-10 carbon atoms. 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 contains 1-4 carbon atoms (e.g., C in the case of a straight chain lower alkyl). 1 ~C 4 ).

[0067] Alkynyl: As used herein, the term "alkynyl" refers to an aliphatic group, as defined herein, having one or more triple bonds.

[0068] Aryl: As used herein, the term "aryl," used alone or as part of a larger moiety such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having, for example, 5-30 total ring members, where at least one ring in the system is aromatic. In some embodiments, the aryl group is a monocyclic, bicyclic, or polycyclic ring system having, for example, 5-14 total ring members, where at least one ring in the system is aromatic, and where each ring in the system contains 3-7 ring members. In some embodiments, the aryl group is a biaryl group. The term "aryl" can 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, and anthracyl, which may bear one or more substituents. Also, as used herein, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, and anthracyl, which may bear one or more substituents. Also included within the scope of the term are aromatic rings fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0069] Equivalent: The term "equivalent" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit a comparison of the results obtained or the phenomena observed. In some embodiments, the equivalent sets of conditions or circumstances are characterized by a number of substantially identical characteristics and one or a small number of dissimilar characteristics. One of skill in the art will understand that sets of conditions are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to provide a basis for a reasonable conclusion that differences in the results obtained or phenomena observed under the various sets of conditions or circumstances are attributable to or indicative of differences in the dissimilar characteristics.

[0070] Alicyclic: The terms "alicyclic", "carbocycle", "carbocyclyl", "carbocyclic radical" and "carbocyclic ring" are used interchangeably and as used herein refer to a non-aromatic, cycloaliphatic monocyclic, bicyclic or polycyclic ring system as described herein that is saturated or partially unsaturated, but has 3 to 30 ring members unless otherwise specified. 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, alicyclic groups have 3 to 6 carbons. In some embodiments, alicyclic groups are saturated and are cycloalkyl. The term "alicyclic" may also include an aliphatic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or 1,2,3,4-tetrahydronaphth-1-yl. In some embodiments, an alicyclic group is bicyclic. In some embodiments, an alicyclic group is tricyclic. In some embodiments, an alicyclic group is polycyclic. In some embodiments, "alicyclic" refers to a C ring that is fully saturated or contains one or more units of unsaturation, but is not aromatic. 3 ~C 6 Monocyclic hydrocarbons or C 8 ~C 10 Bicyclic or polycyclic hydrocarbons or C which are fully saturated or contain one or more unsaturated units, but are not aromatic 9 ~C 16 Refers to polycyclic hydrocarbons.

[0071] Dosing regimen: As used herein, "dosing regimen" or "treatment regimen" refers to a set of unit doses (typically two or more doses) that are administered to a subject individually, typically spaced apart. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each of which is spaced apart by the same amount of time between each other. In some embodiments, a dosing regimen includes multiple doses and at least two different times spaced apart between the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose value. In some embodiments, different doses within a dosing regimen are different values. In some embodiments, a dosing regimen includes a first dose at a first dose value, followed by one or more additional doses at a second dose value that is different from the first dose value. In some embodiments, a dosing regimen includes a first dose at a first dose value, followed by one or more additional doses at a second dose value that is the same as the first dose value.

[0072] Heteroaliphatic: The term "heteroaliphatic" refers to any group of heteroaliphatic groups, including C, CH, CH 2 and C.H. 3 refers to an aliphatic group in which one or more units selected from are independently replaced by one or more heteroatoms. In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0073] Heteroaryl: As used herein, alone or as a larger moiety, e.g., “heteroaryl” refers to any group that is heteroaryl, e.g., The terms "heteroaryl" and "heteroar-" used as part of "heteroaralkyl" or "heteroaralkoxy" refer to heteroaryls having a total of 5 to 30 ring members, for example. "Heteroaryl" refers to a monocyclic, bicyclic or polycyclic ring system 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, heteroaryl groups have 6, 10 or 14 pi electrons shared in a cyclic array; in addition to the carbon atoms, they have 1 to 5 heteroatoms. 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, the heteroaryl is a heterobiaryl group such as bipyridyl. As used herein, the terms "heteroaryl" and "heteroa-" also include aliphatic groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic or polycyclic. The term "heteroaryl" can 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 are independently optionally substituted.

[0074] Heteroatom: The term "heteroatom" refers to an atom that is not carbon or hydrogen. In some embodiments, a heteroatom is oxygen, sulfur, nitrogen, phosphorus, boron, or silicon (any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or substitutable nitrogen of a heterocycle (e.g., N, such as in 3,4-dihydro-2H-pyrrolyl), NH (such as in pyrrolidinyl), or NR + (including, for example, in the case of N-substituted pyrrolidinyl). In some embodiments, the heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, the heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, the heteroatom is nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, the heteroatom is nitrogen, oxygen, or sulfur.

[0075] Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocycle radical" and "heterocycle" are used interchangeably herein and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, the heterocyclyl group is a stable 5-7 membered monocyclic or 7-10 membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1-4, heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0-3 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). The heterocycle can be any heteroatom or ring that results in a stable structure. It can be attached to its pendant group at a carbon atom, and any ring atom can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals 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 radical" are used interchangeably herein and also include heterocyclyl rings 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.

[0076] In vitro: As used herein, the term "in vitro" refers to events that take place not within a living organism (e.g., an animal, a plant, and / or a microorganism), but in an artificial environment, such as a test tube or reaction vessel, a cell culture.

[0077] 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).

[0078] Optionally substituted: Compounds of the present disclosure as described herein, such as oligonucleotides, lipids, carbohydrates, etc., may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise specified, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when multiple positions in any given structure may be substituted with multiple substituents selected from a specified group, the substituents may be either the same or different at each position. The combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are substantially unchanged when exposed to conditions that allow for their production, detection, and in certain embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0079] Suitable monovalent substituents are halogen; -(CH 2 ) 0~4 R ○ ;-(CH 2 ) 0~4 OR ○ ;-O(CH 2 ) 0~4 R o , -O-(CH 2 ) 0~4 C(O)OR°;-(CH 2 ) 0~4 CH(OR ○ ) 2 ;R° may be substituted -(CH 2 ) 0~4 Ph; R° may be substituted with -(CH 2 ) 0~4 O(CH 2 ) 0~1 Ph; may be substituted with R° -CH=CHPh; may be substituted with R° -(CH 2 ) 0~4 O(CH 2 ) 0~1 -Pyridyl; -NO2 ;-CN;-N 3 ;-(CH 2 ) 0~4 N(R ○ ) 2 ;-(CH 2 ) 0~4 N(R ○ )C(O)R ○ ;-N(R ○ )C(S)R ○ ;-(CH 2 ) 0~4 N(R ○ )C(O)N(R ○ ) 2 ;-N(R ○ )C(S)N(R ○ ) 2 ;-(CH 2 ) 0~4 N(R ○ )C(O)OR ○ ;-N(R ○ )N(R ○ )C(O)R ○ ;-N(R ○ )N(R ○ )C(O)N(R ○ ) 2 ;-N(R ○ )N(R ○ )C(O)OR ○ ;-(CH 2 ) 0~4 C(O)R ○ ;-C(S)R ○ ;-(CH 2 ) 0~4 C(O)OR ○ ;-(CH 2 ) 0~4 C(O)SR ○ ;-(CH 2 ) 0~4 C(O)OSi(R ○ ) 3 ;-(CH 2 ) 0~4 OC(O)R ○ ;-OC(O)(CH 2 ) 0~4 SR°、-SC(S)SR°;-(CH 2 ) 0~4 SC(O)R ○ ;-(CH 2 )0~4 C(O)N(R ○ ) 2 ;-C(S)N(R ○ ) 2 ;-C(S)SR°;-SC(S)SR°、-(CH 2 ) 0~4 OC(O)N(R ○ ) 2 ;-C(O)N (OR ○ )R ○ ;-C(O)C(O)R ○ ;-C(O)CH 2 C(O)R ○ ;-C(NOR ○ )R ○ ;-(CH 2 ) 0~4 SSR ○ ;-(CH 2 ) 0~4 S(O) 2 R ○ ;-(CH 2 ) 0~4 S(O) 2 OR ○ ;-(CH 2 ) 0~4 OS(O) 2 R ○ ;-S(O) 2 N(R ○ ) 2 ;-(CH 2 ) 0~4 S(O)R ○ ;-N(R ○ )S(O) 2 N(R ○ ) 2 ;-N(R ○ )S(O) 2 R ○ ;-N(OR ○ )R ○ ;-C(NH)N(R ○ ) 2 ;-Si(R ○ ) 3 ;-OSi(R ○ ) 3 ;-P(R ○ ) 2 ;-P(OR ○ ) 2 ;-P(R ○ )(OR○ );-OP(R ○ ) 2 ;-OP(OR ○ ) 2 ;-OP(R ○ )(OR ○ );-P[N(R ○ ) 2 ] 2 -P(R ○ )[N(R ○ ) 2 ];-P(OR ○ )[N(R ○ ) 2 ];-OP[N(R ○ ) 2 ] 2 ;-OP(R ○ )[N(R ○ ) 2 ];-OP(OR ○ )[N(R ○ ) 2 ];-N(R ○ )P(R ○ ) 2 ;-N(R ○ )P(OR ○ ) 2 ;-N(R ○ )P(R ○ )(OR ○ );-N(R ○ )P[N(R ○ ) 2 ] 2 ;-N(R ○ )P(R ○ )[N(R ○ ) 2 ];-N(R ○ )P(OR ○ )[N(R ○ ) 2 ];-B(R ○ ) 2 ;-B(R ○ )(OR ○ );-B(OR ○ ) 2 ;-OB(R ○ ) 2 ;-OB(R ○ )(OR ○ );-OB(OR ○ ) 2 ;-P(O)(R ○ )2 ;-P(O)(R ○ )(OR ○ );-P(O)(R ○ )(SR ○ );-P(O)(R ○ )[N(R ○ ) 2 ];-P(O)(OR ○ ) 2 ;-P(O)(SR ○ ) 2 ;-P(O)(OR ○ )[N(R ○ ) 2 ];-P(O)(SR ○ )[N(R ○ ) 2 ];-P(O)(OR ○ )(SR ○ );-P(O)[N(R ○ ) 2 ] 2 ;-OP(O)(R ○ ) 2 ;-OP(O)(R ○ )(OR ○ );-OP(O)(R ○ )(SR ○ );-OP(O)(R ○ )[N(R ○ ) 2 ];-OP(O)(OR ○ ) 2 ;-OP(O)(SR ○ ) 2 ;-OP(O)(OR ○ )[N(R ○ ) 2 ];-OP(O)(SR ○ )[N(R ○ ) 2 ];-OP(O)(OR ○ )(SR ○ );-OP(O)[N(R ○ ) 2 ] 2 ;-SP(O)(R ○ ) 2 ;-SP(O)(R ○ )(OR ○ );-SP(O)(R ○ )(SR ○ );-SP(O)(R○ )[N(R ○ ) 2 ];-SP(O)(OR ○ ) 2 ;-SP(O)(SR ○ ) 2 ;-SP(O)(OR ○ )[N(R ○ ) 2 ];-SP(O)(SR ○ )[N(R ○ ) 2 ];-SP(O)(OR ○ )(SR ○ );-SP(O)[N(R ○ ) 2 ] 2 ;-N(R ○ )P(O)(R ○ ) 2 ;-N(R ○ )P(O)(R ○ )(OR ○ );-N(R ○ )P(O)(R ○ )(SR ○ );-N(R ○ )P(O)(R ○ )[N(R ○ ) 2 ];-N(R ○ )P(O)(OR ○ ) 2 ;-N(R ○ )P(O)(SR ○ ) 2 ;-N(R ○ )P(O)(OR ○ )[N(R ○ ) 2 ];-N(R ○ )P(O)(SR ○ )[N(R ○ ) 2 ];-N(R ○ )P(O)(OR ○ )(SR ○ );-N(R ○ )P(O)[N(R ○ ) 2 ] 2 ;-P(R ○ ) 2 [B(R ○ ) 3];-P(OR ○ ) 2 [B(R ○ ) 3 ];-P(NR ○ ) 2 [B(R ○ ) 3 ];-P(R ○ )(OR ○ )[B(R ○ ) 3 ];-P(R ○ )[N(R ○ ) 2 ][B(R ○ ) 3 ];-P(OR ○ )[N(R ○ ) 2 ][B(R ○ ) 3 ];-OP(R ○ ) 2 [B(R ○ ) 3 ];-OP(OR ○ ) 2 [B(R ○ ) 3 ];-OP(NR ○ ) 2 [B(R ○ ) 3 ];-OP(R ○ )(OR ○ )[B(R ○ ) 3 ];-OP(R ○ )[N(R ○ ) 2 ][B(R ○ ) 3 ];-OP(OR ○ )[N(R ○ ) 2 ][B(R ○ ) 3 ];-N(R ○ )P(R ○ ) 2 [B(R ○ ) 3 ];-N(R ○ )P(OR ○ ) 2 [B(R ○ ) 3 ];-N(R ○ )P(NR ○ )2 [B(R ○ ) 3 ];-N(R ○ )P(R ○ )(OR ○ )[B(R ○ ) 3 ];-N(R ○ )P(R ○ )[N(R ○ ) 2 ][B(R ○ ) 3 ];-N(R ○ )P(OR ○ )[N(R ○ ) 2 ][B(R ○ ) 3 ];-P(OR')[B(R') 3 ]-;-(C 1~4 Linear or branched alkylene)ON(R ○ ) 2 ;or-(C 1~4 Linear or branched alkylene)C(O)ON(R ○ ) 2 where each R° can be substituted as defined below and is independently hydrogen, C 1~20 C having 1 to 5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon and phosphorus 1~20 Heteroaliphatic; -CH 2 -(C 6~20 Aryl), -O(CH 2 ) 0~1 (C 6~20 Aryl), -CH 2- (a 5-20 membered heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus), a 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated ring 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° together with their intervening atoms form a ring as defined below. and forming a 3-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated ring or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as desired.

[0080] Suitable monovalent substituents on R° (or the ring formed by two separate occurrences of R° together with their intervening atoms) are independently halogen, -(CH 2 ) 0~2 R ● , -(Halo R ● ), -(CH 2 ) 0~2 OH, -(CH 2 ) 0~2 OR ● , -(CH 2 ) 0~2 CH(OR ● ) 2 ;-O(Halo R ● ), -CN, -N 3 , -(CH 2 ) 0~2 C(O)R ● , -(CH 2 ) 0~2 C(O)OH, -(CH 2 ) 0~2 C(O)OR ● , -(CH 2 ) 0~2 S.R. ● , -(CH 2 ) 0~2 SH, -(CH 2 ) 0~2 NH 2 , -(CH 2 ) 0~2 NHR ● , -(CH 2 ) 0~2 NR ● 2 , -NO 2 , -SiR ● 3 , -OSiR ● 3 , -C(O)SR ● , -(C 1~4 Linear or branched alkylene)C(O)OR ● Or -SSR ● where each R● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and has 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 1~4 Aliphatic, -CH 2 Ph, -O(CH 2 ) 0~1 and independently selected from Ph and a 5- to 6-membered saturated, partially unsaturated ring or an aryl ring. Suitable divalent substituents on a saturated carbon atom of R° include =O and =S.

[0081] For example, suitable divalent substituents on suitable carbon and nitrogen atoms are independently: ═O, ═S, ═CR * 2 , =NNR * 2 , =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O) 2 R * , =NR * , =NOR * , -O(C(R * 2 )) 2~3 O- or -S(C(R * 2 )) 2~3 S-, where each R * may be substituted as defined below and independently represent hydrogen, C 1~20 C having 1 to 5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon and phosphorus 1~20 Heteroaliphatic, -CH 2 -(C 6~20 Aryl), -O(CH 2 ) 0~1 (C 6~20 Aryl), -CH 2 -(5-20 membered heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus), a 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated ring or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, regardless of the above definition, two R *Independent occurrences of together with their intervening atoms form a 3-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated ring or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below. Suitable divalent substituents attached to adjacent substitutable atoms of an "optionally substituted" group include -O(CR * 2 ) 2~3 Contains O-.

[0082] R * The above appropriate monovalent substituent (or two R * The rings formed by the independent occurrences of -(CH) together with their intervening atoms are independently halogen, -(CH 2 ) 0~2 R ● , -(Halo R ● ), -(CH 2 ) 0~2 OH, -(CH 2 ) 0~2 OR ● , -(CH 2 ) 0~2 CH(OR ● ) 2 ;-O(Halo R ● ), -CN, -N 3 , -(CH 2 ) 0~2 C(O)R ● , -(CH 2 ) 0~2 C(O)OH, -(CH 2 ) 0~2 C(O)OR ● , -(CH 2 ) 0~2 S.R. ● , -(CH 2 ) 0~2 SH, -(CH 2 ) 0~2 NH 2 , -(CH 2 ) 0~2 NHR ● , -(CH 2 ) 0~2 NR ● 2 , -NO 2 , -SiR● 3 , -OSiR ● 3 , -C(O)SR ● , -(C 1~4 Linear or branched alkylene)C(O)OR ● Or -SSR ● where each R ● is unsubstituted or, when preceded by "halo", is substituted with only one or more halogens, and independently represents C 1~4 Aliphatic, -CH 2 Ph, -O(CH 2 ) 0~1 R is selected from Ph and a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur. * Suitable divalent substituents on a saturated carbon atom of include ═O and ═S.

[0083] In some embodiments, a suitable nitrogen atom on a substitutable nitrogen of an "optionally substituted" group Suitable substituents include -R † , -NR † 2 , -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH 2 C(O)R † , -S(O) 2 R † , -S(O) 2 NR † 2 , -C(S)NR † 2 , -C(NH)NR † 2 Or -N(R † )S(O) 2 R † where each R † are independently hydrogen, which may be substituted as defined below, 1~6aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, regardless of the above definition, two independent R † occurrences of together with their intervening atoms form an unsubstituted 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0084] In some embodiments, R † Suitable substituents on the aliphatic group are independently halogen, -R ● , -(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH 2 , N.H.R. ● , -NR ● 2 OR -NO 2 where each R ● is unsubstituted or, when preceded by "halo", is substituted with only one or more halogens, and independently represents C 1~4 Aliphatic, -CH 2 Ph, -O(CH 2 ) 0~1 Ph is a 5- to 6-membered saturated, partially unsaturated ring or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0085] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that contains 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.

[0086] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving a controlled therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid dosage forms, including those designed for the following administration purposes: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, such as those targeted for oral, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as subcutaneous, intramuscular, intravenous or epidural injections, such as sterile solutions or suspensions or sustained release formulations; topical application, such as creams, ointments or controlled release patches or sprays applied to the skin, lungs or oral cavity; vaginal or rectal administration, such as pessaries, creams or foams; sublingual; ophthalmic; transdermal; or intranasal, pulmonary and other mucosal surfaces.

[0087] Pharmaceutically acceptable: As used herein, the phrase "pharmacologically 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 impairment or complication, commensurate with a reasonable benefit / risk ratio.

[0088] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulant, involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. 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; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters; polycarbonates and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0089] Pharmaceutically acceptable salts: The term "pharmaceutical acceptable salts" as used herein refers to salts of compounds that are suitable for use in conjunction with pharmaceutical formulations, i.e., salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are known in the art. For example, SM Berge, et al., J. Pharmaceutical Pharmaceutically acceptable salts are described in detail in Sciences, 66: 1-19 (1977). In some embodiments, pharma-ceutically acceptable salts include, but are not limited to, non-toxic acid addition salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, maleic, tartaric, citric, succinic, or malonic acids, or by other methods used in the art, such as ion exchange. In some embodiments, pharma- ceutically acceptable salts include, but are not limited to, adipate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, dihydrogenphosphate, and the like. -hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1-6 carbon atoms, sulfonates, and arylsulfonates, as appropriate.In some embodiments, provided compounds, e.g., oligonucleotides, contain one or more acidic groups (e.g., natural phosphate linkages, phosphorothioate linkages, etc.) and the pharma- ceutically acceptable salts include alkali, alkaline earth metal or ammonium (e.g., N(R). 3 where each R is independently as defined and described in this disclosure). Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. In some embodiments, the pharma- ceutically acceptable salt is a sodium salt. In some embodiments, the pharma- ceutically acceptable salt is a potassium salt. In some embodiments, the pharma- ceutically acceptable salt is a calcium salt. In some embodiments, the pharma- ceutically acceptable salt includes amine cations formed with non-toxic ammonium, quaternary ammonium, and counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1-6 carbon atoms, sulfonates, and arylsulfonates, as appropriate. In some embodiments, provided compounds contain more than one acidic group, e.g., oligonucleotides may contain two or more acidic groups (e.g., at natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, the pharma- ceutical moieties of such compounds may be substituted or unsubstituted. A pharma- ceutically acceptable salt, or salt in general, comprises two or more cations, which may be the same or different. In some embodiments, in a pharma-ceutically acceptable salt (or salt in general), each acidic group having sufficient acidity is present independently in its salt form (e.g., in an oligonucleotide comprising a natural phosphate linkage and a phosphorothioate internucleotide linkage, each of the natural phosphate linkage and the phosphorothioate internucleotide linkage is present independently in its salt form). In some embodiments, a pharma-ceutically acceptable salt of an oligonucleotide, such as an oligonucleotide provided, is a sodium salt of the oligonucleotide provided. In some embodiments, a pharma-ceutically acceptable salt of an oligonucleotide, such as an oligonucleotide, is a sodium salt of such an oligonucleotide, where each acidic linkage, such as each natural phosphate linkage and phosphorothioate internucleotide linkage, is present in sodium salt form (all sodium salts).

[0090] Protecting Group: The term "protecting group" as used herein is known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference in their entirety. In addition, protecting groups specifically designed for nucleoside and nucleotide chemistry, such as those described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012, Chapter 2, entirely incorporated herein by reference, are also included. Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthy]methyl carbamate (DBD-Tmoc), 4-methoxyphenacylcarbamate, and the like. Bamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (T CBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-biphenylcarbamate)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Cat), Carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-Dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethyl-thiophenyl Carbamate (Bmpc), 2-phosphinoethyl 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-nitrophenoxy, Nyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amyl carbamate, S-benzylthiocarbamate, p -Cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethyl-carboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethyl-carboxamido)propyl carbamate, 1,1-dimethyl- Propynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-Trimethylbenzylcarbamate, 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)acetamide, o-phenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine 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)-2-phenylpropane, N-isopropyl ... Propyl-4-nitro-2-oxo-3-pyrroloyl-3-yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-Dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboron, Phosphonic acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidates, diphenyl phosphoramidates, benzenesulfenamides, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl -4-Methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide and phenacylsulfonamide.

[0091] 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, 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.

[0092] 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, tetramethylsil ... Hydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl , 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzohydrin , 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 3-(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-butylsilyl Triphenyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoyl formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate (levulinate), 4,4-(Ethylenedithio)pentanoate (levulinoyl dithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (TMSEC), 2-(triphenylphosphonio)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-benzylthiocarbonate, 4-ethoxy-1-naphthothyl carbonate, methyl di Thiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid Acid, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate and tosylate (Ts). When protecting 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 keta, benzylidene acetal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzyl butylsiloxane orthoesters, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoesters, di-t-butylsilylene groups (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronic acid salts, ethyl borate, and phenyl borate.

[0093] In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl, (D 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (Pixyl) or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting groups are selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.

[0094] In some embodiments, the phosphorus protecting group is a group that is added to the internucleotide phosphorus bond throughout oligonucleotide synthesis. In some embodiments, the phosphorus protecting group is added to the sulfur atom of the internucleotide phosphorothioate bond. In some embodiments, the phosphorus protecting group is added to the oxygen atom of the internucleotide phosphorothioate bond. In some embodiments, the phosphorus protecting group is added to the oxygen atom of the internucleotide phosphate bond. In some embodiments, the phosphorus protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl, N-methyl)aminoethyl, 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl. be.

[0095] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a sequence of at least two amino acids linked together by a peptide bond). In some embodiments, a protein contains only naturally occurring amino acids. In some embodiments, a protein contains one or more non-naturally occurring amino acids (e.g., a moiety that forms one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues in a protein chain contain a non-amino acid moiety (e.g., a glycan, etc.). In some embodiments, a protein contains two or more polypeptide chains, for example, linked by one or more disulfide bonds or linked by other means. In some embodiments, a protein contains L-amino acids, D-amino acids, or both; in some embodiments, a protein contains one or more amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0096] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes. Exemplary subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates and humans; insects; worms; etc.) and plants. In some embodiments, the subject may suffer from and / or be susceptible to a disease, disorder and / or condition, such as muscular dystrophy.

[0097] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting all or nearly all of the extent or degree of a feature or characteristic of interest. Those skilled in the art will appreciate that biological and chemical phenomena rarely, if ever, reach and / or proceed to completion or achieve or avoid absolute consequences. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0098] Suffering from: An individual "suffering from" a disease, disorder and / or condition, e.g., muscular dystrophy, has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder and / or condition, e.g., muscular dystrophy.

[0099] Susceptible: An individual who is "susceptible" to a disease, disorder and / or condition, such as muscular dystrophy, is an individual who is at a higher risk of developing the disease, disorder and / or condition than the general population. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition, such as muscular dystrophy, 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, such as muscular dystrophy, 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, such as muscular dystrophy, 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, such as muscular dystrophy, will develop the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition, such as a muscular dystrophy, does not develop the disease, disorder and / or condition.

[0100] Systemic: The phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally," as used herein, refer to a compound or composition that enters the entire body of the recipient. It refers to administration of something and has its art-recognized meaning.

[0101] Tautomeric forms: The phrase "tautomeric forms" is used herein and as generally understood in the art to describe different isomeric forms of an organic compound capable of easy interconversion. Tautomers may be characterized by the formal migration of a hydrogen atom or a proton accompanied by the inversion of a single bond and an adjacent double bond. In some embodiments, tautomers may result from prototropic tautomerism (i.e., rearrangement of a proton). In some embodiments, tautomers may result from valence tautomerism (i.e., rapid rearrangement of bond electrons). All such tautomeric forms are intended to be included within the scope of the present disclosure. In some embodiments, tautomeric forms of a compound exist in mobile equilibrium with one another, such that attempts to prepare separate substances would result in the formation of a mixture. In some embodiments, tautomeric forms of a compound are separable and isolatable compounds. In some embodiments of the present disclosure, chemical compositions may be provided that are or include pure preparations of a single tautomeric form of the compound. In some embodiments of the present disclosure, chemical compositions may be provided as mixtures of two or more tautomeric forms of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomeric forms; in certain embodiments, such mixtures contain at least two different tautomeric forms of a compound in different amounts. In some embodiments of the present disclosure, the chemical composition may contain all tautomeric forms of a compound. In some embodiments of the present disclosure, the chemical composition may contain less than all tautomeric forms of a compound. In some embodiments of the present disclosure, the chemical composition may contain one or more tautomeric forms of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the present disclosure, the tautomer is keto-enol tautomerism. Those skilled in the chemical arts will recognize that the keto-enol tautomer can be "trapped" (i.e., chemically modified so that it remains in the "enol" form) using any suitable reagent known in the chemical art to provide an enol derivative, which can then be separated using one or more suitable techniques known in the art.Unless otherwise indicated, the present disclosure encompasses all tautomeric forms of the relevant compounds, whether in pure form or in admixture with each other.

[0102] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, reduce, 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, such as muscular dystrophy.

[0103] 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 induces 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, such as muscular dystrophy, when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be appreciated by those skilled in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition, such as muscular dystrophy, is an amount that alleviates, improves, relieves, 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 utilized to deliver a therapeutically effective amount.

[0104] Treat: As used herein, the terms "treat", "treatment" or "treating" refer to any method used to partially or completely alleviate, ameliorate, reduce, 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, such as muscular dystrophy. Treatment may be administered to subjects who do not show signs of a disease, disorder, and / or condition, such as muscular dystrophy. In some embodiments, treatment may be administered to subjects who show only early signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing a condition associated with the disease, disorder, and / or condition.

[0105] Unit dose: The term "unit dose" as used herein refers to an amount administered as a single dose of a pharmaceutical composition and / or administered in a physically separate unit. In many embodiments, a unit dose comprises a predetermined amount of an active agent. In some embodiments, a unit dose comprises an entire single dose of an agent. In some embodiments, two or more unit doses are administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required or expected to be required to achieve an intended effect. A unit dose can be, for example, a volume of a liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, or a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents. It is understood that a unit dose can be in a formulation that includes any of a variety of components in addition to a therapeutic agent. For example, an acceptable carrier (e.g., a pharma- ceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, and the like can be included as described below. Those skilled in the art will understand that in many embodiments, an appropriate total daily dosage of a particular therapeutic agent may include a portion or multiple unit doses, which may be determined by the attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dosage level for any particular subject or organism may vary depending on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific active compound used; the specific composition used; the age, weight, health, sex, and diet of the subject; the time of administration of the specific active compound used and its excretion rate; the duration of treatment; drugs and / or additional therapies used in combination or simultaneously with the specific compound used, and similar factors known in the medical arts.

[0106] Unsaturated: The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0107] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning of referring to an entity that has structure and / or activity as found in a naturally "normal" (as opposed to mutant, diseased, altered, etc.) state or context. One of skill in the art will understand that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0108] Nucleic Acid: The term "nucleic acid" includes any nucleotide, analogs thereof, 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 analogs thereof. These terms refer to the primary structure of the molecule, and include double- and single-stranded DNA as well as double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made with nucleotide analogs, and modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. These terms include polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and phosphate bridges and / or modified phosphorus atom bridges (referred to herein as "internucleobases"). The term encompasses nucleic acids derived from a nucleotide sequence (also referred to as a "nucleotide linkage"). The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, natural phosphate internucleotide linkages or non-natural internucleotide linkages. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both a ribose moiety and a deoxyribose moiety, and nucleic acids containing a ribose moiety and a modified ribose moiety. Unless otherwise specified, the prefix poly refers to a nucleic acid containing from 2 to about 10,000 nucleotide monomer units, where the prefix oligo refers to a nucleic acid containing from 2 to about 200 nucleotide monomer units.

[0109] Nucleotide: The term "nucleotide" as used herein refers to a monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more phosphate or phosphorus-containing internucleotide linkages. Naturally occurring bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are derivatives of purines or pyrimidines, but it should be understood that naturally occurring and non-naturally occurring base analogs are also included. Naturally occurring sugars include the pentose (five-carbon sugar) deoxyribose (which is found in natural DNA) or ribose (which is found in natural RNA), but it should be understood that naturally occurring and non-naturally occurring sugar analogs are also included, such as sugars with 2'-modifications, sugars in locked nucleic acids (LNAs) and phosphorodiamidate morpholino oligomers (PMOs), and the like. Nucleotides are linked by internucleotide linkages to form nucleic acids or polynucleotides. Numerous internucleotide linkages are known in the art, including but not limited to natural phosphate linkages, phosphorothioate linkages, boranophosphate linkages, etc. Artificial nucleic acids include PNAs (peptide nucleic acids), phosphate triesters, phosphorothioates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids. In some embodiments, the nucleotides are natural nucleotides that include naturally occurring nucleobases, naturally occurring sugars, and natural phosphate linkages. In some embodiments, the nucleotides are modified nucleotides or nucleotide analogs that are structural analogs that can be used in place of natural nucleotides.

[0110] Modified Nucleotide: The term "modified nucleotide" includes any chemical moiety that is structurally different from a naturally occurring nucleotide but is capable of performing at least one function of a naturally occurring nucleotide. In some embodiments, a modified nucleotide comprises a modification in 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 performing at least one function of a nucleotide, e.g., forming a subunit in a polymer that is capable of base pairing with a nucleic acid that comprises at least a complementary base sequence.

[0111] Analog: The term "analog" includes any chemical moiety that is structurally different from a reference chemical moiety or class of chemical moieties, but is capable of performing at least one function of such reference chemical moiety or class of chemical moieties. As non-limiting examples, a nucleotide analog is structurally different from a nucleotide, but performs at least one function of a nucleotide; a nucleobase analog is structurally different from a nucleobase, but performs at least one function of a nucleobase; a sugar analog is structurally different from a nucleobase, but performs at least one function of a sugar, etc.

[0112] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar.

[0113] Modified nucleosides: The term "modified nucleoside" refers to a chemical moiety that is chemically different from a natural nucleoside but has the ability to perform at least one function of a nucleoside. In some embodiments, a modified nucleoside is derived from or chemically similar to a natural nucleoside, but contains a chemical modification that distinguishes it from a natural nucleoside. Non-limiting examples of modified nucleosides include those that contain base and / or sugar modifications. Non-limiting examples of modified nucleosides include those that have a 2' modification on the sugar. Further non-limiting examples of modified nucleosides include abasic nucleosides (nucleobases are missing). In some embodiments, modified nucleosides are capable of at least one function of a nucleoside and can form a moiety in a polymer that can base pair with a nucleic acid that contains at least a complementary base sequence.

[0114] Nucleoside analog: The term "nucleoside analog" refers to a chemical moiety that is chemically different from a natural nucleoside but is capable of performing at least one function of a nucleoside. In some embodiments, a nucleoside analog includes a sugar analog and / or a nucleobase analog. In some embodiments, a modified nucleoside is capable of performing at least one function of a nucleoside, e.g., forming a moiety in a polymer that is capable of base pairing with a nucleic acid that contains a complementary base sequence.

[0115] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in a closed and / or open state. In some embodiments, the sugar is a monosaccharide. In some embodiments, the 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, for example, glycols, polymers of which form the backbone of nucleic acid analogs such as glycol nucleic acid (GNA). As used herein, the term "sugar" also includes structural analogs that are used in place of natural or naturally occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, the sugar is D-2-deoxyribose. In some embodiments, the sugar is β-D-deoxyribofuranose. In some embodiments, the sugar moiety is a β-D-deoxyribofuranose moiety. In some embodiments, the sugar is D-ribose. In some embodiments, the sugar is β-D-ribofuranose. In some embodiments, the sugar moiety is a β-D-ribofuranose moiety. In some embodiments, the sugar is an optionally substituted β-D-deoxyribofuranose or β-D-ribofuranose. In some embodiments, the sugar moiety is an optionally substituted β-D-deoxyribofuranose or β-D-ribofuranose moiety. In some embodiments, the sugar moiety / unit in an oligonucleotide, e.g., a DMD oligonucleotide, a nucleic acid, etc., is a sugar comprising one or more carbon atoms each independently linked to an internucleotide linkage, e.g., an optionally substituted β-D-deoxyribofuranose or β-D-ribofuranose whose 5'-C and / or 3'-C are each independently linked to an internucleotide linkage (e.g., a natural phosphate linkage, a modified internucleotide linkage, a chiral controlled internucleotide linkage, etc.).

[0116] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. In some embodiments, the modified sugar is a substituted β-D-deoxyribofuranose or β-D-ribofuranose. In some embodiments, the modified sugar comprises a 2'-modification. In some embodiments, the modified sugar comprises a linker (e.g., an optionally substituted divalent heteroaliphatic) connecting two sugar carbon atoms (e.g., C2 and C4), e.g., as found in LNA. In some embodiments, the linker is -O-CH(R)-, where R is as described in this disclosure. In some embodiments, the linker is -O-CH(R)-, where O is linked to C2 of the sugar, -CH(R)- is linked to C4, and R is As described in the present disclosure. In some embodiments, R is methyl. In some embodiments, R is -H. In some embodiments, -CH(R)- is in the S configuration. In some embodiments, -CH(R)- is in the R configuration.

[0117] Nucleic acid base: The term "nucleic acid base" refers to the portion of a nucleic acid that participates in hydrogen bonds that bind one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common natural nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C) and thymine (T). In some embodiments, modified nucleobases are substituted nucleobases whose nucleobases are selected from A, T, C, G, U and tautomers thereof. In some embodiments, natural nucleobases are modified adenine, guanine, uracil, cytosine or thymine. In some embodiments, natural nucleobases are methylated adenine, guanine, uracil, cytosine or thymine. In some embodiments, nucleobases are "modified nucleobases", e.g., nucleobases other than adenine (A), guanine (G), uracil (U), cytosine (C) and thymine (T). In some embodiments, modified nucleobases are methylated adenine, guanine, uracil, cytosine or thymine. In some embodiments, modified nucleobases mimic the spatial arrangement, electronic properties, or any other physicochemical properties of nucleobases and retain the hydrogen bond properties that bind one nucleic acid strand to another in a sequence-specific manner. In some embodiments, modified nucleobases can pair with all five natural bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of oligonucleotide duplex strands. As used herein, the term "nucleobase" also includes structural analogs that are used in place of natural or naturally occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, nucleobases are A, T, C, G, or U, which are optionally substituted, or substituted nucleobases, whose nucleobases are selected from A, T, C, G, U, and tautomers thereof.

[0118] Modified nucleobase: The term "modified nucleobase", "modified base" and the like refers 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 and can form a moiety, for example in a polymer, that can base pair with a nucleic acid that comprises at least a complementary base sequence. In some embodiments, a modified nucleobase is a substituted nucleobase whose nucleobase is selected from A, T, C, G, U and tautomers thereof.

[0119] Chiral Ligand: The term "chiral ligand" or "chiral auxiliary" refers to a moiety that is chiral and can be incorporated into a reaction such that the reaction can be carried out with a certain stereoselectivity. In some embodiments, the term can also refer to a compound that contains such a moiety.

[0120] Blocking group: The term "blocking group" refers to a group that masks the reactivity of a functional group. The functional group may be subsequently unmasked by removal of the blocking group. In some embodiments, a blocking group is a protecting group.

[0121] Moiety: The term "moiety" refers to a specific segment of a molecule's functionality. Chemical moieties are commonly recognized chemical entities embedded in or appended to a molecule. In some embodiments, a moiety of a compound is a monovalent, divalent, or polyvalent group formed from the compound by removing one or more -H and / or its equivalents from the compound. In some embodiments, depending on the context, "moiety" can also refer to the compound or entity from which the moiety is derived.

[0122] Reading Frame: The term "reading frame" refers to one of six possible reading frames, three in each direction, of a double-stranded DNA molecule. A coding frame determines which codons are used to code for amino acids within the coding sequence of a DNA molecule.

[0123] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotide monomers containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, natural phosphate linkages or non-natural internucleotide linkages.

[0124] The oligonucleosides of the present disclosure can be of various lengths. In particular embodiments, the oligonucleosides can range from about 20 to about 200 nucleosides in length. In various related embodiments, the single-stranded, double-stranded, and triple-stranded oligonucleosides can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In some embodiments, the oligonucleosides are from about 9 to about 39 nucleosides in length. In some embodiments, the oligonucleosides are at least 15 nucleosides in length. In some embodiments, the oligonucleosides are at least 20 nucleosides in length. In some embodiments, the oligonucleosides are at least 25 nucleosides in length. In some embodiments, the oligonucleoside is at least 30 nucleosides in length. In some embodiments, the oligonucleoside is a duplex of complementary strands at least 18 nucleosides in length. In some embodiments, the oligonucleoside is a duplex of complementary strands at least 21 nucleosides in length. In some embodiments, for purposes of oligonucleotide length, each nucleoside considered comprises an optionally substituted nucleobase independently selected from A, T, C, G, U and tautomers thereof.

[0125] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to a linkage, typically a phosphorus-containing linkage, between nucleotide units of a nucleic acid or oligonucleotide, and is synonymous with "intersugar linkage," "internucleoside linkage," and "phosphorus atom bridge," as used above and herein. As will be understood by those of skill in the art, naturally occurring DNA and RNA contain natural phosphate linkages. In some embodiments, the internucleotide linkage is a natural phosphate linkage (-OP(O)(OH)O-, typically in its anionic form, e.g., at a pH of about 7.4, -OP(O)(OH)O-, as found in naturally occurring DNA and RNA molecules. - )O-). In some embodiments, the internucleotide linkage is a modified internucleotide linkage (or a non-natural internucleotide linkage) that is structurally different from, but may be substituted for, a natural phosphate linkage, such as a phosphorothioate internucleotide linkage, a PMO linkage, etc. In some embodiments, the internucleotide linkage is a modified internucleotide linkage in which one or more oxygen atoms of a natural phosphodiester linkage are independently replaced by one or more organic or inorganic moieties. In some embodiments, such organic or inorganic moieties include, but are not limited to, =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 below. In some embodiments, the internucleotide linkage is a phosphotriester linkage. In some embodiments, the internucleotide linkage is a phosphorothioate diester linkage (phosphorothioate internucleotide linkage, [ka] , typically in its anionic form -OP(O)(S - )O- It will be appreciated by those skilled in the art that an internucleotide linkage may exist as an anion or a cation at a given pH due to the presence of an acid or base moiety in the linkage.

[0126] Unless otherwise specified, the Rp / Sp designation preceding an oligonucleotide sequence describes the configuration of the linked phosphorus in the chiral controlled internucleotide linkage in the 5' to 3' order of the oligonucleotide sequence.

[0127] Oligonucleotide type: As used herein, the phrase "oligonucleotide type" is used to define oligonucleotides having a particular base sequence, pattern of backbone linkages (i.e., internucleotide linkage types, such as natural phosphate linkages, phosphorothioate internucleotide linkages, negatively charged internucleotide linkages, neutral internucleotide linkages, etc.), pattern of backbone chiral centers (i.e., pattern of linkage phosphorus stereochemistry (Rp / Sp)), and pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides of a common designated "type" are structurally identical to each other.

[0128] Those skilled in the art will appreciate that the synthesis methods of the present disclosure provide a degree of control during oligonucleotide (e.g., DMD oligonucleotide) chain synthesis, whereby each nucleotide unit of the oligonucleotide chain can be pre-designed and / or selected to have a specific stereochemistry at the binding phosphorus and / or a specific modification at the binding phosphorus, and / or a specific base, and / or a specific sugar. In some embodiments, the oligonucleotide chain is pre-designed and / or selected to have a specific combination of stereocenters at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the structural features described above. The present disclosure provides compositions (e.g., chiral controlled oligonucleotide compositions) that include or consist of a plurality of oligonucleotide molecules. In some embodiments, such molecules are all of the same type. In some embodiments, such molecules are all structurally identical to one another. In some embodiments, the provided compositions include a plurality of oligonucleotides of different types, typically in predetermined (non-random) relative amounts. In some embodiments, the oligonucleotide is a DMD oligonucleotide as described herein.

[0129] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical assignment of the chiral linkage phosphorus in the chiral internucleotide linkage in an oligonucleotide (e.g., DMD oligonucleotide). In some embodiments, the control is achieved through chiral elements absent from the sugar and base moieties of the oligonucleotide, e.g., in some embodiments, the control is achieved through one or more chiral auxiliary groups during the oligonucleotide preparation process, as exemplified in the present disclosure, where the chiral auxiliary is often part of the chiral phosphoramidite used in the oligonucleotide preparation process. In contrast to chiral control, those skilled in the art will understand that conventional oligonucleotide synthesis without the use of a chiral auxiliary 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 linkage phosphorus in the chiral internucleotide linkage in an oligonucleotide is controlled.

[0130] Chiral controlled oligonucleotide composition: The term "chiral controlled (stereocontrolled or stereodefined) oligonucleotide composition" refers to a chiral (stereocontrolled or stereodefined) oligonucleotide composition. As used herein, "predetermined or stereodefined nucleic acid composition" and the like refer to a composition comprising a plurality of oligonucleotides (or nucleic acids, chiral controlled oligonucleotides or chiral controlled nucleic acids) (a particular type of oligonucleotides) that share 1) a common base sequence, 2) a common pattern of backbone linkages, 3) a common pattern of backbone chiral centers, and 4) a common pattern of backbone phosphorus modifications, where the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chiral controlled internucleotide linkages, where the chiral linkage phosphorus is Rp or Sp, not a random Rp and Sp mixture as in non-chiral controlled internucleotide linkages). The level of the plurality of oligonucleotides (or nucleic acids) in a chiral controlled oligonucleotide composition is non-random (predetermined, controlled). Chiral controlled oligonucleotide compositions are typically prepared through the stereoselective formation of one or more chiral internucleotide bonds, for example, by chiral controlled oligonucleotide preparation (e.g., using chiral auxiliary groups as exemplified in the present disclosure, compared to non-chiral controlled (stereorandom, non-stereoselective, racemic) oligonucleotide synthesis, such as conventional phosphoramidite-based oligonucleotide synthesis, which does not use chiral auxiliary groups or chiral catalysts to purposefully control stereoselectivity). Chiral controlled oligonucleotide compositions are enriched for a plurality of oligonucleotides compared to substantially racemic preparations of oligonucleotides having a common base sequence, a common backbone bond pattern, and a common backbone phosphorus modification pattern. In some embodiments, chiral controlled oligonucleotide compositions include a plurality of oligonucleotides of a particular oligonucleotide type defined by 1) base sequence; 2) backbone bond pattern; 3) backbone chiral center pattern; and 4) backbone phosphorus modification pattern, where the oligonucleotides of the particular oligonucleotide type are enriched compared to substantially racemic preparations of oligonucleotides having the same base sequence, backbone bond pattern, and backbone phosphorus modification pattern.As one skilled in the art will readily appreciate, such enhancement may be characterized by a higher level of the bonded phosphorus having the desired stereochemistry in each chiral controlled internucleotide linkage, as compared to a substantially racemic preparation. In some embodiments, each chiral controlled internucleotide linkage independently has a diastereomeric purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with respect to its chiral bonded phosphorus. In some embodiments, each independently has a diastereomeric purity of at least 90%. In some embodiments, each independently has a diastereomeric purity of at least 95%. In some embodiments, each independently has a diastereomeric purity of at least 97%. In some embodiments, each independently has a diastereomeric purity of at least 98%. In some embodiments, the oligonucleotides have the same chemical structure. In some embodiments, the oligonucleotides have the same chemical structure and stereochemistry, and are structurally identical.

[0131] In some embodiments, multiple oligonucleotides in a chiral controlled oligonucleotide composition share the same base sequence, the same nucleobase, sugar and internucleotide linkage modifications, if present, and independently the same stereochemistry (Rp or Sp) at the linking phosphorus chiral center of one or more chiral controlled internucleotide linkages, although the stereochemistry of a particular linking phosphorus chiral center may differ. In some embodiments, about 0.1% to 100% (e.g., about 1% to 100%, 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%, 10 ... %, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 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 multiple oligonucleotides. In some embodiments, about 0.1% to 100% (e.g., about 1% to 100%, 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 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 0.1% to 100% (e.g., about 1% to 100%, 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%, 10 ... 100%, 50% to 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 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, all of the oligonucleotides in the chiral controlled oligonucleotide composition or all of the oligonucleotides in the composition that share a common base sequence (e.g., among multiple oligonucleotides or oligonucleotide types) share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern (e.g., among multiple oligonucleotides or oligonucleotide types) or all of the oligonucleotides in the composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and / or a common internucleotide linkage modification pattern (e.g., among multiple oligonucleotides or oligonucleotide types). or about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, %, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 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 percentage is at least (DP). NCIwhere DP is a percentage selected from 85% to 100% and NCI is the number of chiral controlled internucleotide linkages. In some embodiments, DP is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, DP is at least 85%. In some embodiments, DP is at least 90%. In some embodiments, DP is at least 95%. In some embodiments, DP is at least 96%. In some embodiments, DP is at least 97%. In some embodiments, DP is at least 98%. In some embodiments, DP is at least 99%. In some embodiments, DP reflects the diastereopurity of the linking phosphorus chiral center of the chiral controlled internucleotide linkage. In some embodiments, the internucleotide linkage is at least 97%. In some embodiments, the internucleotide linkage is at least 97%. In some embodiments, the internucleotide linkage is at least 99%. In some embodiments, the internucleotide linkage is at least 97 ... The diastereomeric purity of the linker chiral center of the oligonucleotide linkage can typically be assessed using an appropriate dimer comprising such an internucleotide linkage and two nucleoside units linked by the internucleotide linkage. In some embodiments, the oligonucleotides have about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30 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 with the same stereochemistry. In some embodiments, the plurality of oligonucleotides comprises between about 0.1% and 100% (e.g., between about 1% and 100%, between about 5% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 95% and 100%, between 50% and 90%, or between about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 50%, 60%, 70%, 80%, 90%, 100%, 95%, 10%, 5 ... %, 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% have the same stereochemistry. 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, not all of the chiral internucleotide linkages are chiral controlled internucleotide linkages and the composition is a partially chiral controlled oligonucleotide composition. In some embodiments, the chiral controlled oligonucleotide composition comprises a predetermined level of each oligonucleotide or nucleic acid type.For example, in some embodiments, the chiral controlled oligonucleotide composition comprises one oligonucleotide type at a predetermined level (e.g., as described above). In some embodiments, the chiral controlled oligonucleotide composition comprises two or more oligonucleotide types, each independently at a predetermined level. In some embodiments, the chiral controlled oligonucleotide composition comprises multiple oligonucleotide types, each independently at a predetermined level. In some embodiments, the chiral controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which comprises a plurality of oligonucleotides of that oligonucleotide type at a predetermined level. In some embodiments, the chiral controlled oligonucleotide composition is a chiral controlled DMD oligonucleotide composition comprising a plurality of DMD oligonucleotides. In some embodiments, the chiral controlled oligonucleotide composition is a composition of oligonucleotides of a DMD oligonucleotide type.

[0132] Chirally pure: As used herein, the phrase "chirally pure" is used to describe an oligonucleotide or composition thereof in which all or nearly all of the oligonucleotide molecules (the remainder being impurities) are present in a single diastereomeric form with respect to the bound phosphorus atom. In many embodiments, as will be understood by those skilled in the art, a chirally pure oligonucleotide composition is substantially pure in that substantially all of the oligonucleotides in the composition are structurally identical (the same stereoisomer).

[0133] Linked phosphorus: As defined herein, the phrase "linked phosphorus" is used to refer to the particular phosphorus atom being referred to as being a phosphorus atom present in an internucleotide linkage, which corresponds to the phosphorus atom of a natural phosphate linkage as present in naturally occurring DNA and RNA. In some embodiments, the linked phosphorus atom is in a modified internucleotide linkage. In some embodiments, the linked phosphorus atom is chiral.

[0134] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to a linkage, typically a phosphorus-containing linkage, between nucleotide units of a nucleic acid or oligonucleotide, and is synonymous with "intersugar linkage," "internucleotide linkage," and "phosphorus atom bridge," as used above and herein. As will be understood by those of skill in the art, naturally occurring DNA and RNA contain natural phosphate linkages. In some embodiments, the internucleotide linkage is a natural phosphate linkage (-OP(O)(OH)O-, typically in its anionic form, e.g., at a pH of about 7.4, -OP(O)(OH)O-, as found in naturally occurring DNA and RNA molecules. - )O-). In some embodiments, the internucleotide linkage is a modified internucleotide linkage (or a non-natural internucleotide linkage) that is structurally different from, but may be substituted for, a natural phosphate linkage, such as a phosphorothioate internucleotide linkage, a PMO linkage, etc. In some embodiments, the internucleotide linkage is a modified internucleotide linkage in which one or more oxygen atoms of a natural phosphodiester linkage are independently replaced by one or more organic or inorganic moieties. In some embodiments, such organic or inorganic moieties include, but are not limited to, =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 below. In some embodiments, the internucleotide linkage is a phosphotriester linkage. In some embodiments, the internucleotide linkage is a phosphorothioate diester linkage (phosphorothioate internucleotide linkage, [ka] , typically in its anionic form -OP(O)(S -)O-). It will be understood by those skilled 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 internucleotide linkage is a non-negatively charged internucleotide linkage at the given pH. In some embodiments, the internucleotide linkage is a neutral internucleotide linkage at the given pH. In some embodiments, the given pH is about pH 7.4. In some embodiments, the given pH is in the range of about pH 0, 1, 2, 3, 4, 5, 6, or 7 to about pH 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the given pH is in the range of pH 5 to 9. In some embodiments, the given pH is in the range of pH 6 to 8. In some embodiments, the internucleotide linkage is, for example, one of a PNA (peptide nucleic acid) linkage or a PMO (phosphorodiamidate morpholino oligomer) linkage. In some embodiments, the internucleotide linkage comprises a chiral phosphorus linkage. In some embodiments, the internucleotide linkage is a chiral controlled internucleotide linkage. In some embodiments, the internucleotide linkage is selected from s(phosphorothioate), s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, or s18, where each of s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, and s18 is independently as described in WO2017 / 062862.

[0135] Unless otherwise specified, salts, e.g., pharma- ceutically acceptable acid or base addition salts, stereoisomeric forms, and tautomeric forms of a compound (e.g., DMD oligonucleotide, drug, etc.) are included. Unless otherwise specified, the singular forms "a," "an," and "the" are used. includes plural references (and vice versa) unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" may include a plurality of such compounds. [Brief description of the drawings]

[0136] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] An example of an HELISA assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0137] Detailed Description of Specific Embodiments Synthetic oligonucleotides provide useful molecular tools in a wide variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research and novel nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their vulnerability to endonucleases and exonucleases. Therefore, various synthetic counterparts have been developed to circumvent these drawbacks. This includes synthetic oligonucleotides that contain chemical modifications, such as base modifications, sugar modifications, backbone modifications, etc., that among other things make such molecules less susceptible to degradation and improve other properties of oligonucleotides, such as DMD oligonucleotides. Chemical modifications may also lead to certain undesirable effects, such as increased toxicity. From a structural standpoint, modifications to the natural phosphate linkages can introduce chirality, and certain properties of oligonucleotides can be influenced by the configuration of the phosphorus atoms that form the backbone of the oligonucleotide.

[0138] In some embodiments, the present disclosure relates to DMD oligonucleotides or DMD oligonucleotide compositions having a sequence at least partially complementary to a DMD target nucleic acid, and in some embodiments, capable of mediating skipping of a DMD exon. In some embodiments, the DMD oligonucleotides or DMD oligonucleotide compositions are capable of mediating skipping of DMD exon 51.

[0139] In some embodiments, a DMD oligonucleotide or DMD oligonucleotide composition comprises any of a variety of modifications to the internucleotide linkages (eg, backbone), sugars and / or nucleobases.

[0140] In some embodiments, the DMD oligonucleotide or DMD oligonucleotide composition is any DMD oligonucleotide or DMD oligonucleotide composition disclosed herein (eg, Table A1).

[0141] In some embodiments, the chirality of the backbone (e.g., the configuration of the phosphorus atom) or the inclusion of natural phosphate linkages or non-natural internucleotide linkages in the backbone and / or sugar and / or nucleobase modifications and / or addition of chemical moieties can affect the properties and activity of the DMD oligonucleotide, such as the ability of a DMD oligonucleotide (e.g., a DMD oligonucleotide antisense to a dystrophin (DMD) DMD transcript sequence) to skip DMD exon 51 and / or other properties of the DMD oligonucleotide, including, but not limited to, increased stability, improved pharmacokinetics, and / or reduced immunogenicity, etc. Assays suitable for evaluating the properties and / or activity of the provided compounds, such as DMD oligonucleotides and compositions thereof, are widely known in the art and can be utilized in the present disclosure.

[0142] In some embodiments, the DMD transcript is a pre-mRNA. In some embodiments, the splicing product is a mature RNA. In some embodiments, the splicing product is an mRNA. In some embodiments, the modulation or alteration of splicing comprises skipping of DMD exon 51.

[0143] In some embodiments, the provided DMD oligonucleotide, e.g., a plurality of DMD oligonucleotides in a provided composition, comprises a base modification, a sugar modification, and / or an internucleotide linkage modification. In some embodiments, the provided DMD oligonucleotide comprises a base modification and a sugar modification. In some embodiments, the provided DMD oligonucleotide comprises a base modification and an internucleotide linkage modification. In some embodiments, the provided DMD oligonucleotide comprises a sugar modification and an internucleotide linkage modification. In some embodiments, the provided composition comprises a base modification, a sugar modification, and an internucleotide linkage modification. Exemplary chemical modifications, such as base modifications, sugar modifications, and internucleotide linkage modifications, are widely known in the art, including, but not limited to, those described in this disclosure. In some embodiments, the modified base is a substituted A, T, C, G, or U. In some embodiments, the sugar modification is a 2'-modification. In some embodiments, the 2'-modification is a 2-F modification. In some embodiments, the 2'-modification is a 2'-OR 1 where R 1 is not hydrogen. In some embodiments, the 2'-modification is 2'-OR 1 where R 1 is an optionally substituted alkyl. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring having 5-20 ring atoms, where one or more ring atoms are optionally and independently a heteroatom. Exemplary cyclic structures are widely known in the art, such as those found in BNAs, LNAs, etc.

[0144] In some embodiments, the DMD oligonucleotides provided include one or more modified internucleotide linkages. In some embodiments, the DMD oligonucleotides provided include one or more chiral modified internucleotide linkages. In some embodiments, the DMD oligonucleotides provided include one or more chiral controlled chiral modified internucleotide linkages. In some embodiments, the DMD oligonucleotides provided include one or more natural phosphate linkages. In some embodiments, the DMD oligonucleotides provided include one or more modified internucleotide linkages and one or more natural phosphate linkages. In some embodiments, the modified internucleotide linkages are phosphorothioate linkages. In some embodiments, each modified internucleotide linkage is a phosphorothioate linkage.

[0145] In some embodiments, the DMD oligonucleotides provided contain both one or more modified internucleotide linkages and one or more natural phosphate linkages. In some embodiments, the DMD oligonucleotides and compositions thereof containing both modified internucleotide linkages and natural phosphate linkages provide improved properties, such as exon 51 skipping and toxicity. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the modified internucleotide linkage is a substituted phosphorothioate linkage.

[0146] In particular, the present disclosure encompasses the recognition that stereorandom DMD oligonucleotide formulations include multiple individual chemical entities that differ from one another, for example, in terms of the stereochemical configuration of individual backbone-linked phosphorus chiral centers within the DMD oligonucleotide chain. In the absence of control of the stereochemistry of the backbone chiral centers, stereorandom DMD oligonucleotide formulations provide uncontrolled compositions that include indeterminate levels of DMD oligonucleotide stereoisomers with respect to the uncontrolled chiral centers, e.g., the chiral linking phosphorus. Even though these stereoisomers may have the same base sequence, they are different chemical entities at least due to their different backbone stereochemistry, and they have different characteristics, as demonstrated herein. The present disclosure may have various properties, such as skipping of exon 51, toxicity, etc. Among other things, the present disclosure provides novel DMD oligonucleotide compositions in which the stereochemistry of one or more linker phosphorus chiral centers is independently controlled (e.g., at a chiral controlled internucleotide linkage). In some embodiments, the present disclosure provides chiral controlled DMD oligonucleotide compositions that are or contain a particular stereoisomer of a DMD oligonucleotide of interest.

[0147] In some embodiments, the pattern of backbone chiral centers in DMD oligonucleotides can result in improved one or more activities or improved one or more properties, including, but not limited to, improved skipping of DMD exon 51, increased stability, increased activity, reduced toxicity, reduced immune response, improved protein binding profile, increased binding to specific proteins, and / or enhanced delivery.

[0148] In some embodiments, the provided DMD oligonucleotides comprise one or more non-negatively charged internucleotide linkages. In some embodiments, the non-negatively charged internucleotide linkages are positively charged internucleotide linkages. In some embodiments, the non-negatively charged internucleotide linkages are neutral internucleotide linkages. In some embodiments, the modified internucleotide linkages (e.g., non-negatively charged internucleotide linkages) comprise an optionally substituted guanidine moiety. In some embodiments, the modified internucleotide linkages comprise an optionally substituted cyclic guanidine moiety. In some embodiments, the modified internucleotide linkages comprise an optionally substituted cyclic guanidine moiety, [ka] where W is O. In some embodiments, a non-negatively charged internucleotide linkage (e.g., a neutral internucleotide linkage) has the structure: [ka] wherein each variable is independently as described herein. In some embodiments, two R 1 (either on the same or different nitrogen atoms) is R and, together with their intervening atoms, forms a ring that is optionally substituted as described herein. In some embodiments, a non-negatively charged internucleotide linkage (e.g., a neutral internucleotide linkage) is [ka] wherein each variable is independently as described herein. In some embodiments, W is O. In some embodiments, such internucleotide linkages are chiral controlled. Various variables, such as R 1 , R', R sUseful embodiments such as those described in U.S. Patent Application No. 62 / 776,432, WO 2019 / 200185 and WO 2019 / 217784, the descriptions of which, including embodiments of each variable, are independently incorporated by reference into this specification.

[0149] In some embodiments, the non-negatively charged internucleotide linkage is stereochemically controlled.

[0150] In some embodiments, provided DMD oligonucleotides can bind to a DMD transcript and alter the splicing pattern of the DMD transcript by inducing (e.g., mediating) skipping of exon 51. In some embodiments, provided DMD oligonucleotides provide exon skipping of an exon with increased efficiency compared to a comparable DMD oligonucleotide under one or more suitable conditions, e.g., as described herein. In some embodiments, the skipping efficiency provided is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more times greater than a comparable DMD oligonucleotide under one or more suitable conditions, e.g., as described herein.

[0151] In some embodiments, the chiral controlled DMD oligonucleotide compositions provided are surprisingly effective when compared to a reference condition. In some embodiments, the change is measured by an increase in desirable mRNA levels compared to a reference condition. In some embodiments, the change is measured by a decrease in undesirable mRNA levels compared to a reference condition. In some embodiments, the reference condition is the absence of DMD oligonucleotide treatment. In some embodiments, the reference condition is a stereorandom composition of DMD oligonucleotides with the same base sequence and chemical modification.

[0152] In some embodiments, the provided DMD oligonucleotide composition is characterized in that when it is contacted with a DMD transcript in a DMD transcript splicing system, splicing of the DMD transcript is altered (e.g., exon 51 is skipped) relative to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof. In some embodiments, the desired splicing product (e.g., one lacking exon 51) 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. In some embodiments, the desired splicing reference is not present under the reference conditions (e.g., cannot be reliably detected by quantitative PCR). In some embodiments, as exemplified in the present disclosure, the level of the plurality of DMD oligonucleotides, eg, a plurality of DMD oligonucleotides, in a provided composition is predetermined.

[0153] In some embodiments, DMD oligonucleotides that share a common base sequence may have the same pattern of nucleoside modifications, such as sugar modifications, base modifications, etc. In some embodiments, the pattern of nucleoside modifications may be represented by a combination of positions and modifications. In some embodiments, non-chiral linkages (e.g., PO) may be omitted altogether. In some embodiments, DMD oligonucleotides having the same base sequence have the same chemical constitution.

[0154] In some embodiments, the DMD oligonucleotide compositions are chiral controlled.

[0155] In some embodiments, the DMD oligonucleotide compositions are not stereorandom and are not racemic preparations of diastereomers.

[0156] As will be appreciated by those skilled in the art, stereorandom or racemic formulations of DMD oligonucleotides are typically prepared by non-stereoselective and / or low stereoselective coupling of nucleotide monomers without the use of any chiral auxiliary, chiral modifying reagent and / or chiral catalyst. In some embodiments, in a substantially racemic (or non-chiral controlled) formulation of a DMD oligonucleotide, all or most of the coupling steps are not chirally controlled in that the coupling steps are not specifically performed to provide enhanced stereoselectivity. An exemplary substantially racemic formulation of a DMD oligonucleotide is the formulation of a phosphorothioate DMD oligonucleotide through sulfurization of a phosphite triester with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) from the commonly used phosphoramidite DMD oligonucleotide synthesis, a method well known in the art. In some embodiments, a substantially racemic formulation of a DMD oligonucleotide provides a substantially racemic DMD oligonucleotide composition (or a non-chiral controlled DMD oligonucleotide composition). In some embodiments, at least one coupling of the nucleotide monomers has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, each internucleotide linkage independently has a diastereoselectivity of less than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, the diastereoselectivity is less than about 60:40. In some embodiments, the diastereoselectivity is less than about 70:30. In some embodiments, the diastereoselectivity is less than about 80:20. In some embodiments, the diastereoselectivity is less than about 90:10. In some embodiments, the diastereoselectivity is less than about 91:9. In some embodiments, at least one internucleotide linkage has a diastereoselectivity of less than about 90:10.In some embodiments, each internucleotide linkage independently has a diastereoselectivity of less than about 90:10. In some embodiments, the non-chiral controlled internucleotide linkage has a diastereomeric purity of 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 55% or less. In some embodiments, the purity is 90% or less. In some embodiments, the purity is 85% or less. In some embodiments, the purity is 80% or less.

[0157] In contrast, in chiral controlled DMD oligonucleotide compositions, such as those of the DMD oligonucleotides of the chiral controlled DMD oligonucleotide compositions, at least one, and typically each, chiral controlled internucleotide linkage independently has a diastereomeric purity about the chiral linkage phosphorus of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater. In some embodiments, the diastereomeric purity is 95% or greater. In some embodiments, the diastereomeric purity is 96% or greater. In some embodiments, the diastereomeric purity is 97% or greater. In some embodiments, the diastereomeric purity is 98% or greater. In some embodiments, the diastereomeric purity is 98% or greater. In embodiments, the diastereomeric purity is 99% or greater. Notably, the techniques of the present disclosure routinely provide chiral controlled internucleotide linkages of high diastereomeric purity.

[0158] As will be appreciated by those skilled in the art, the diastereoselectivity of coupling or the diastereomeric purity of the internucleotide linkage (diastereomeric purity) can be assessed by the diastereoselectivity of dimer formation / diastereomeric purity of the internucleotide linkage of the formed dimer under the same or equivalent conditions, where the dimers have the same 5'- and 3'-nucleoside and internucleotide linkages.

[0159] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides comprises: 1) a common base sequence, and 2) independently, the same bond phosphorus stereochemistry in 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, 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages"). Share.

[0160] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides comprises: 1) a common base sequence, and 2) independently, the same bond phosphorus stereochemistry in 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, 25 or more) chiral internucleotide linkages ("chiral controlled internucleotide linkages"). Share; The composition is enriched for a plurality of oligonucleotides as compared to substantially racemic preparations of oligonucleotides sharing a common base sequence.

[0161] 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 ... 0%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc.) are 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%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc., are 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%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (e.g., 80%-90%, 85%-90%, 95% or 100%) are chiral controlled. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage 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.

[0162] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, which are enriched for the oligonucleotide and / or its pharma- ceutically acceptable salt form as compared to substantially racemic preparations of the oligonucleotide.

[0163] In some embodiments, at least about 5% to 100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition are multiple oligonucleotides. In some embodiments, at least about 5% to 100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition that are the same base sequence (e.g., a common base sequence) are multiple oligonucleotides. In some embodiments, the enrichment compared to a substantially racemic formulation is that at least about 5%-100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of all oligonucleotides in the composition that are the same base sequence (e.g., a common base sequence) are ...). In some embodiments, the disclosure provides compositions of oligonucleotides, wherein at least about 5% to 100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 10 ... 7%, 98%, or 99% are each independently an oligonucleotide in one or more of its various forms (e.g., acid, base, various salt forms, etc.). In some embodiments, the disclosure provides compositions of oligonucleotides, wherein at least about 5%-100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition are each independently an oligonucleotide or a pharma- ceutically acceptable salt thereof. In some embodiments, the disclosure provides compositions of oligonucleotides, wherein at least about 5%-100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition that are the same base sequence as the oligonucleotide are each independently an oligonucleotide in one or more of its various forms (e.g., acid, base, various salt forms, etc.). In some embodiments, the disclosure provides a composition of oligonucleotides, wherein at least about 5%-100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition that are the same base sequence as the oligonucleotide are each independently an oligonucleotide or a pharma- ceutically acceptable salt thereof. In some embodiments, the disclosure provides a composition of oligonucleotides, wherein at least about 5%-100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition that are of the same base sequence and have the same pattern of nucleobase, sugar and / or internucleotide linkage modifications (if present) as the oligonucleotide, are each independently an oligonucleotide in one or more of its various forms (e.g., acid, base, various salt forms, etc.).In some embodiments, the disclosure provides a composition of oligonucleotides, wherein at least about 5%-100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition that are of the same base sequence and have the same nucleobase, sugar and / or internucleotide linkage modification (if present) pattern as the oligonucleotide are each independently an oligonucleotide or a pharma- ceutically acceptable salt thereof. In some embodiments, at least about 5%-100%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of all oligonucleotides in the composition that share one or more characteristics (e.g., as described above) with the oligonucleotide are one or more pharma- ceutically acceptable salts of the oligonucleotide. In some embodiments, the composition comprises only one pharma- ceutically acceptable salt of the oligonucleotide. In some embodiments, the composition comprises two or more pharma- ceutically acceptable salts of the oligonucleotide. In some embodiments, the composition is a liquid composition, and the oligonucleotide and / or one or more salt forms thereof are dissolved. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage 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 95%. In some embodiments, the base sequence of the oligonucleotide is or includes a sequence in Table A1. In some embodiments, the oligonucleotide includes one or more natural phosphate linkages, one or more phosphorothioate internucleotide linkages, and one or more neutral internucleotide linkages.In some embodiments, the oligonucleotides are those described in Table A1, where each chiral oligonucleotide is independently Rp or Sp.

[0164] In some embodiments, the present disclosure provides: 1) Common base sequence; 2) a common backbone bond pattern; and 3) Patterns of common skeletal chiral centers and wherein the oligonucleotides are provided herein (e.g., Table A1).

[0165] In some embodiments, the present disclosure provides chiral controlled DMD oligonucleotide compositions of a plurality of DMD oligonucleotides, wherein the compositions are enriched for DMD oligonucleotides of a single DMD oligonucleotide type, as compared to substantially racemic preparations of the same DMD oligonucleotide. In some embodiments, the present disclosure provides chiral controlled DMD oligonucleotide compositions of a plurality of DMD oligonucleotides, wherein the compositions are enriched for DMD oligonucleotides of a single DMD oligonucleotide type, as compared to substantially racemic preparations of the same DMD oligonucleotide. 1) Nucleotide sequence; 2) pattern of skeletal bonds; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification The DMD oligonucleotides are enriched for a single DMD oligonucleotide type defined by:

[0166] In some embodiments, the present disclosure provides: 1) Nucleotide sequence; 2) pattern of skeletal bonds; 3) the pattern of backbone chiral centers; and 4) Pattern of backbone phosphorus modification and a chiral controlled DMD oligonucleotide composition comprising a plurality of DMD oligonucleotides of a particular DMD oligonucleotide type defined by: wherein the composition is enriched for a DMD oligonucleotide of a particular DMD oligonucleotide type relative to a substantially racemic preparation of a DMD oligonucleotide having the same base sequence and length, where the oligonucleotide is provided herein (e.g., Table A1).

[0167] In some embodiments, the DMD oligonucleotides of a certain DMD oligonucleotide type have a common pattern of backbone phosphorus modifications and a common sugar modification pattern. In some embodiments, the DMD oligonucleotides of a certain DMD oligonucleotide type have a common pattern of backbone phosphorus modifications and a common base modification pattern. In some embodiments, the DMD oligonucleotides of a certain DMD oligonucleotide type have a common pattern of backbone phosphorus modifications and a common nucleoside modification pattern. In some embodiments, the DMD oligonucleotides of a particular type have the same chemical composition. In some embodiments, the DMD oligonucleotides of a certain DMD oligonucleotide type are identical.

[0168] In some embodiments, the chiral controlled DMD oligonucleotide composition comprises It is a substantially pure preparation of a DMD oligonucleotide type in that the DMD oligonucleotides in the composition that are not DMD oligonucleotide types are impurities from the preparation process of said DMD oligonucleotide types, in some cases following certain purification procedures.

[0169] In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the DMD oligonucleotides in the composition have a common base sequence, a common backbone bond pattern and a common pattern of backbone chiral centers.

[0170] In some embodiments, DMD oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications. In some embodiments, DMD oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, DMD oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, DMD oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, DMD oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are identical.

[0171] In some embodiments, the purity of a chiral controlled DMD oligonucleotide composition of a certain DMD oligonucleotide type is expressed as the percentage of DMD oligonucleotides of that DMD oligonucleotide type in the composition. In some embodiments, at least about 10% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of that DMD oligonucleotide type. In some embodiments, at least about 20% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of that DMD oligonucleotide type. In some embodiments, at least about 30% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of that DMD oligonucleotide type. In some embodiments, at least about 40% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of that DMD oligonucleotide type. In some embodiments, at least about 50% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of that DMD oligonucleotide type. In some embodiments, at least about 60% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of that DMD oligonucleotide type. In some embodiments, at least about 70% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type.In some embodiments, at least about 80% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type.In some embodiments, at least about 90% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type.In some embodiments, at least about 92% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type.In some embodiments, at least about 94% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type.In some embodiments, at least about 95% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type.In some embodiments, at least about 96% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are the same. DMD oligonucleotide type. In some embodiments, at least about 97% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type. In some embodiments, at least about 98% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type. In some embodiments, at least about 99% of the DMD oligonucleotides in the chiral controlled DMD oligonucleotide composition are of the DMD oligonucleotide type.

[0172] In some embodiments, the purity of the chiral controlled DMD oligonucleotide composition can be controlled by the stereoselectivity of each coupling step in its preparation. In some embodiments, the coupling step has a stereoselectivity (e.g., diastereoselectivity) of 60% (60% of the new internucleotide linkages formed from the coupling step have the intended stereochemistry). After such a coupling step, the new internucleotide linkages formed can be said to have 60% purity. In some embodiments, each coupling step has a stereoselectivity of at least 60%. In some embodiments, each coupling step has a stereoselectivity of at least 70%. In some embodiments, each coupling step has a stereoselectivity of at least 80%. In some embodiments, each coupling step has a stereoselectivity of at least 85%. In some embodiments, each coupling step has a stereoselectivity of at least 90%. In some embodiments, each coupling step has a stereoselectivity of at least 91%. In some embodiments, each coupling step has a stereoselectivity of at least 92%. In some embodiments, each coupling step has a stereoselectivity of at least 93%. In some embodiments, each coupling step has at least 94% stereoselectivity. In some embodiments, each coupling step has at least 95% stereoselectivity. In some embodiments, each coupling step has at least 96% stereoselectivity. In some embodiments, each coupling step has at least 97% stereoselectivity. In some embodiments, each coupling step has at least 98% stereoselectivity. In some embodiments, each coupling step has at least 99% stereoselectivity. In some embodiments, each coupling step has at least 99.5% stereoselectivity. In some embodiments, each coupling step has virtually 100% stereoselectivity.

[0173] In some embodiments, in the provided compositions, at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% of the DMD oligonucleotides having a base sequence of a particular DMD oligonucleotide type (defined by 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications) are DMD oligonucleotides of that particular DMD oligonucleotide type. In some embodiments, at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% of the DMD oligonucleotides having a base sequence, backbone linkage pattern and backbone phosphorus modification pattern of a particular DMD oligonucleotide type are DMD oligonucleotides of that particular DMD oligonucleotide type.

[0174] In some embodiments, the DMD oligonucleotides provided include one or more chiral Modified phosphate linkages. In some embodiments, chiral controlled (and / or stereochemically pure) preparations are provided of DMD oligonucleotides that contain one or more modified backbone linkages, bases and / or sugars.

[0175] In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 80% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 85% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 90% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 91% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 92% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 93% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 94% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 95% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 96% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 97% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 98% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) formulations provided are of greater than about 99% stereochemical purity.

[0176] In some embodiments, one or more is 1. In some embodiments, one or more is 2. In some embodiments, one or more is 3. In some embodiments, one or more is 4. In some embodiments, one or more is 5. In some embodiments, one or more is 6. In some embodiments, one or more is 7. In some embodiments, one or more is 8. In some embodiments, one or more is 9. In some embodiments, one or more is 10. In some embodiments, one or more is at least 1. In some embodiments, one or more is at least 2. In some embodiments, one or more is at least 3. In some embodiments, one or more is at least 4. In some embodiments, one or more is at least 5. In some embodiments, one or more is at least 6. In some embodiments, one or more is at least 7. In some embodiments, one or more is at least 8. In some embodiments, one or more is at least 9. In some embodiments, one or more is at least 10.

[0177] In some embodiments, a base sequence, e.g., a common base sequence of multiple DMD oligonucleotides, a base sequence of a particular DMD oligonucleotide type, etc., comprises or is a sequence complementary to a gene or DMD transcript (e.g., of dystrophin or DMD). In some embodiments, the common base sequence comprises or is a sequence that is 100% complementary to a gene.

[0178] In some embodiments, the linking phosphorus of the chiral internucleotide bond is chiral controlled. In some embodiments, the chiral internucleotide bond is a phosphorothioate internucleotide bond. In some embodiments, each chiral internucleotide bond in the DMD oligonucleotide of the provided composition is a phosphorothioate internucleotide bond.

[0179] As will be appreciated by those of skill in the art, internucleotide linkages, natural phosphate linkages, phosphorothioate internucleotide linkages, and the like, may exist in their salt form depending on the pH of the environment, and unless otherwise indicated, when such internucleotide linkages are referred to, such salt forms are encompassed herein.

[0180] In some embodiments, the DMD oligonucleotides of the present disclosure comprise one or more modified sugar moieties. In some embodiments, the DMD oligonucleotides of the present disclosure comprise one or more modified base moieties. As known to those skilled in the art and described herein, various modifications can be introduced into the sugar and base moieties. For example, in some embodiments, the modifications are those described in U.S. Pat. No. 9,006,198, WO 2014 / 012081, WO 2015 / 107425 and WO 2017 / 062862 (each of which sugar and base modifications are incorporated herein by reference).

[0181] As used herein in this disclosure, unless otherwise clear from the context, (i) the word "a" or "an" may be understood to mean "at least one"; (ii) the word "or" may be understood to mean "and / or"; (iii) the words "including", "including", "including" (whether used in "without limitation") and "including" (whether used in "without limitation") may be understood to encompass the itemized component or step, whether presented alone or with one or more additional components or steps; (iv) the word "another" may be understood to mean at least an additional / second one or more; (v) the words "about" and "approximately" may be understood to allow for standard variations that will be understood by one of ordinary skill in the art; and (vi) when ranges are specified, the endpoints are included.

[0182] Unless otherwise stated, descriptions of oligonucleotides and their elements (e.g., base sequence, sugar modifications, internucleotide linkages, bond phosphorus stereochemistry, their patterns, etc.) are from 5' to 3'. As one of skill in the art will appreciate, in some embodiments, oligonucleotides can be provided and / or utilized as salt forms, particularly pharma- ceutically acceptable salt forms, such as sodium salts. As one of skill in the art will also appreciate, in some embodiments, individual oligonucleotides within a composition can be considered to be of the same composition and / or structure, even though within such a composition (e.g., a liquid composition), a particular such oligonucleotide may be in different salt forms at a particular moment (and may be dissolved, for example, in a liquid composition, and the oligonucleotide chain may be present in an anionic form). For example, one of skill in the art will appreciate that at a given pH, individual internucleotide linkages along an oligonucleotide chain may be in the acid (H) form or one of multiple possible salt forms (e.g., sodium salts or salts of different cations, depending on which ions may be present in the formulation or composition), and may be in their acid form (e.g., all cations, if present, may be H). + It will be understood that so long as the first and second oligonucleotides (replaced with ) are of the same composition and / or structure, such individual oligonucleotides may appropriately be considered to be of the same composition and / or structure.

[0183] In some embodiments, nucleobases, sugars, internucleotide linkages, and the like that can be utilized in the provided technology are described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, U.S. Patent Application Publication Nos. 2013 / 0178612, 20150211006, 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 160741, WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 055951, WO 2019 ...55951, WO 2019 / 055951, WO 2019 / 055951, WO 2019 / 055951, WO 2019 / 055951, WO 2019 / 055951, WO 2019 / 055951, WO 2019 / 055951, WO 2019 / 05 Nos. 62 / 776,432, 2019 / 200185, and / or 2019 / 217784, each of which is independently incorporated herein by reference, and the nucleobases, sugars, and internucleotide linkages. In some embodiments, various useful techniques (e.g., nucleobases, sugars, internucleotide linkages, stereochemistry and patterns thereof, base sequences, oligonucleotides, compositions, methods, etc.) are described in U.S. Patent Application No. 62 / 776,432, WO 2019 / 200185, and WO 2019 / 217784, each of which is independently incorporated herein by reference.

[0184] Dystrophin In some embodiments, the disclosure provides techniques, such as DMD oligonucleotides, compositions, methods, etc., relating to the dystrophin (DMD) gene or products encoded thereby, such as DMD transcripts, proteins (e.g., various variants of dystrophin protein), etc.

[0185] In some embodiments, the present disclosure provides technology including DMD oligonucleotides and their compositions and methods of use for the treatment of muscular dystrophies, including but not limited to Duchenne muscular dystrophy (also abbreviated as DMD) and Becker muscular dystrophy (BMD). In some embodiments, DMD comprises one or more mutations. In some embodiments, such mutations are associated with reduced biological function of dystrophin protein in subjects suffering from or susceptible to muscular dystrophy.

[0186] In some embodiments, the dystrophin (DMD) gene or a product thereof or a variant or portion thereof is selected from the group consisting of DMD, BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85 or dystrophin; External ID: OMIM:300377MGI:94909; HomoloGene:20856; GeneCards:DMD; In humans: Entrez:1 756;Ensembl:ENSG00000198947;UniProt:P11532;RefSeq(mRNA):NM_000109;NM_004006;NM_004007;NM_00 4009;NM_004010;RefSeq(Protein):NP_000100;NP_003997;NP_004000;NP_004001;NP_004002;Position (UCSC):Chr X:31.1-33.34Mb; in mouse: Entrez:13405; Ensembl:ENSMUSG00000045103; UniProt:P11531; RefSeq(mRNA):NM_007868; NM_001314034; NM_001314035; NM_001314036; NM_001314037; RefSeq(protein):NP_001300963; NP_001300964; NP_001300965; NP_001300966; NP_001300967; location (UCSC):Chr X:82.95-85.21Mb.

[0187] DMD gene reportedly contains 79 exons distributed over 2.3 million bp of gene space on X chromosome. However, it is reported that only about 14,000 bp (less than 1%) is used for protein translation (coding sequence). It is reported that about 99.5% of the gene sequence of the initial heterogeneous nuclear RNA DMD transcript of 2.3 million bp is spliced ​​out to provide a mature 14,000 bp mRNA that contains all the key information for dystrophin protein production. In some embodiments, DMD patients have one or more mutations in this DMD gene that prevent the proper assembly of wild-type DMD mRNA and / or the production of wild-type dystrophin protein, and DMD patients often show significant dystrophin deficiency in their muscles.

[0188] In some embodiments, the dystrophin DMD transcript, e.g., mRNA or protein, The qualities include those related to or resulting from alternative splicing. For example, after analysis of the splicing pattern of the DMD gene in skeletal muscle, brain and heart tissue, 16 alternative DMD transcripts of the dystrophin gene were reported. Sironi et al. 2002 FEBS Letters 517: 163-166.

[0189] Dystrophin has been reported to have several isoforms. In some embodiments, dystrophin refers to a particular isoform. At least three full-length dystrophin isoforms, each controlled by a tissue-specific promoter, have been reported. Klamut et al. 1990 Mol. Cell. Biol. 10: 193-205; Nudel et al. 1989 Nature 337: 76-78; Gorecki et al. 1992 Hum. Mol. Genet. 1: 505-510. Muscle Isoforms The dystrophin form is reportedly expressed primarily in skeletal muscle, but also in smooth and cardiac muscle [Bies, RD, Phelps, SF, Cortez, MD, Roberts, R., Caskey, CT and Chamberlain, JS 1992 Nucleic Acids Res. 20: 1725-1731], and brain dystrophin is reportedly expressed in cortical and cardiac muscle [Bies, RD, Phelps, SF, Cortez, MD, Roberts, R., Caskey, CT and Chamberlain, JS 1992 Nucleic Acids Res. 20: 1725-1731]. Dystrophin is specific to stromal neurons but can also be detected in cardiac and cerebellar neurons, while the Purkinje cell type reportedly accounts for nearly all of the cerebellar dystrophin [Gorecki et al. 1992 Hum. Mol. Genet. 1: 505-510]. Alternative splicing has been reported to If successful, this gene could provide a means of dystrophin diversification: alternative splicing of the 3' region of the gene reportedly produces tissue-specific DMD transcripts in brain neurons, cardiac Purkinje fibers, and smooth muscle cells [Bies et al. 1992 Nucleic Acids Res. 20: 1725-1731; and Feener et al. 1989 Nature 338: 509-511], whereas 12 patterns of alternative splicing have been reported in the 5' region of this gene in skeletal muscle [Surono et al. 1997 Biochem. Biophys. Res. Commun. 239: 895-899].

[0190] In some embodiments, the dystrophin mRNA, gene or protein is a revertant version. In particular, for revertant dystrophin, see, for example: Hoffman et al. 1990 J. Neurol. Sci. 99:9-25; Klein et al. 1992 Am. J. Hum. Genet. 50: 950-959; and Chelly et al. 1990 Cell 63: 1239-1348; Arahata et al. 1998 Nature 333: 861-863; Bonilla et al. 1988 Cell 54: 447-452; Fanin et al. 1992 Neur. Disord. 2: 41-45; Nicholson et al. 1989 J. Neurol. Sci. 94: 137-146; Shimizu et al. 1988 Proc. Jpn. Acad. Sci. 64: 205-208; Sicinzki et al. 1989 Science 244: 1578-1580; and Sherratt et al. Am. J. Hum. Genet. 53: 1007-1015.

[0191] All documents cited herein include supplementary data, if present.

[0192] Various mutations in the DMD gene have been reported that can and / or do cause muscular dystrophy, including some in exon 51.

[0193] Muscular dystrophies Compositions comprising one or more DMD oligonucleotides described herein can be used to treat or delay the onset of muscular dystrophy or at least one symptom thereof. In some embodiments, muscular dystrophy (MD) is any of a group of muscle conditions, diseases or disorders that cause (progressive) weakness and destruction of skeletal muscles over time. These conditions, diseases or disorders differ in which muscles are primarily affected, the degree of weakness when symptoms appear, and how quickly symptoms worsen. Many MD patients eventually have difficulty walking. In many cases, muscular dystrophy is fatal. Some types are also associated with damage to other organs, including the central nervous system. In some embodiments, muscular dystrophy is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0194] In some embodiments, the symptoms of Duchenne muscular dystrophy are reportedly: Muscle weakness with muscle wasting, affecting voluntary muscles first, especially those of the hip joint, pelvic region, thigh, shoulder, and calf. Muscle weakness may reportedly occur later in the arms, neck, and other areas. The calves are reportedly often enlarged. Symptoms reportedly usually appear by age 6 and may appear in early infancy. Other physical symptoms reportedly include awkward gait, stepping, or running (in some cases patients tend to walk on their forefoot due to increased calf muscle tone), frequent falls, fatigue, impaired motor skills (e.g., running, jumping, hopping), lumbar hyperlordosis that may lead to shortening of the hip flexors, general posture and / or abnormal gait, stepping, or running, Achilles and hamstring muscle contractures impairing function, These include progressive difficulty walking, deformities of muscle fibres, pseudohypertrophy (swelling) of the tongue and calf muscles, increased risk of neurobehavioural disorders (e.g. ADHD), learning disabilities (e.g. dyslexia) and non-progressive decline of certain cognitive skills (e.g. short-term verbal memory) that are thought to be the result of loss or dysfunction of dystrophin in the brain, eventual loss of the ability to walk (usually lost by age 12), skeletal deformities (including scoliosis in some cases) and difficulty rising from a lying or sitting position.

[0195] In some embodiments, Becker muscular dystrophy (BMD) is reportedly caused by a mutation that results in a shortened, but in-frame, DMD transcript, resulting in the production of a truncated but partially functional protein or proteins. Such partially functional protein or proteins have been reported to retain critical amino-terminal, cysteine-rich and C-terminal domains, but generally lack elements of the central rod domain that are reported to be of little functional significance. England et al. 1990 Nature, 343, 180-182.

[0196] In some embodiments, the BMD phenotype ranges from mild DMD to virtually asymptomatic depending on the details of the mutation and the level of dystrophin production. Yin et al. 2008 Hum. Mol. Genet. 17: 3909-3918.

[0197] In some embodiments, dystrophic patients with out-of-frame mutations are generally diagnosed with more severe Duchenne muscular dystrophy, and dystrophic patients with in-frame mutations are generally diagnosed with less severe Becker muscular dystrophy. However, a minority of patients with in-frame deletions are diagnosed with Duchenne muscular dystrophy, including patients with deletion mutations beginning or ending with exon 50 or 51, which encodes a portion of the hinge region, such as deletions of exons 47-51, 48-51, and 49-53. Without wishing to be bound by any particular theory, the present disclosure points out that inter-patient variability in disease severity despite the presence of the same exon deletions may reportedly be related to the effect of the specific deletion breakpoints on mRNA splicing efficiency and / or pattern; translation or DMD transcription efficiency after genome rearrangement; and stability or function of the truncated protein structure. Yokota et al. 2009 Arch. Neurol. 66: 32.

[0198] Exon skipping as a treatment for muscular dystrophies In some embodiments, the treatment of muscular dystrophy comprises the use of a DMD oligonucleotide having the ability to mediate skipping of dystrophin (DMD) exon 51. In some embodiments, the present disclosure provides a method of treating muscular dystrophy comprising administering to a subject suffering from or susceptible thereto a DMD oligonucleotide or a composition comprising a DMD oligonucleotide. In particular, among other things, the present disclosure demonstrates that chiral controlled DMD oligonucleotides / chiral controlled DMD oligonucleotide compositions are unexpectedly effective in modulating exon skipping as compared to otherwise identical but non-chiral controlled DMD oligonucleotides / oligonucleotide compositions. In some embodiments, the present disclosure provides a method of treating muscular dystrophy comprising administering to a subject suffering from or susceptible thereto a DMD oligonucleotide or a composition comprising a DMD oligonucleotide. We demonstrate that incorporation of one or more non-negatively charged internucleotide linkages into a tide can greatly improve delivery and / or overall exon skipping efficiency.

[0199] In some embodiments, treatment of muscular dystrophy utilizes the use of DMD oligonucleotides, where the DMD oligonucleotides are capable of mediating (e.g., directing) skipping of DMD exon 51. In some embodiments, the DMD oligonucleotides are capable of mediating skipping of an exon that contains a mutation (e.g., a frameshift, insertion, deletion, missense or nonsense mutation, or other mutation), where translation of the exon-skipped mRNA results in a truncated but functional (or nearly functional) DMD protein.

[0200] In some embodiments, compositions comprising DMD oligonucleotides are useful for treating dystrophin-related disorders of the central nervous system. In some embodiments, the present disclosure relates to a method for treating dystrophin-related disorders of the central nervous system, comprising administering a therapeutically effective amount of a DMD oligonucleotide to a patient suffering from a dystrophin-related disorder of the central nervous system. In some embodiments, the DMD oligonucleotide is administered outside the central nervous system (for non-limiting examples, intravenously or intramuscularly) to a patient suffering from a dystrophin-related disorder of the central nervous system, and the DMD oligonucleotide has the ability to cross the blood-brain barrier and enter the central nervous system. In some embodiments, the DMD oligonucleotide is administered directly to the central nervous system (for non-limiting examples, by intrathecal, intraventricular, intracranial, etc. delivery).

[0201] In some embodiments, the dystrophin-related disorder of the central nervous system or symptoms thereof may be any one or more of: reduced intelligence, reduced long-term memory, reduced short-term memory, language impairment, epilepsy, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder, learning disability, behavioral disorder, reduced brain volume, reduced gray matter volume, low white matter anisotropy, high white matter radial diffusivity, abnormalities in skull shape, or deleterious changes in the volume or structure of the hippocampus, globus pallidus, caudate putamen, hypothalamus, anterior commissure, periaqueductal gray matter, internal capsule, amygdala, corpus callosum, septal nucleus, nucleus accumbens, fimbria, ventricles, or midbrain thalamus. In some embodiments, patients who exhibit muscle-related symptoms of muscular dystrophy also exhibit symptoms of a dystrophin-related disorder of the central nervous system.

[0202] In some embodiments, the dystrophin-related disorder of the central nervous system is associated with, accompanied by, and / or caused by abnormalities in the level, activity, expression, and / or distribution of gene products of the dystrophin gene, such as full-length dystrophin or small isoforms of dystrophin, including but not limited to Dp260, Dp140, Dp116, Dp71, or Dp40. In some embodiments, the DMD oligonucleotide is administered to the central nervous system of a patient with muscular dystrophy for the improvement of one or more systems of dystrophin-related disorder of the central nervous system. In some embodiments, the dystrophin-related disorder of the central nervous system is associated with, accompanied by, and / or caused by abnormalities in the level, activity, expression, and / or distribution of gene products of the dystrophin gene, such as full-length dystrophin or small isoforms of dystrophin, including but not limited to Dp260, Dp140, Dp116, Dp71, or Dp40. In some embodiments, administration of a DMD oligonucleotide to a patient suffering from a dystrophin-related disorder of the central nervous system increases the level, activity and / or expression and / or improves distribution of the gene product of the dystrophin gene.

[0203] In some embodiments, the disclosure provides techniques to modulate dystrophin pre-mRNA splicing, whereby exon 51 is excised and the mutation is removed.

[0204] In some embodiments, in a DMD patient, the DMD gene contains an exon that contains a mutation, and the disorder is treated, at least in part, by skipping of DMD exon 51.

[0205] In some embodiments, the DMD patient has a mutation in one or more exons of the DMD gene or DMD transcript, which is a missense or nonsense mutation and / or a deletion, insertion, inversion, translocation or duplication.

[0206] In some embodiments, the treatment of muscular dystrophy involves skipping an exon (e.g., exon 51) of DMD, where the exon encodes a sequence of amino acids that is not essential for DMD protein function, or where the skipping can result in a fully or at least partially functional DMD protein.

[0207] In some embodiments, in the treatment of muscular dystrophy, the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, thereby generating an mRNA which can be translated into an artificial internally truncated DMD protein mutant, which results in at least partially improved or fully restored biological activity.

[0208] In some embodiments, an internally truncated DMD protein mutant produced from a dystrophin DMD transcript in which exon 51 has been skipped is more functional than a terminally truncated DMD protein produced from a dystrophin DMD transcript having an out-of-frame deletion.

[0209] In some embodiments, internally truncated DMD protein mutants produced from dystrophin DMD transcripts in which exon 51 has been skipped are highly resistant to nonsense-mediated decay mechanisms that can degrade terminally truncated DMD proteins produced from dystrophin DMD transcripts with, for example, out-of-frame deletions.

[0210] In some embodiments, the treatment of muscular dystrophy utilizes the use of a DMD oligonucleotide, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51.

[0211] In some embodiments, the present disclosure encompasses the recognition that the nature and location of a DMD mutation can be utilized to design an exon skipping strategy, in some embodiments, if a DMD patient has a mutation in an exon, skipping of that mutated exon can produce an internally truncated, but at least partially functional DMD protein variant.

[0212] In some embodiments, DMD patients have mutations that alter the splicing of DMD transcripts, for example, by inactivating sites required for splicing, or by activating cryptic sites so that they become active during splicing, or by creating alternative (e.g., non-natural) splice sites. In some embodiments, such mutations cause the production of proteins with low activity or inactivity. In some embodiments, splicing modulation, such as exon skipping, suppression of such mutations, etc., can be used to eliminate or reduce the effect of such mutations, for example, by restoring proper splicing to produce proteins with restored activity, or by producing internally truncated dystrophin protein variants with improved or restored activity.

[0213] In some embodiments, restoring the reading frame can convert an out-of-frame mutation to an in-frame mutation; in some embodiments, in humans, such an alteration can convert severe Duchenne muscular dystrophy to the milder Becker muscular dystrophy.

[0214] In some embodiments, a DMD patient or a patient suspected of having DMD is analyzed for DMD genotype prior to administration of a composition comprising a DMD oligonucleotide.

[0215] In some embodiments, a DMD patient or a patient suspected of having DMD is analyzed for a DMD phenotype prior to administration of a composition comprising a DMD oligonucleotide.

[0216] In some embodiments, DMD patients are analyzed for genotype and phenotype to determine the relationship between DMD genotype and DMD phenotype prior to administration of a composition comprising a DMD oligonucleotide.

[0217] In some embodiments, the patient is genetically confirmed to have a dystrophy prior to administration of a composition comprising a DMD oligonucleotide.

[0218] In some embodiments, analysis of the DMD genotype or genetic confirmation of a DMD or patient comprises determining whether the patient has one or more deleterious mutations in DMD.

[0219] In some embodiments, analyzing the DMD genotype or genetic confirmation of DMD or a patient comprises determining whether the patient has one or more deleterious mutations in DMD, and / or analyzing DMD splicing, and / or detecting splice variants of DMD, where the splice variants are produced by aberrant DMD splicing.

[0220] In some embodiments, analysis of the DMD genotype or genetic confirmation of DMD informs the selection of compositions comprising DMD oligonucleotides useful for treatment.

[0221] In some embodiments, the abnormal or mutant DMD gene or a portion thereof is removed or copied from a patient or one or more cells or one or more tissues of the patient, and the abnormal or mutant DMD gene or a portion thereof or copy thereof containing the abnormality or mutation is inserted into the cell. In some embodiments, the cell can be used to test various compositions containing DMD oligonucleotides to predict whether such compositions may be useful as a treatment for the patient. In some embodiments, the cell is a myoblast or a myotube.

[0222] In some embodiments, an individual or patient may produce one or more splice variants of DMD prior to treatment with a DMD oligonucleotide, often with each variant produced at very low levels, and in some embodiments, any suitable method may be used to detect low levels of splice variants produced in a patient prior to, during, or after administration of a DMD oligonucleotide.

[0223] In some embodiments, patients and / or their tissues are analyzed for the production of various splice variants of the DMD gene prior to administration of a composition comprising a DMD oligonucleotide.

[0224] In some embodiments, the disclosure provides methods for designing DMD oligonucleotides (e.g., DMD oligonucleotides capable of mediating skipping of DMD exon 51). In some embodiments, the present disclosure utilizes rational design and optionally sequence walking as described herein to design DMD oligonucleotides, e.g., for testing exon skipping in one or more assays and / or conditions. In some embodiments, effective DMD oligonucleotides are developed after rational design, including using various information for a given biological system.

[0225] In some embodiments, in the method of DMD oligonucleotide development, a DMD oligonucleotide is designed to anneal to one or more potential splicing-associated motifs and then tested for its ability to mediate exon skipping.

[0226] Exemplary Techniques for Evaluating Oligonucleotides and Oligonucleotide Compositions In the present disclosure, various techniques can be used to evaluate the properties and / or activity of DMD oligonucleotides, such as those described in U.S. Patent Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 015575, WO 2017 / 192664, WO 2017 / 062862, WO 2017 / 192679, WO 2017 / 210647, and the like.

[0227] For example, DMD oligonucleotides can be assessed for their ability to mediate exon skipping in a variety of assays, including in vitro and in vivo assays according to the present disclosure. In vitro assays can be performed in a variety of test cells described herein or known in the art, including, but not limited to, Δ48-50 patient-derived myoblasts. In vivo tests can be performed in test animals described herein or known in the art, including, but not limited to, mice, rats, cats, pigs, dogs, monkeys, or non-human primates.

[0228] As non-limiting examples, a number of assays for evaluating the properties / activity of DMD oligonucleotides are described below. A variety of other suitable assays are available and can be used to evaluate DMD oligonucleotide properties / activity, including those of DMD oligonucleotides that are not designed for exon skipping (e.g., for DMD oligonucleotides that may involve RNase H in reducing target DMD transcript levels, assays described in U.S. Patent Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 015575, WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, etc.).

[0229] DMD oligonucleotides can be assessed on dystrophin RNA for their ability to mediate exon 51 skipping, which can be tested using, as non-limiting examples, nested PCR, qRT-PCR and / or sequencing.

[0230] DMD oligonucleotides can be assessed for their ability to mediate protein restoration (e.g., production of an internally truncated dystrophin protein variant lacking the amino acid corresponding to the codon encoded in the skipped exon, which has improved function compared to the protein (if any) produced prior to exon skipping), which can be assessed by any number of protein detection and / or quantification methods, such as Western blot, immunostaining, etc. Antibodies against dystrophin are commercially available or can be developed as needed for the desired purpose.

[0231] DMD oligonucleotides can be assessed for their ability to mediate the production of a stable restored protein. The stability of the restored protein can be tested, in non-limiting examples, in assays for serum and tissue stability.

[0232] DMD oligonucleotides can be assessed for their ability to bind to proteins such as albumin. Exemplary related techniques include those described in, for example, WO 2017 / 015555, WO 2017 / 015575, etc.

[0233] DMD oligonucleotides can be assessed for immune activity, for example through assays for cytokine activation, complement activation, TLR9 activity, etc. Exemplary related techniques include those described in, for example, WO 2017 / 015555, WO 2017 / 015575, WO 2017 / 192679, WO 2017 / 210647, etc.

[0234] In some embodiments, the efficacy of DMD oligonucleotides can be tested, for example, by in silico analysis and prediction, cell-free extracts, cells transfected with artificial constructs, animals such as mice carrying the human dystrophin transgene or a portion thereof, normal and dystrophic human myogenic cell lines, and / or clinical trials. Normal and dystrophic human myogenic cell lines can sometimes produce different efficacy results under certain conditions, so it may be desirable to utilize more than one assay (Mitrpant et al. 2009 Mol. Ther. 17: 1418).

[0235] In some embodiments, the DMD oligonucleotides can be tested in cells in vitro. In some embodiments, the in vitro testing in cells involves delivery of one or more DMD oligonucleotides by gymnosin or delivery using a delivery agent or transfectant, many of which are known in the art and may be utilized in the present disclosure.

[0236] In some embodiments, DMD oligonucleotides can be tested in normal human skeletal muscle cells (hSkMCs) in vitro. See, e.g., Arechavala et al. 2007 Hum. Gene Ther. 18: 798-810.

[0237] In some embodiments, DMD oligonucleotides can be tested on muscle explants from DMD patients, as described, for example, in Fletcher et al. 2006 J. Gene Med. 8: 207-216; McClorey et al. 2006 Neur. Dis. 16: 583-590; and and Arechavala et al. 2007 Hum. Gene Ther. 18: 798-810.

[0238] In some embodiments, the cells are or comprise cultured muscle cells from a DMD patient, see, e.g., Aartsma-Rus et al. 2003 Hum. Mol. Genet. 8: 907-914.

[0239] In some embodiments, an individual DMD oligonucleotide may, under certain circumstances, exhibit inter-experiment variability in its ability to skip exon 51. In some embodiments, an individual DMD oligonucleotide may exhibit variability in its ability to skip one or more exons 51, depending on which cells are used, growth conditions, and other experimental factors. To control for variability, the DMD oligonucleotide to be tested and a control DMD oligonucleotide are typically evaluated under the same or substantially the same conditions.

[0240] In vitro experiments include those performed with patient-derived myoblasts. Certain results of such experiments are described herein. In certain such experiments, cells were cultured in skeletal growth medium to keep the cells in a dividing / immature myoblast state. The medium was then changed to "differentiation" medium (containing insulin and 2% horse serum) and DMD oligonucleotides were spiked into the medium for simultaneous dosing. The cells were differentiated into myotubes as they received treatment for a suitable period of time, e.g., a total of 4 days for RNA experiments and 6 days for protein experiments (such conditions were referred to as "0 day pre-differentiation" (day 0+4 for RNA, day 0+6 for protein)). (These are referred to as

[0241] Without wishing to be bound by any particular theory, the present disclosure points out that it may be desirable to know whether DMD oligonucleotides can enter mature myotubes and induce skipping in these cells as well as "immature" cells. In some embodiments, the present disclosure provides an assay to test the effect of DMD oligonucleotides in myotubes. In some embodiments, a different dosing schedule than "0-day pre-differentiation" is used, in which myoblasts are pre-differentiated into myotubes in differentiation medium for several days (4 days or 7 days or 10 days) and then DMD oligonucleotide is administered. Specific related protocols are described in Example 19.

[0242] In some embodiments, the present disclosure has demonstrated that in pre-differentiation experiments, DMD oligonucleotides (except those that are PMOs) generally result in approximately the same level of RNA skipping and dystrophin protein restoration, regardless of the number of days that cells are cultured in differentiation medium before administration. In some embodiments, the present disclosure provides DMD oligonucleotides that may be capable of entering and becoming active in myoblasts and myotubes. In some embodiments, DMD oligonucleotides are tested in vitro in Δ45-52 DMD patient cells (also referred to as D45-52 or del45-52) or Δ52 DMD patient cells (also referred to as D52 or del52) with 0, 4 or 7 days of pre-differentiation.

[0243] In some embodiments, DMD oligonucleotides can be tested in any one or more of a variety of animal models, including non-mammalian and mammalian models; non-limiting examples include Caenorhabditis, Drosophila, zebrafish, mouse, rat, cat, dog, and pig. See, for example, the review in McGreevey et al. 2015 Dis. Mod. Mech. 8: 195-213.

[0244] Exemplary uses of mdx mice include, for example, Lu et al. 2003 Nat. Med. 9: 1009; Jearawiriyapaisarn et al. 2008 Mol. Ther., 16, 1624-1629; Yin et al. 2008 Hum. Mol. Genet., 17, 3909-3918; Wu et al. 2009 Mol. Ther., 17, 864-871; Wu et al. 2008 Proc. Natl Acad. Sci. USA, 105, 14814-14819; Mann et al. 2001 Proc. Nat. Acad. Sci. USA 98: 42-47; and Gebski et al. 2003 Hum. Mol. Gen. 12: Reported in 1801-1811.

[0245] The efficacy of DMD oligonucleotides can be tested in the Golden Retriever Muscular Dystrophy (GRMD) animal model in dogs. Lu et al. 2005 Proc. Natl. Acad. Sci. USA 102:198-203; Alter et al. 2006 Nat. Med. 12:175-7; McClorey et al. 2006 Gene Ther. 13:1373-81; and Yokota et al. 2012 Nucl. Acid Ther. 22: 306.

[0246] DMD oligonucleotides can be determined in vivo in test animals for efficient delivery to various tissues (e.g., skeletal muscle, cardiac muscle and / or diaphragm muscle); in non-limiting examples, this can be tested by hybridization ELISA and testing for distribution in animal tissues.

[0247] DMD oligonucleotides can be determined in vivo in test animals for plasma PK; this can be tested by assaying, as non-limiting examples, AUC (area under the curve) and half-life.

[0248] In some embodiments, DMD oligonucleotides can be tested in vivo by intramuscular administration into a muscle of a test animal.

[0249] In some embodiments, DMD oligonucleotides can be tested in vivo by intramuscular administration into the gastrocnemius muscle of a test animal.

[0250] In some embodiments, DMD oligonucleotides can be tested in vivo by intramuscular administration into the gastrocnemius muscle of mice.

[0251] In some embodiments, DMD oligonucleotides can be tested in vivo by intramuscular administration to the gastrocnemius muscle of a mouse model transgenic for the entire human dystrophin locus. See, e.g., Bremmer-Bout et al. 2004 Mol. Ther. 10, 232-240. Please refer to.

[0252] Additional tests that can be performed to determine the effectiveness of DMO DMD oligonucleotides include central core fiber count and dystrophin positive fiber count and functional grip strength analysis.For non-limiting examples, see the experimental protocol reported in Yin et al. 2009 Hum. Mol. Genet. 18: 4405-4414.

[0253] Additional testing methods for DMD oligonucleotides include, by way of non-limiting example, those reported in Kinali et al. 2009 Lancet 8: 918; Bertoni et al. 2003 Hum. Mol. Gen. 12: 1087-1099.

[0254] Specific Examples of Oligonucleotides and Compositions In some embodiments, the present disclosure provides DMD oligonucleotides and / or DMD oligonucleotide compositions useful for various purposes, such as modulating skipping, reducing levels of DMD transcripts, improving levels of beneficial proteins, treating pathologies, diseases and disorders, etc. In some embodiments, the present disclosure provides DMD oligonucleotide compositions with improved properties, such as increased skipping of exon 51, reduced toxicity, etc. In particular, the DMD oligonucleotides of the present disclosure include chemical modifications, stereochemistry and / or combinations thereof that can improve various properties and activities of the DMD oligonucleotides. Non-limiting examples are provided in Table A1. In some embodiments, the DMD oligonucleotide types are as defined by the base sequence, backbone bond pattern, backbone chiral center pattern and backbone phosphorus modification pattern of the DMD oligonucleotides of Table A1, where the DMD oligonucleotides include at least one chiral controlled internucleotide bond (at least one R or S in "stereochemistry / bond").

[0255] In some embodiments, the disclosure relates to DMD oligonucleotides described herein, eg, in Table A1.

[0256] In the tables below, "ID" refers to the identification or oligonucleotide number; and "Description" refers to the modified sequence.

[0257] [Table 1]

[0258] [Table 2]

[0259] [Table 3]

[0260] [Table 4]

[0261]

Table 5

[0262]

Table 6

[0263]

Table 7

[0264]

Table 8

[0265]

Table 9

[0266]

Table 10

[0267]

Table 11

[0268]

Table 12

[0269]

Table 13

[0270]

Table 14

[0271] [Table 15]

[0272] [Table 16]

[0273] [Table 17]

[0274] [Table 18]

[0275] [Table 19]

[0276] [Table 20]

[0277] [Table 21]

[0278] [Table 22]

[0279] In Table A1, spaces are used to improve formatting and readability. For example, OXXX XX XXXXX XXXXX XXXX indicates the same stereochemistry as OXXXXXXXXXXXXXXXX; * S and * S denotes a phosphorothioate internucleotide linkage in which both linking phosphorus have the Sp configuration, etc.

[0280] The DMD oligonucleotides listed in Table A1 are all single stranded. As described herein, they may be used as single strands or as strands complexed with one or more other strands.

[0281] Some sequences are split onto multiple lines due to their length.

[0282] As will be understood by one of skill in the art, the nucleoside units are unmodified and, unless otherwise indicated (e.g., r, m, m5, eo, etc.), comprise unmodified nucleobases and 2'-deoxy sugars (two 2'-H); the linkages, unless otherwise indicated, are natural phosphate linkages; and the acidic / basic groups may, independently, be present in their salt forms.

[0283] ID: Oligonucleotide identification number WV-13405, WV-13406 and WV-13407 are fully PMOs (morpholino oligonucleotides). n001: Non-negative charge bond [ka] (This is stereo random unless otherwise indicated (e.g. n001R or n001S)); n001R: n001 which is chiral controlled and has the Rp configuration; n001S: n001 that is chiral controlled and has the Sp configuration; nX: In "Bond / Stereochemistry", nO or nX indicates stereorandom n001; nR: In "Bonding / Stereochemistry", nR indicates n001 which is chiral controlled and has the Rp configuration; nS: In "Bonding / Stereochemistry", nS indicates n001 which is chiral controlled and has the Sp configuration; F, f: 2'-F modification on the following nucleoside (e.g., fA( [ka] where BA is nucleobase A); m: 2'-OMe modification on the following nucleoside (e.g., mA( [ka] where BA is nucleobase A); * , PS: phosphorothioate; * R, R, Rp: phosphorothioate in the Rp conformation; * S, S, Sp: phosphorothioate in Sp conformation; X: stereorandom phosphorothioate; O, PO: phosphodiester (phosphate). If no internucleotide linkage is specified between two nucleoside units, the internucleotide linkage is a phosphodiester linkage (a natural phosphate linkage).

[0284] In some embodiments, each phosphorothioate internucleotide linkage of the DMD oligonucleotide is independently a chiral controlled internucleotide linkage. In some embodiments, the DMD oligonucleotide composition provided is a chiral controlled DMD oligonucleotide composition of the DMD oligonucleotide type listed in Table A1, wherein each phosphorothioate internucleotide linkage of the DMD oligonucleotide is independently a chiral controlled internucleotide linkage.

[0285] In some embodiments, the present disclosure provides a composition (e.g., a chiral controlled DMD oligonucleotide composition) that comprises or consists of a plurality of the provided DMD oligonucleotides. In some embodiments, all of the DMD oligonucleotides of the plurality are of the same type, i.e., all have the same base sequence, pattern of backbone linkages, pattern of backbone chiral centers, and pattern of backbone phosphorus modifications. In some embodiments, the DMD oligonucleotides of the same type are all structurally identical. In some embodiments, the provided composition comprises DMD oligonucleotides of a plurality of DMD oligonucleotide types, typically in controlled amounts. In some embodiments, the provided chiral controlled DMD oligonucleotide composition comprises a combination of two or more of the provided DMD oligonucleotide types.

[0286] In some embodiments, the DMD oligonucleotide compositions of the disclosure are chiral controlled DMD oligonucleotide compositions, wherein the sequence of a plurality of the DMD oligonucleotides comprises or consists of a base sequence listed in Table A1.

[0287] In some embodiments, the base sequence of the oligonucleotide is or comprises a sequence set forth in Table A1. In some embodiments, the base sequence is or comprises AGUUUCCUUAGUAACCACAG. In some embodiments, the base sequence is or comprises UGGCAUUUCUAGUUUGGAGA. In some embodiments, the base sequence is or comprises GGUAAGUUCUGUCCAAGCCC. In some embodiments, the base sequence is or comprises GGUAAGUUCUGUCCAAGCCC. In some embodiments, the base sequence is or comprises AUGGCAUUUCUAGUUUGGAG. In some embodiments, the base sequence is or comprises GCAUUUCUAGUUUGGAGAUG. In some embodiments, the base sequence is or comprises CAGUUUCCUUAGUAACCACA. In some embodiments, the base sequence is or comprises UUCCUUAGUAACCACAGGUU. In some embodiments, the base sequence is GUACCUCCAA In some embodiments, the base sequence is or comprises CAUCAAGGAA. In some embodiments, the base sequence is or comprises GGCAUUUCUAGUUUGGAGAU. In some embodiments, the base sequence is or comprises UGGCAGUUUCCUUAGUAACC. In some embodiments, the base sequence is or comprises GGUAAGUUCUGUCCAAGCCC. In some embodiments, the base sequence is or comprises CAACAUCAAGGAAGAUGGCA. In some embodiments, the base sequence is or comprises AUGGCAUUUCUAGUUUGGAG.

[0288] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20011.

[0289] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20052.

[0290] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20059.

[0291] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20072.

[0292] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20073.

[0293] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20074.

[0294] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20075.

[0295] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20076.

[0296] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20096.

[0297] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20097.

[0298] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20101.

[0299] In some embodiments, the disclosure provides a DMD oligonucleotide composition, wherein the DMD oligonucleotide is WV-20119.

[0300] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20011.

[0301] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20052.

[0302] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20059.

[0303] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20072.

[0304] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20073.

[0305] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20074.

[0306] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20075.

[0307] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20076.

[0308] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20096.

[0309] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20097.

[0310] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20101.

[0311] In some embodiments, the disclosure provides chiral controlled compositions of the DMD oligonucleotide WV-20119.

[0312] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20011.

[0313] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20052.

[0314] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20059.

[0315] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20072.

[0316] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20073.

[0317] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20074.

[0318] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20075.

[0319] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20076.

[0320] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20096.

[0321] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20097.

[0322] In some embodiments, the disclosure provides chiral controlled compositions of DMD oligonucleotide WV-20101.

[0323] In some embodiments, the disclosure provides chiral controlled compositions of the DMD oligonucleotide WV-20119.

[0324] As described herein, in some embodiments, the disclosure provides oligonucleotides and compositions (e.g., chiral controlled oligonucleotide compositions, pharma- ceutically acceptable compositions, etc.) useful for preventing and / or treating conditions, disorders, or diseases (e.g., BMD, DMD, etc.) that are amenable to exon skipping, e.g., exon 51 skipping. In some embodiments, the disclosure provides methods for preventing and / or treating conditions, disorders, or diseases (e.g., BMD, DMD, etc.) that are amenable to exon skipping, e.g., exon 51 skipping, comprising administering to a subject susceptible to or afflicted therewith a therapeutically effective amount of an oligonucleotide or a pharma- ceutically acceptable salt or composition thereof. In some embodiments, the oligonucleotide can be administered in a composition comprising the oligonucleotide in various forms, e.g., a liquid composition comprising one or more dissolved acid forms and / or one or more salt forms of the oligonucleotide in a buffer system. In some embodiments, the salt is a sodium salt. In some embodiments, the oligonucleotide is WV-31582 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31565 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31568 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31561 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31576 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31567 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31569 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31583 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31562 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31578 or a pharma- ceutically acceptable salt thereof.In some embodiments, the oligonucleotide is WV-31580 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31573 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31563 or a pharma- ceutically acceptable salt thereof. In some embodiments, the oligonucleotide is WV-31564 or a pharma- ceutically acceptable salt thereof. In some embodiments, the salt is a sodium salt. In some embodiments, the oligonucleotide provided has high diastereomeric purity, e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the diastereomeric purity is at least 10%. In some embodiments, In some embodiments, the diastereopurity is at least 20%. In some embodiments, the diastereopurity is at least 30%. In some embodiments, the diastereopurity is at least 40%. In some embodiments, the diastereopurity is at least 50%. In some embodiments, the diastereopurity is at least 60%. In some embodiments, the diastereopurity is at least 70%. In some embodiments, the diastereopurity is at least 80%. In some embodiments, the diastereopurity is at least 90%.

[0325] As described herein, in some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, wherein a level of all oligonucleotides in the composition (e.g., at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) each independently have the structure of a single oligonucleotide or salt thereof. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, wherein a level of all oligonucleotides sharing a common base sequence in the composition (e.g., at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) each independently have the structure of a single oligonucleotide or salt thereof. In some embodiments, the level is at least 10%. In some embodiments, the level is at least 20%. In some embodiments, the level is at least 30%. In some embodiments, the level is at least 40%. In some embodiments, the level is at least 50%. In some embodiments, the level is at least 60%. In some embodiments, the level is at least 70%. In some embodiments, the level is at least 80%. In some embodiments, the level is at least 90%. In some embodiments, each salt is independently a pharma- ceutically acceptable salt. In some embodiments, the salt is a sodium salt. In some embodiments, the single oligonucleotide is WV-31582. In some embodiments, the single oligonucleotide is WV-31565. In some embodiments, the single oligonucleotide is WV-31568. In some embodiments, the single oligonucleotide is WV-31561. In some embodiments, the single oligonucleotide is WV-31576. In some embodiments, the single oligonucleotide is WV-31567.In some embodiments, the single oligonucleotide is WV-31569. In some embodiments, the single oligonucleotide is WV-31583. In some embodiments, the single oligonucleotide is WV-31562. In some embodiments, the single oligonucleotide is WV-31578. In some embodiments, the single oligonucleotide is WV-31580. In some embodiments, the single oligonucleotide is WV-31573. In some embodiments, the single oligonucleotide is WV-31563. In some embodiments, the single oligonucleotide is WV-31564. In some embodiments, the chiral controlled oligonucleotide composition is a pharmaceutical composition comprising a therapeutically effective amount of a single oligonucleotide that may exist in various forms (e.g., an acid form and / or one or more pharma-ceutically acceptable salt forms). In some embodiments, the pharmaceutical composition may further comprise a pharma-ceutically acceptable carrier and other ingredients as described herein. In some embodiments, the pharmaceutical composition is a liquid composition, such as a buffer having an appropriate pH (eg, about 7, about 7-8, about 7.4, etc.), containing one or more dissolved oligonucleotides.

[0326] In some embodiments, such provided oligonucleotide compositions can be chiral controlled and include a plurality of such oligonucleotides, where one or more (e.g., 1, 2, 3 In some embodiments, at least one of the chiral internucleotide linkages is independently chiral controlled. ...

[0327] In some embodiments, the disclosure provides chiral controlled DMD oligonucleotide compositions, where the DMD oligonucleotide has the ability to mediate skipping of a DMD exon and is a DMD oligonucleotide.

[0328] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20011.

[0329] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20052.

[0330] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20059.

[0331] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20072.

[0332] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20073.

[0333] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20074.

[0334] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20075.

[0335] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20076.

[0336] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20096.

[0337] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20097.

[0338] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20101.

[0339] In some embodiments, the disclosure provides a chiral controlled DMD oligonucleotide composition, wherein the DMD oligonucleotide has the ability to mediate skipping of DMD exon 51, and the DMD oligonucleotide is WV-20119.

[0340] In some experiments, the provided DMD oligonucleotides can unexpectedly provide high exon 51 skipping when compared to, for example, drisapersen and / or eteplirsen. For example, various chiral controlled DMD oligonucleotide compositions each show superior, in some embodiments many-fold, ability to mediate exon 51 skipping in dystrophin compared to drisapersen and / or eteplirsen. Specific data are provided as examples in the present disclosure.

[0341] In some embodiments, when assaying exemplary DMD oligonucleotides in mice, the DMD oligonucleotides are injected intravenously into the tail vein of male C57BL / 10ScSndmdmdx mice (4-5 weeks old) at a test amount, e.g., 10 mg / kg, 30 mg / kg, etc. In some embodiments, tissues are harvested at test time points, e.g., days, e.g., 2, 7, and / or 14 days after injection, and in some embodiments, are fresh frozen in liquid nitrogen and stored at -80°C until analysis.

[0342] In the present disclosure, various assays can be used to assess DMD oligonucleotide levels. In some embodiments, hybrid ELISA is used to quantify DMD oligonucleotide levels in tissues using test article serial dilutions as standard curve: For example, in an exemplary procedure, maleic anhydride-activated 96-well plate (Pierce 15110) is coated with 50 μl of capture probe in 2.5% NaHCO3 (Gibco, 25080-094) at 500 nM for 2 hours at 37°C. The plate is then washed three times with PBST (PBS + 0.1% Tween-20) and blocked with 5% nonfat milk-PBST for 1 hour at 37°C. Test article DMD oligonucleotide is serially diluted in matrix. The standard is diluted with lysis buffer (4M guanidine; 0.33% N-lauryl sarcosine; 25 mM sodium citrate; 10 mM DTT) together with the original sample, so that the amount of DMD oligonucleotide in all samples is less than 100 ng / mL. 20 μl of diluted sample was mixed with 180 μl of 333 nM detection probe diluted in PBST and then denatured in a PCR machine (65°C, 10 min, 95°C, 15 min, 4C ∞). 50 μl of denatured sample was distributed in triplicate to blocked ELISA plates and incubated overnight at 4°C. After washing three times with PBST, 1:2000 streptavidin-AP in PBST was added at 50 μl per well and incubated for 1 h at room temperature. After washing with a large amount of PBST, 100 μl of AttoPhos (Promega S1000) was added and incubated for 10 min at room temperature in the dark. The plate was then read using a plate reader (Molecular Device, M5) with fluorescence channels: Ex 435 nm, Em 555 nm. The oligonucleotides in the samples were calculated using a four-parameter regression equation based on the standard curve.

[0343] In some embodiments, provided DMD oligonucleotides are stable in both plasma and tissue homogenates.

[0344] Exemplary dystrophin oligonucleotides and compositions for exon skipping of exon 51 In some embodiments, the present disclosure provides a DMD oligonucleotide for mediating exon 51 skipping in DMD (e.g., mouse, human, etc.), Nucleotide compositions and methods of use thereof are provided.

[0345] In some embodiments, the DMD oligonucleotides and / or compositions provided are capable of mediating exon 51 skipping.

[0346] In some embodiments, non-limiting examples of such DMD oligonucleotides and compositions include those of WV-20011, WV-20052, WV-20059, WV-20072, WV-20073, WV-20074, WV-20075, WV-20076, WV-20096, WV-20097, WV-20101, and WV-20119, as well as other DMD oligonucleotides having a base sequence comprising at least 15 contiguous bases of any of these DMD oligonucleotides.

[0347] In some embodiments, the sequence of the region of interest for exon 51 skipping differs between mouse and human.

[0348] In the present disclosure, various assays can be utilized to evaluate DMD oligonucleotides for exon skipping. In some embodiments, to test the efficacy of a particular combination of chemistry and stereochemistry of DMD oligonucleotides intended for exon 51 skipping in humans, a corresponding DMD oligonucleotide with the mouse sequence can be prepared and then tested in mice. The present disclosure recognizes that several differences exist between the human and mouse homologs of exon 51 (underlined below). [ka] where M is nt 7571 to 7630 in mouse; and H is nt 7665 to 7724 in human.

[0349] Due to these differences, slightly different DMD oligonucleotides can be prepared for mouse and human testing with respect to skipping of exon 51. As a non-limiting example, the following DMD oligonucleotide sequences can be used for human and mouse testing: [ka] [ka] Mismatches between humans and mice underline As shown in.

[0350] DMD oligonucleotides intended for treatment of human subjects may comprise the base sequence [ka] ) with a particular pattern of chemistry, internucleotide linkages, stereochemistry, and additional chemical moieties (if any). Such DMD oligonucleotides can be tested in vitro in human cells or in vivo in human subjects. However, their suitability for testing in mice may be limited, e.g., due to mismatches in the two base sequences.

[0351] The corresponding DMD oligonucleotides were [ka] and can be constructed with the same pattern of chemistry, internucleotide linkages, stereochemistry, and additional chemical moieties (if present). Such DMD oligonucleotides can be tested in vivo in mice. Several DMD oligonucleotides containing mouse base sequences have been constructed and tested.

[0352] In some embodiments, human DMD exon skipping DMD oligonucleotides can be tested in mice modified to contain a DMD gene containing the human sequence.

[0353] Various DMD oligonucleotides containing various modification patterns are described herein. The following table shows the results of testing certain DMD oligonucleotides. Generally, the numbers indicate the amount of skipping, where 100 indicates 100% skipping and 0 indicates no skipping unless otherwise indicated. To assay for exon skipping of DMD, DMD oligonucleotides were tested in vitro in Δ52 human patient-derived myoblasts and / or Δ45-52 human patient-derived myoblasts (human cells with exon 52 or exons 45-52 already deleted). In various experiments, DMD oligonucleotides were delivered by Gymnosis unless otherwise noted.

[0354] [Table 23]

[0355] [Table 24]

[0356] [Table 25]

[0357] [Table 26]

[0358] [Table 27]

[0359] Various oligonucleotides shown to induce exon 51 skipping in DMD transcripts were further tested for their ability to promote the production of the corresponding internally truncated DMD protein. Experiments measured the production of a protein recognized by an anti-dystrophin antibody (Abcam, Cambridge, MA) and of a size corresponding to that theoretically generated by transcription of the DMD transcript in which exon 51 was skipped. Experiments were performed in vitro on δ48-50 cells treated naked with 5 μM oligonucleotides for 7 days. Oligonucleotide WV-3152 (5 μM) positively increased wild-type dystrophin levels observed in wild-type (healthy) human immortalized myoblasts. When normalized, it produced 18% internally truncated DMD protein; WV-15860 (5 μM) produced 31%.

[0360] [Table 28]

[0361] [Table 29]

[0362] [Table 30]

[0363] [Table 31]

[0364] [Table 32]

[0365] [Table 33]

[0366]

Table 34

[0367]

Table 35

[0368]

Table 36

[0369]

Table 37

[0370]

Table 38

[0371]

Table 39

[0372]

Table 40

[0373]

Table 41

[0374]

Table 42

[0375]

Table 43

[0376]

Table 44

[0377] [Table 45]

[0378] [Table 46]

[0379] [Table 47]

[0380] [Table 48]

[0381] [Table 49]

[0382] [Table 50]

[0383] Additional information relating to DMD oligonucleotides, their activity, their synthesis and uses, as well as other aspects, can be found in WO 2019 / 200185 and WO 2019 / 217784, the DMD oligonucleotides of which are incorporated herein by reference.

[0384] Exemplary Methods for Preparing Oligonucleotides and Compositions In particular, the present disclosure provides techniques (methods, reagents, conditions, purification methods, etc.) for preparing oligonucleotides and oligonucleotide compositions, including chiral controlled oligonucleotides and chiral controlled oligonucleotide nucleotides. The preparation of oligonucleotides and compositions thereof may be carried out using any of the techniques described herein, including, but not limited to, those disclosed in U.S. Pat. No. 9,695,211, U.S. Pat. No. 9,605,019, U.S. Pat. No. 9,598,458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Patent Application Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 160741, WO 2017 / 192664, WO 2017 / 192664, WO 2017 / 192665, WO 2017 / 192666, WO 2017 / 192667, WO 2017 / 192668, WO 2017 / 192669 ... A variety of techniques (methods, reagents, conditions, purification methods, etc.) as described herein can be utilized, including those described in International Publication No. WO 2017 / 192679, International Publication No. WO 2017 / 210647, International Publication No. WO 2018 / 223056, International Publication No. WO 2018 / 237194, International Publication No. WO 2019 / 055951, International Publication No. WO 2019 / 200185 and / or International Publication No. WO 2019 / 217784 (the preparation techniques of each of which are incorporated herein by reference).

[0385] In some embodiments, the present disclosure provides chiral controlled oligonucleotides, such as chiral controlled DMD oligonucleotides. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than 50% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 55% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 60% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 65% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 70% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 75% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 80% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 85% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 90% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 91% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 92% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 93% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 94% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 95% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 96% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 97% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 98% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 99% pure.In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 99.5% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 99.6% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 99.7% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 99.8% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are greater than about 99.9% pure. In some embodiments, the chiral controlled DMD oligonucleotides provided are at least greater than about 99% pure.

[0386] In some embodiments, chiral controlled oligonucleotide compositions, such as chiral controlled DMD oligonucleotide compositions, are compositions designed to contain a single oligonucleotide type. In certain embodiments, such compositions contain about 50% dimers. In some embodiments, such compositions are about 50% diastereomeric purity. In some embodiments, such compositions are about 50% diastereomeric purity. In some embodiments, such compositions are about 55% diastereomeric purity. In some embodiments, such compositions are about 60% diastereomeric purity. In some embodiments, such compositions are about 65% diastereomeric purity. In some embodiments, such compositions are about 70% diastereomeric purity. In some embodiments, such compositions are about 75% diastereomeric purity. In some embodiments, such compositions are about 80% diastereomeric purity. In some embodiments, such compositions are about 85% diastereomeric purity. In some embodiments, such compositions are about 90% diastereomeric purity. In some embodiments, such compositions are about 91% diastereomeric purity. In some embodiments, such compositions are about 92% diastereomeric purity. In some embodiments, such compositions are about 93% diastereomeric purity. In some embodiments, such compositions are about 94% diastereomeric purity. In some embodiments, such compositions are about 95% diastereomeric purity. In some embodiments, such compositions are about 96% diastereomeric purity. In some embodiments, such compositions are about 97% diastereomeric pure. In some embodiments, such compositions are about 98% diastereomeric pure. In some embodiments, such compositions are about 99% diastereomeric pure. In some embodiments, such compositions are about 99.5% diastereomeric pure. In some embodiments, such compositions are about 99.6% diastereomeric pure. In some embodiments, such compositions are about 99.7% diastereomeric pure. In some embodiments, such compositions are about 99.8% diastereomeric pure. In some embodiments, such compositions are about 99.9% diastereomeric pure. In some embodiments, such compositions are at least about 99% diastereomeric pure.

[0387] In particular, the present disclosure recognizes the problems of stereoselective (non-stereorandom or racemic) preparation of oligonucleotides, such as DMD oligonucleotides. In particular, the present disclosure provides methods and reagents for stereoselective preparation of oligonucleotides containing multiple (e.g., 5, 6, 7, 8, 9, or 10 or more) internucleotide linkages, and especially for DMD oligonucleotides containing multiple (e.g., 5, 6, 7, 8, 9, or 10 or more) chiral internucleotide linkages. In some embodiments, in stereorandom or racemic formulations of oligonucleotides, such as DMD oligonucleotides, at least one chiral internucleotide linkage is formed with a diastereoselectivity of less than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral controlled formulations of oligonucleotides, such as DMD oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral controlled formulations of DMD oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 95:5. In some embodiments, for stereoselective or chiral controlled formulations of DMD oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 96:4. In some embodiments, for stereoselective or chiral controlled formulations of DMD oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 97:3. In some embodiments, for stereoselective or chiral controlled formulations of DMD oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 98:2. In some embodiments, for stereoselective or chiral controlled formulations of DMD oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 99:1. In some embodiments, the diastereoselectivity of the chiral internucleotide linkage in oligonucleotides, e.g., DMD oligonucleotides, is determined using model reactions, e.g. This can be measured through the formation of a dimer under essentially the same or equivalent conditions, where the dimer has the same internucleotide linkage as the chiral internucleotide linkage, the 5'-nucleoside of the dimer is the same as the nucleoside to the 5' terminus of the chiral internucleotide linkage, and the 3'-nucleoside of the dimer is the same as the nucleoside to the 3' terminus of the chiral internucleotide linkage.

[0388] In some embodiments, chiral controlled DMD oligonucleotide composition is a composition designed to contain multiple DMD oligonucleotide types.In some embodiments, the method of the present disclosure allows the creation of a library of chiral controlled DMD oligonucleotides, and allows any one or more chiral controlled DMD oligonucleotide types to be mixed with any one or more other chiral controlled DMD oligonucleotide types in a preselected amount to create chiral controlled DMD oligonucleotide composition.In some embodiments, the preselected amount of DMD oligonucleotide types is a composition having any one of the diastereomeric purities described above.

[0389] In some embodiments, the present disclosure provides a method for making a chiral controlled oligonucleotide (e.g., a DMD oligonucleotide), the method comprising: (1) coupling step; (2) capping step; (3) an optional modification step; (4) a deblocking step; and (5) Repeat steps (1) to (4) until the desired length is achieved. Includes.

[0390] In some embodiments, the disclosure provides a method for preparing, e.g., a DMD oligonucleotide, the method comprising one or more cycles, each of which independently comprises: (1) coupling step; (2) an optional pre-modification capping step; (3) modification step; (4) an optional post-modification capping step; and (5) Optional Deblocking Step Includes.

[0391] In some embodiments, the cycle includes one or more pre-modification capping steps. In some embodiments, the cycle includes one or more post-modification capping steps. In some embodiments, the cycle includes one or more pre- and post-modification capping steps. In some embodiments, the cycle includes one or more deprotection steps. In some embodiments, the cycle includes a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a deprotection step. In some embodiments, the cycle includes a coupling step, a pre-modification capping step, a modification step, and a deprotection step. In some embodiments, the cycle includes a coupling step, a modification step, a post-modification capping step, and a deprotection step. In some embodiments, the cycle includes a coupling step, a modification step, a post-modification capping step, and a deprotection step. In some embodiments, the one or more cycles include a coupling step, a pre-modification capping step, a modification step, and a deprotection step. In some embodiments, the one or more cycles include a coupling step, a pre-modification capping step, a modification step, and a deprotection step. In some embodiments, the one or more cycles include a coupling step, a modification step, a post-modification capping step, and a deprotection step.

[0392] In describing the methods provided, the term "cycle" refers to the process as understood by one of skill in the art. It has its usual meaning. In some embodiments, one round of steps (1) to (4) is referred to as a cycle. In some embodiments, some cycles include modifying. In some embodiments, some cycles do not include modifying. In some embodiments, some cycles include modifying and some cycles do not include modifying. In some embodiments, each cycle independently includes a modifying step. In some embodiments, each cycle does not include a cycling step.

[0393] In some embodiments, chirally pure phosphoramidites containing chiral auxiliaries are utilized to stereoselectively form chiral controlled internucleotide linkages. In the present disclosure, various phosphoramidites and chiral auxiliaries are used, such as those described in U.S. Pat. No. 9,695,211, U.S. Pat. No. 9,605,019, U.S. Pat. No. 9,598,458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Patent Application Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 160741, WO 2017 / 170742 ... In some embodiments, the phosphoramidites and chiral auxiliaries described in WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 055951, WO 2019 / 200185 and / or WO 2019 / 217784 (each of which phosphoramidites and chiral auxiliaries are incorporated herein by reference) may be utilized.

[0394] In some embodiments, such internucleotide linkages are neutral internucleotide linkages. In some embodiments, such internucleotide linkages are chiral controlled internucleotide linkages. In some embodiments, such internucleotide linkages include a chiral auxiliary moiety. In some embodiments, such internucleotide linkages do not include a chiral auxiliary moiety. In some embodiments, the chiral auxiliary moiety falls off during modification.

[0395] The provided technology offers a variety of advantages. In particular, as demonstrated herein, the provided technology can significantly improve oligonucleotide synthesis crude purity and yield, particularly for modified and / or chirally pure oligonucleotides, such as DMD oligonucleotides, that provide a number of properties and activities critical for therapeutic purposes. Due to its ability to provide unexpectedly high crude purity and yield for therapeutically important DMD oligonucleotides, the provided technology can significantly reduce manufacturing costs (e.g., through simplified purification, greatly improved overall yields, etc.). In some embodiments, the provided technology can be easily scaled up to produce DMD oligonucleotides in sufficient quantities and quality for clinical purposes. In some embodiments, the G 2 The provided technology, which includes chiral auxiliaries that include electron-withdrawing groups (e.g., PSM chiral auxiliaries), is particularly useful for the preparation of chiral controlled internucleotide linkages that include PN linkages (e.g., n001 non-negatively charged internucleotide linkages), which can greatly simplify manufacturing operations, reduce costs, and / or facilitate downstream formation.

[0396] In some embodiments, the provided techniques provide improved reagent compatibility. For example, as demonstrated in this disclosure, the provided techniques provide the flexibility to use different reagent systems for oxidation, sulfurization and / or azide reactions, particularly for chiral controlled DMD oligonucleotide synthesis.

[0397] In particular, the present disclosure provides DMD oligonucleotide compositions of high crude purity. In some embodiments, the present disclosure provides chiral controlled DMD oligonucleotide compositions of high crude purity. In some embodiments, the present disclosure provides chiral controlled DMD oligonucleotide compositions of high crude purity. In some embodiments, the present disclosure provides DMD oligonucleotides of high crude purity and / or high stereopurity.

[0398] Supports and Linkers In some embodiments, oligonucleotides can be prepared in solution.In some embodiments, oligonucleotides can be prepared using a support.In some embodiments, oligonucleotides are prepared using a solid support.Suitable supports that can be used in the present disclosure include, for example, U.S. Patent No. 9695211, U.S. Patent No. 9605019, U.S. Patent No. 9598458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Patent Application Publication No. 20170037399, International Publication No. 2017 / 015555, International Publication No. 2017 / 062862, International Publication No. 2017 / 160741, Examples of solid supports include those described in WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 055951, WO 2019 / 200185 and / or WO 2019 / 217784 (each of which solid supports is incorporated herein by reference).

[0399] In some embodiments, a linker moiety is used to link the oligonucleotide chain to the support during synthesis. Suitable linkers are widely used in the art, and are described in U.S. Patent No. 9695211, U.S. Patent No. 9605019, U.S. Patent No. 9598458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Patent Application Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 160741, WO 2017 / 170742 ... Linkers include those described in Publication No. WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 055951, WO 2019 / 200185 and / or WO 2019 / 217784 (each of which linkers are incorporated herein by reference).

[0400] In some embodiments, the linking moiety is a succinamic acid linker, or a succinic acid linker (-CO-CH 2 -CH 2 In some embodiments, the linking moiety and the nucleoside are linked together by an ester bond. In some embodiments, the linking moiety and the nucleoside are linked together by an amide bond. In some embodiments, the linking moiety links the nucleoside to another nucleotide or nucleic acid. Suitable linkers are described, for example, in Oligonucleotides And Analogues A Practical and Solid-Phase Supports for Oligonucleotide Synthesis, Pon, RT, Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28. In some embodiments, the oligonucleotide is attached to the solid support using a universal linker (UnyLinker) (Ravikumar et al., Org. Process Res. Dev., 2008, 12 (3), 399-410). In some embodiments, other universal linkers are used (Pon, RT, Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, various orthogonal linkers (such as disulfide linkers) are used (Pon, RT, Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

[0401] In particular, the present disclosure recognizes that linkers may be selected or designed to be compatible with the set of reaction conditions used in oligonucleotide synthesis. In some embodiments, the auxiliary groups are selectively removed prior to deprotection to avoid degradation of the oligonucleotide and to avoid desulfurization. In some embodiments, the DPSE group is F - Selective removal by ions In some embodiments, the present disclosure provides a method for the preparation of 0.1 M TBAF in MeCN, 0.5 M HF-Et in THF or MeCN. 3 Provided are linkers that are stable under DPSE deprotection conditions, such as N. In some embodiments, the provided linkers are as illustrated below. [ka]

[0402] solvent Synthesis of oligonucleotides is generally carried out in aprotic organic solvent. In some embodiments, the solvent is a nitrile solvent, such as acetonitrile. In some embodiments, the solvent is a basic amine solvent, such as pyridine. In some embodiments, the solvent is an ether solvent, such as tetrahydrofuran. In some embodiments, the solvent is a halogenated hydrocarbon, such as dichloromethane. In some embodiments, a mixture of solvents is used. In certain embodiments, the solvent is a mixture of any one or more of the above-mentioned solvent classes.

[0403] In some embodiments, when the aprotic organic solvent is not basic, a base is present in the reaction step. In some embodiments in which a base is present, the base is an amine base, such as pyridine, quinoline, or N,N-dimethylaniline. Exemplary other amine bases include pyrrolidine, piperidine, N-methylpyrrolidine, pyridine, quinoline, N,N-dimethylaminopyridine (DMAP), or N,N-dimethylaniline.

[0404] In some embodiments, the base is other than an amine base.

[0405] In some embodiments, the aprotic organic solvent is anhydrous. In some embodiments, the anhydrous aprotic organic solvent is freshly distilled. In some embodiments, the freshly distilled anhydrous aprotic organic solvent is a basic amine solvent, such as pyridine. In some embodiments, the freshly distilled anhydrous aprotic organic solvent is an ether solvent, such as tetrahydrofuran. In some embodiments, the freshly distilled anhydrous aprotic organic solvent is a nitrile solvent, such as acetonitrile.

[0406] Chiral Reagents / Chiral Auxiliaries In some embodiments, chiral reagents (sometimes referred to as chiral auxiliaries) are used to impart stereoselectivity in the creation of chiral controlled oligonucleotides. Many chiral reagents, also referred to herein as chiral auxiliaries by those skilled in the art, may be used in the methods of the present disclosure. Examples of such chiral reagents are described herein and in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Patent Application Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 160741, WO 2017 / 17 ... and / or WO 2019 / 217784 (each of which chiral auxiliaries are incorporated by reference).

[0407] In some embodiments, the chiral reagent has formula 3-AA: [ka] wherein each variable is independently as described herein. It is a compound of the formula:

[0408] In some embodiments of Formula 3-AA, W 1 and W 2 are independently, -NG 5 -, -O- or -S-; G 1 , G 2 , G 3 , G 4 and G 5are independently hydrogen or an optionally substituted group selected from aliphatic, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaliphatic, heterocyclyl, heteroaryl, or aryl; or G 1 , G 2 , G 3 , G 4 and G 5 Two of them are G 6 (which together form a monocyclic or polycyclic, fused or unfused, optionally substituted saturated, partially unsaturated or unsaturated carbocyclic or heteroatom-containing ring of up to about 20 ring atoms); 1 , G 2 , G 3 , G 4 and G 5 Four or less are G 6 As in the compound of formula 3-I, G 1 , G 2 , G 3 , G 4 Or G 5 Any of the is optionally substituted with an oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moiety. In some embodiments, such substitutions result in stereoselectivity in the creation of chiral controlled oligonucleotides. In some embodiments, the heteroatom-containing moiety, such as heteroaliphatic, heterocyclyl, heteroaryl, etc., has 1-5 heteroatoms. In some embodiments, the heteroatoms are selected from nitrogen, oxygen, sulfur, and silicon. In some embodiments, at least one heteroatom is nitrogen. In some embodiments, the aliphatic, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaliphatic, heterocyclyl, heteroaryl, or aryl group has 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, or 1-6 carbon atoms.

[0409] In some embodiments, W 1 -NG 5 In some embodiments, W 1 -NG 5In some embodiments, W is -O-, where -O- is bonded to -H. 1 -NG 5 In some embodiments, G 5 And, G 3 and G 4 Together with one of the two, it contains 0 to 3 heteroatoms W 1 In some embodiments, G forms an optionally substituted 3-10 membered ring having, in addition to the nitrogen atom, 5 and G 3 Together, W and W are 0 to 3 heteroatoms. 1 In addition to the nitrogen atom, optionally substituted 3 to In some embodiments, G forms a 10-membered ring. 5 and G 4 Together, W and W are 0 to 3 heteroatoms. 1 In some embodiments, the ring formed is an optionally substituted 4-, 5-, 6-, 7-, or 8-membered ring. In some embodiments, the ring formed is an optionally substituted 4-membered ring. In some embodiments, the ring formed is an optionally substituted 5-membered ring. In some embodiments, the ring formed is an optionally substituted 6-membered ring. In some embodiments, the ring formed is an optionally substituted 7-membered ring.

[0410] In some embodiments, the chiral reagent provided is [ka] In some embodiments, the chiral reagent provided has the structure: [ka] In some embodiments, the chiral reagent provided has the structure: [ka] In some embodiments, the chiral reagent provided has the structure: [ka] In some embodiments, the chiral reagent provided has the structure: [ka] In some embodiments, the chiral reagent provided has the structure: [ka] In some embodiments, the chiral reagent provided has the structure: [ka] In some embodiments, the chiral reagent provided has the structure: [ka] It has the structure:

[0411] In some embodiments, W 1 -NG 5 And W 2 is O and G 1 and G 3 Each of is independently hydrogen or C 1~10 an optionally substituted group selected from aliphatic, heterocyclyl, heteroaryl, and aryl; 2 is -C(R) 2 Si(R) 3 and G 4 and G 5and together form a monocyclic or polycyclic, fused or unfused, optionally substituted saturated, partially unsaturated, or unsaturated heteroatom-containing ring of up to about 20 ring atoms. In some embodiments, each R is independently hydrogen or C 1 ~C 6 is an optionally substituted group selected from aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. 2 is -C(R) 2 Si(R) 3 wherein -C(R) 2 - is an optionally substituted -CH 2 - and -Si(R) 3 Each R is independently 1~10 is an optionally substituted group selected from aliphatic, heterocyclyl, heteroaryl, and aryl. In some embodiments, -Si(R) 3 At least one R is independently an optionally substituted C 1~10 In some embodiments, -Si(R) is alkyl. 3 At least one R is independently an optionally substituted phenyl. In some embodiments, -Si(R) 3 One R is independently an optionally substituted phenyl, and each of the other two R is independently an optionally substituted C 1~10 In some embodiments, -Si(R) is alkyl. 3 One R is independently an optionally substituted C 1~10 In some embodiments, G is alkyl, and each of the other two R is independently an optionally substituted phenyl. 2 is an optionally substituted -CH 2 Si(Ph)(Me) 2 In some embodiments, G 2 is an optionally substituted -CH 2 Si(Me)(Ph) 2 In some embodiments, G 2 -CH 2Si(Me)(Ph) 2 In some embodiments, G 4 and G 5 and together, one nitrogen atom (and G 5 In some embodiments, G forms an optionally substituted saturated 5-6 membered ring containing 4 and G 5 and together form an optionally substituted saturated 5-membered ring containing one nitrogen atom. 1 is hydrogen. 3 is hydrogen. 1 and G 3 are both hydrogen.

[0412] In some embodiments, W 1 -NG 5 And W 2 is O and G 1 and G 3 Each of the groups independently represents R 1 and G 2 -R 1 and G 4 and G 5 and together form a monocyclic or polycyclic, fused or unfused, optionally substituted saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms, wherein R 1 is C 1~20 Aliphatic, C with 1-5 heteroatoms 1~20 aliphatic, C 6~20 Aryl, C with 1-5 heteroatoms 5~20 Heteroaryl and combinations thereof (e.g., aliphatic-aryl / heteroaryl, heteroaliphatic-aryl / heteroaryl). In some embodiments, G 1 and G 3 Each of is independently R. In some embodiments, G 1 and G 3 Each of is independently -H.2 is linked to the remainder of the molecule by a carbon atom that is substituted with one or more electron-withdrawing groups. 2 is methyl substituted with one or more electron withdrawing groups. 2 is methyl substituted with one and only one electron withdrawing group. 2 is a methyl substituted with two or more electron-withdrawing groups. 2 Chiral auxiliaries having the following structure can be readily removed by base (e.g., are base-labile under substantially water-free anhydrous conditions; in many cases, oligonucleotides containing internucleotide linkages that preferably contain such chiral auxiliaries can be easily removed under conditions / reagent systems that contain significant amounts of water (e.g., NH 4 OH) resulting in various advantages as described herein, such as high crude purity, high yield, high stereoselectivity, simpler handling, fewer steps, further manufacturing cost reduction and / or simpler downstream formulation (e.g., lower amount of salt(s) after cleavage). In some embodiments, as described in the Examples, such auxiliary groups may provide alternative or additional chemical compatibility with other functional groups and / or protecting groups. In some embodiments, as demonstrated in the Examples, base-labile chiral auxiliary groups are particularly useful for constructing chirally controlled non-negatively charged internucleotide linkages (e.g., neutral internucleotide linkages such as n001); in some instances, as demonstrated in the Examples, when utilized with removal with base under, for example, anhydrous conditions, it can result in greatly improved yield and / or crude purity with high stereoselectivity. In some embodiments, such chiral auxiliary groups are bonded to the phosphorus bond via an oxygen atom (e.g., which corresponds to an -OH group in the corresponding chiral auxiliary compound), the carbon atom in the chiral auxiliary to which that oxygen is bonded (the α carbon) is also bonded to -H (in addition to other groups; in some embodiments, a secondary carbon), and the next carbon atom in the chiral auxiliary (the β carbon) is bonded to one or two electron-withdrawing groups.2 In some embodiments, G 1 is -H. In some embodiments, G 2 may contain one or two electron withdrawing groups or otherwise facilitate removal of the chiral auxiliary by base. 1 is -H and G 2 contains one or two electron-withdrawing groups, -W 2 In some embodiments, G 1 is -H and G 2 contains one or two electron-withdrawing groups, -W 2 -H is -OH, -W 1 -H is -NG 5 -H and G 3 and G 4 One of them is G 5 together with their intervening atoms to form a ring as described herein (e.g., G 5 is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms in addition to the nitrogen atom thereon (e.g., G 5 forms an optionally substituted 3-, 4-, 5- or 6-membered monocyclic saturated ring) which has no other heteroatoms in addition to the nitrogen atom thereon.

[0413] As one of ordinary skill in the art will appreciate, a variety of electron-withdrawing groups are known in the art and can be utilized in the present disclosure. In some embodiments, the electron-withdrawing group contains a carbon atom and / or is, for example, -S(O)-, -S(O) 2 -, -P(O)(R 1 )-, -P(S)R 1 In some embodiments, the electron withdrawing group is linked to a carbon atom via -CN, -NO 2 , halogen, -C(O)R 1 , -C(O)OR', -C(O)N(R') 2 , -S(O)R 1 , -S(O) 2 R 1 , -P(W)(R 1 ) 2, -P(O)(R 1 ) 2 , -P(O)(OR') 2 Or -P(S)(R 1 ) 2 In some embodiments, the electron withdrawing group is -CN, -NO 2 , halogen, -C(O)R 1 , -C(O)OR', -C(O)N(R') 2 , -S(O)R 1 , -S(O) 2 R 1 , -P(W)(R 1 ) 2 , -P(O)(R 1 ) 2 , -P(O)(OR') 2 Or -P(S)(R 1 ) 2 1 of 1 and n is 0 or 1. The radicals are preferably aryl or heteroaryl, substituted with one or more alkyl groups, such as phenyl.

[0414] In some embodiments, G 2 is -L'-L"-R', where L' is -C(R) 2 - or optionally substituted -CH 2 -, and L" is -P(O)(R')-, -P(O)(R')O-, -P(O)(OR')-, -P(O)(OR')O-, -P(O)[N(R')]-, -P(O)[N(R')]O-, -P(O)[N(R')][N(R')]-, -P(S)(R')-, -S(O) 2 -, -S(O) 2 -, -S(O) 2 -O-, -S(O)-, -C(O)-, -C(O)N(R')-, or -S-, where each R' is independently R as described herein. 1 In some embodiments, L' is -C(R) 2 In some embodiments, L' is optionally substituted -CH 2 -It is.

[0415] In some embodiments, L' is -C(R) 2In some embodiments, each R is independently hydrogen or C 1 ~C 6 In some embodiments, L' is an optionally substituted group selected from aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. 2 In some embodiments, L″ is —P(O)(R′)—, —P(S)(R′)—, —S(O) 2 In some embodiments, G 2 -L'-C(O)N(R') 2 In some embodiments, G 2 -L'-P(O)(R') 2 In some embodiments, G 2 -L'-P(S)(R') 2 In some embodiments, each R' is independently an optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl as described herein (e.g., embodiments described for R). In some embodiments, each R' is independently an optionally substituted phenyl. In some embodiments, each R' is independently an optionally substituted phenyl, where one or more substituents are independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, each R' is independently a substituted phenyl, where one or more substituents are independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, each R' is independently a substituted phenyl, where the substituents are independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, each R' is independently a monosubstituted phenyl, where the substituents are independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, the two R's are the same. In some embodiments, the two R's are different. 2 is -L'-S(O)R'. In some embodiments, G 2 -L'-C(O)N(R') 2 In some embodiments, G 2-L'-S(O) 2 R'. In some embodiments, R' is an optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl as described herein (e.g., embodiments described for R). In some embodiments, R' is an optionally substituted phenyl. In some embodiments, R' is an optionally substituted phenyl, where one or more substituents are independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, R' is a substituted phenyl, where one or more substituents are independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, R' is a substituted phenyl, where each substituent is independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, R' is a monosubstituted phenyl. In some embodiments, R' is a monosubstituted phenyl, where the substituents are independently selected from -CN, -OMe, -Cl, -Br, and -F. In some embodiments, the substituent is an electron-withdrawing group. In some embodiments, the electron withdrawing group is -CN, -NO 2 , halogen, -C(O)R 1 , -C(O)OR', -C(O)N(R') 2 , -S(O)R 1 , -S(O) 2 R 1 , -P(W)(R 1 ) 2 , -P(O)(R 1 ) 2 , -P(O)(OR') 2 Or -P(S)(R 1 ) 2 It is.

[0416] In some embodiments, G 2 is an optionally substituted -CH 2 -L″-R, where each of L″ and R is independently as described herein. In some embodiments, G 2 is an optionally substituted -CH(-L"-R) 2wherein each of L″ and R is independently as described herein. In some embodiments, G 2 is an optionally substituted -CH(-SR) 2 In some embodiments, G 2 is an optionally substituted -CH 2 In some embodiments, two R groups together with their intervening atoms form a ring. In some embodiments, the ring formed is an optionally substituted 5-, 6-, or 7-membered ring having 0-2 heteroatoms in addition to the intervening heteroatoms. In some embodiments, G 2 is optionally replaced [ka] In some embodiments, G 2 teeth, [ka] In some embodiments, -S- can be converted to -S(O)- or -S(O)-, e.g., by oxidation, to facilitate removal, e.g., by base. 2 - can be converted to

[0417] In some embodiments, G 2 is -L'-R', where each variable is as described herein. 2 Ha-CH 2 In some embodiments, G 2 is -CH(R') 2 In some embodiments, G 2 is -C(R') 3 In some embodiments, R' is an optionally substituted aryl or heteroaryl. In some embodiments, R' is a substituted aryl or heteroaryl, where one or more of the substituents are independently an electron withdrawing group. In some embodiments, -L'- is an optionally substituted -CH 2- and R' is R, where R is an optionally substituted aryl or heteroaryl. In some embodiments, R is a substituted aryl or heteroaryl, where one or more of the substituents are independently an electron-withdrawing group. In some embodiments, R is a substituted aryl or heteroaryl, where each of the substituents is independently an electron-withdrawing group. In some embodiments, R is an aryl or heteroaryl substituted with two or more substituents, where each of the substituents is independently an electron-withdrawing group. In some embodiments, the electron-withdrawing group is -CN, -NO 2 , halogen, -C(O)R 1 , -C(O)OR', -C(O)N(R') 2 , -S(O)R 1 , -S(O) 2 R 1 , -P(W)(R 1 ) 2 , -P(O)(R 1 ) 2 , -P(O)(OR') 2 Or -P(S)(R 1 ) 2 In some embodiments, R' is [ka] In some embodiments, R' is p-NO 2 In some embodiments, And R' is [ka] In some embodiments, R' is [ka] In some embodiments, R' is [ka] In some embodiments, R' is [ka] In some embodiments, R' is [ka] In some embodiments, G 2 teeth, [ka] In some embodiments, R' is [ka] In some embodiments, R' is [ka] In some embodiments, R' is 2,4,6-trichlorophenyl. In some embodiments, R' is 2,4,6-trifluorophenyl. In G 2 -CH(4-chlorophenyl) 2 In some embodiments, G 2 is -CH(R') 2 where each R' is [ka] In some embodiments, G 2 is -CH(R') 2 where each R' is [ka] In some embodiments, R' is -C(O)R. In some embodiments, R' is CH 3 It is C(O)-.

[0418] In some embodiments, G 2 -L'-S(O) 2 R′, where each variable is as described herein. In some embodiments, G 2 Ha-CH 2 -S(O) 2 R'. In some embodiments, G 2 is -L'-S(O)R', where each variable is as described herein. 2 Ha-CH 2 In some embodiments, G 2 -L'-C(O) 2 R′, where each variable is as described herein. In some embodiments, G 2 Ha-CH 2 -C(O) 2 R'. In some embodiments, G 2 is -L'-C(O)R', where each variable is as described herein. 2 Ha-CH 2 In some embodiments, -L'- is an optionally substituted -CH 2 - and R' is R. In some embodiments, R is an optionally substituted aryl or heteroaryl. In some embodiments, R is an optionally substituted aliphatic. In some embodiments, R is an optionally substituted heteroaliphatic. In some embodiments, R is an optionally substituted heteroaryl. In some embodiments, R is an optionally substituted aryl. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is not phenyl, or is a mono-, di-, or tri-substituted phenyl, where each substituent is -NO. 2 , halogen, -CN, -C 1~3 Alkyl and C 1~3In some embodiments, R is selected from alkyloxy. In some embodiments, R is a substituted aryl or heteroaryl, where one or more of the substituents are independently an electron-withdrawing group. In some embodiments, R is a substituted aryl or heteroaryl, where each of the substituents is independently an electron-withdrawing group. In some embodiments, R is an aryl or heteroaryl substituted with two or more substituents, where each of the substituents is independently an electron-withdrawing group. In some embodiments, the electron-withdrawing group is selected from -CN, -NO 2 , halogen, -C(O)R 1 , -C(O)OR', -C(O)N(R') 2 , -S(O)R 1 , -S(O) 2 R 1 , -P(W)(R 1 ) 2 , -P(O)(R 1 ) 2 , -P(O)(OR') 2 Or -P(S)(R 1 ) 2 In some embodiments, R' is phenyl. In some embodiments, R' is substituted phenyl. In some embodiments, R' is [ka] In some embodiments, R' is [ka] In some embodiments, R' is [ka] In some embodiments, R' is an optionally substituted C 1~6 In some embodiments, R' is aliphatic. In some embodiments, R' is t-butyl. In some embodiments, R' is isopropyl. In some embodiments, R' is methyl. In some embodiments, G 2 Ha-CH 2C(O)OMe. In some embodiments, G 2 Ha-CH 2 C(O)Ph. In some embodiments, G 2 Ha-CH 2 C(O)-tBu.

[0419] In some embodiments, G 2 HA-L'-NO 2 In some embodiments, G 2 Ha-CH 2 -NO 2 In some embodiments, G 2 -L'-S(O) 2 N(R') 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(R') 2 In some embodiments, G 2 -L'-S(O) 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 In some embodiments, R' is methyl. In some embodiments, G 2 Ha-CH 2 -S(O) 2 NH(CH 3 In some embodiments, R' is -CH 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 NH(CH 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(CH 2 Ph) 2 In some embodiments, R' is phenyl. In some embodiments, G 2 Ha-CH 2 -S(O) 2 In some embodiments, the G 2 Ha-CH2 -S(O) 2 N(CH 3 ) Ph. In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(CH 3 ) 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 NH(CH 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 In some embodiments, the G 2 Ha-CH 2 -S(O) 2 NH(CH 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(CH 3 ) 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(CH 3 ) Ph. In some embodiments, G 2 -L'-S(O) 2 N(R')(OR'). In some embodiments, G 2 Ha-CH 2 -S(O) 2 In some embodiments, each R' is methyl. 2 Ha-CH 2 -S(O) 2 N(CH 3 )(OCH 3 In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(Ph)(OCH 3 In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(CH 2Ph)(OCH 3 In some embodiments, G 2 Ha-CH 2 -S(O) 2 N(CH 2 Ph)(OCH 3 In some embodiments, G 2 -L'-S(O) 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 In some embodiments, G 2 Ha-CH 2 -S(O) 2 OCH 3 In some embodiments, G 2 Ha-CH 2 -S(O) 2 OCH 2 It is Ph.

[0420] In some embodiments, G 2 -L'-P(O)(R') 2 In some embodiments, G 2 Ha-CH 2 -P(O)(R') 2 In some embodiments, G 2 is -L'-P(O)[N(R') 2 ] 2 In some embodiments, G 2 Ha-CH 2 -P(O)[N(R') 2 ] 2 In some embodiments, G 2 is -L'-P(O)[O(R') 2 ] 2 In some embodiments, G 2 Ha-CH 2 -P(O)[O(R') 2 ] 2 In some embodiments, G 2is -L'-P(O)(R')[N(R') 2 ] 2 In some embodiments, G 2 Ha-CH 2 -P(O)(R')[N(R') 2 In some embodiments, G 2 is -L'-P(O)(R')[O(R')]. In some embodiments, G 2 Ha-CH 2 -P(O)(R')[O(R')]. In some embodiments, G 2 is -L'-P(O)(OR')[N(R') 2 In some embodiments, G 2 Ha-CH 2 -P(O)(OR')[N(R') 2 In some embodiments, G 2 -L'-C(O)N(R') 2 wherein each variable is as described herein. In some embodiments, G 2 Ha-CH 2 -C(O)N(R') 2 In some embodiments, each R' is independently R. In some embodiments, one R' is an optionally substituted aliphatic and one R is an optionally substituted aryl. In some embodiments, one R' is an optionally substituted C 1~6 In some embodiments, R is an optionally substituted phenyl. In some embodiments, R' is an optionally substituted phenyl. 1~6 In some embodiments, G 2 Ha-CH 2 -P(O)(CH 3 ) Ph. In some embodiments, G 2 Ha-CH 2 -P(O)(CH 3 ) 2 In some embodiments, G 2 Ha-CH 2 -P(O)(Ph) 2 In some embodiments, G 2Ha-CH 2 -P(O)(OCH 3 ) 2 In some embodiments, G 2 Ha-CH 2 -P(O)(CH 2 Ph) 2 In some embodiments, G 2 Ha-CH 2 -P(O)[N(CH 3 )Ph] 2 In some embodiments, G 2 Ha-CH 2 -P(O)[N(CH 3 ) 2 ] 2 In some embodiments, G 2 Ha-CH 2 -P(O)[N(CH 2 Ph) 2 ] 2 In some embodiments, G 2 Ha-CH 2 -P(O)(OCH 3 ) 2 In some embodiments, G 2 Ha-CH 2 -P(O)(OPh) 2 It is.

[0421] In some embodiments, G 2 is -L'-SR'. In some embodiments, G 2 Ha-CH 2 -SR'. In some embodiments, R' is optionally substituted phenyl. In some embodiments, R' is phenyl.

[0422] In some embodiments, the chiral reagent provided is [ka] wherein each R 1 are independently as described herein. In some embodiments, the chiral reagent provided is [ka] wherein each R 1 is independently as described herein. In some embodiments, each R 1 is independently R as described herein. In some embodiments, each R 1 is independently R, where R is an optionally substituted aliphatic, aryl, heteroaliphatic, or heteroaryl as described herein. 1 is phenyl. In some embodiments, R 1 is -L-R'. In some embodiments, R 1 is -L-R', where L is -O-, -S- or -N(R'). In some embodiments, the chiral reagent provided is [ka] wherein each X 1 are independently -H, an electron-withdrawing group, -NO 2 , -CN, -OR, -Cl, -BR or -F, and W is O or S. In some embodiments, the chiral reagent provided is [ka] wherein each X 1 are independently -H, an electron-withdrawing group, -NO 2 , -CN, -OR, -Cl, -BR or -F, and W is O or S. In some embodiments, each X 1 is independently -CN, -OR, -Cl, -BR, or -F, where R is not -H. In some embodiments, R is an optionally substituted C 1~6 In some embodiments, R is an optionally substituted C 1~6 In some embodiments, R is -CH 3In some embodiments, one or more X 1 are independently an electron withdrawing group (e.g., -CN, -NO 2 , halogen, -C(O)R 1 , -C(O)OR', -C(O)N(R') 2 , -S(O)R 1 , -S(O) 2 R 1 , -P(W)(R 1 ) 2 , -P(O)(R 1 ) 2 , -P(O)(OR') 2 , -P(S)(R 1 ) 2 etc.).

[0423] In some embodiments, the chiral reagent provided is [ka] wherein R 1 is as described herein. In some embodiments, the chiral reagent provided is [ka] wherein R 1 is as described herein. In some embodiments, R 1 is R as described in this disclosure. In some embodiments, R 1 is R, where R is an optionally substituted aliphatic, aryl, heteroaliphatic, or heteroaryl as described herein. In some embodiments, R 1 is -L-R'. In some embodiments, R 1 is -L-R', where L is -O-, -S- or -N(R'). In some embodiments, the chiral reagent provided is [ka] wherein X1 -H, electron withdrawing group, -NO 2 , -CN, -OR, -Cl, -BR or -F, and W is O or S. In some embodiments, the chiral reagent provided is [ka] wherein X 1 -H, electron withdrawing group, -NO 2 , -CN, -OR, -Cl, -BR or -F, and W is O or S. 1 is -CN, -OR, -Cl, -BR or -F, where R is not -H. In some embodiments, R is an optionally substituted C 1~6 In some embodiments, R is an optionally substituted C 1~6 In some embodiments, R is -CH 3 In some embodiments, X 1 is an electron-withdrawing group (e.g., -CN, -NO 2 , halogen, -C(O)R 1 , -C(O)OR', -C(O)N(R') 2 , -S(O)R 1 , -S(O) 2 R 1 , -P(W)(R 1 ) 2 , -P(O)(R 1 ) 2 , -P(O)(OR') 2 , -P(S)(R 1 ) 2 In some embodiments, X 1 -CN, -NO 2 or a non-halogen electron withdrawing group. 1 -H, -CN, -NO 2 , halogen or C 1~3 Not alkyloxy.

[0424] In some embodiments, G 2 -CH(R 21 )-CH(R22 )=C(R 23 )(R 24 ) in which R 21 , R 22 , R 23 and R 24 Each of is independently R. In some embodiments, R 22 and R 23 are both R, and these two R groups together with their intervening atoms form an aryl or heteroaryl ring, which is optionally substituted as described herein. In some embodiments, one or more of the substituents are independently an electron-withdrawing group. In some embodiments, R 21 and R 24 are both R, and these two R groups together with their intervening atoms form a ring, which is optionally substituted as described herein. R 21 and R 24 are both R, and these two R groups together with their intervening atoms form a saturated or partially saturated ring, which is optionally substituted as described herein. 22 and R 23 are both R, and these two R groups together with their intervening atoms form an aryl or heteroaryl ring, which is optionally substituted as described herein, and R 21 and R 24 are both R, and these two R groups together with their intervening atoms form a partially saturated ring, which is optionally substituted as described herein. 21 is -H. In some embodiments, R 24 is -H. In some embodiments, G 2 is optionally replaced [ka] In some embodiments, G 2 is optionally replaced [ka] wherein each ring A 2 is independently a 3-15 membered monocyclic, bicyclic or polycyclic ring as described herein. 2 is an optionally substituted 5-10 membered monocyclic aryl or heteroaryl ring having 1-5 heteroatoms as described herein. 2 is a phenyl ring, optionally substituted as described herein. In some embodiments, G 2 is optionally replaced [ka] In some embodiments, G 2 teeth, [ka] In some embodiments, G 2 teeth, [ka] In some embodiments, G 2 teeth, [ka] It is.

[0425] In some embodiments, the chiral auxiliary is a DPSE auxiliary. In some embodiments, the chiral auxiliary is a PSM auxiliary.

[0426] In some embodiments, the corresponding compound is a compound of formula 3-I or formula 3-AA, e.g., the chiral auxiliary portion of the internucleotide linkage upon contact with a base undergoes the loss of a water molecule (G 2 -W 2 In some embodiments, such an alkene may be released as an alkene having the same structure as the product formed by the elimination of -H=-OH and α-H. 2 =C(R 1 )-LN(R 5 )(R 6 ), (electron-withdrawing group)H=C(R 1 )-LN(R 5 )(R 6 ), CH(-L”-R’)=C(R 1 )-LN(R 5 )(R 6 ) (wherein the CH group is optionally substituted) or C x =C(R 1 )-LN(R 5 )(R 6 )(wherein, C x is optionally replaced [ka] and optionally fused to one or more optionally substituted rings, and each other variable is independently as described herein. x is optionally replaced [ka] In some embodiments, C x teeth, [ka] In some embodiments, the alkene is [ka] In some embodiments, the alkene is [ka] In some embodiments, the alkene is [ka] It is.

[0427] In some embodiments, the chiral reagent is an amino alcohol. In some embodiments, the chiral reagent is an amino thiol. In some embodiments, the chiral reagent is an amino phenol. In some embodiments, the chiral reagent is (S)- and (R)-2-methylamino-1-phenylethanol, (1R,2S)-ephedrine, or (1R,2S)-2-methylamino-1,2-diphenylethanol.

[0428] In some embodiments of the present disclosure, the chiral reagent is a compound of one of the following formulas: [ka]

[0429] As will be appreciated by those of skill in the art, chiral reagents are typically stereochemically pure or substantially stereochemically pure, and are typically utilized as a single stereoisomer substantially free of other stereoisomers. In some embodiments, the compounds of the present disclosure are stereochemically pure or substantially stereochemically pure.

[0430] As demonstrated herein, stereochemically pure chiral reagents are generally utilized to obtain stereoselectivity when used to prepare chiral internucleotide linkages. In particular, the present disclosure provides stereochemically pure chiral reagents, including those having the structures depicted.

[0431] Selection of a chiral reagent, such as an isomer of formula Q or its stereoisomer, formula R, allows specific control of the chirality at the bound phosphorus. Thus, selection of either the Rp or Sp configuration in each synthesis cycle can allow control of the overall three-dimensional structure of the chiral-controlled DMD oligonucleotide. In some embodiments, the chiral controlled DMD oligonucleotide has all Rp stereocenters. In some embodiments of the present disclosure, the chiral controlled DMD oligonucleotide has all Sp stereocenters. In some embodiments of the present disclosure, each binding phosphorus in the chiral controlled DMD oligonucleotide is independently Rp or Sp. In some embodiments of the present disclosure, each binding phosphorus in the chiral controlled DMD oligonucleotide is independently Rp or Sp, with at least one being Rp and at least one being Sp. In some embodiments, the Rp and Sp centers are selected to provide the chiral controlled DMD oligonucleotide with a particular three-dimensional superstructure. Examples of such selections are described in more detail herein.

[0432] In some embodiments, the DMD oligonucleotides provided include a chiral auxiliary moiety, for example at the internucleotide linkage. In some embodiments, the chiral auxiliary is linked to the linking phosphorus. In some embodiments, the chiral auxiliary is W 2 In some embodiments, the chiral auxiliary is linked to the phosphorus by W 2 is linked to the bound phosphorus by 2 is O. Optionally, W 1 For example, W 1 -NG 5 - is capped during synthesis of the DMD oligonucleotide. In some embodiments, the W in the chiral auxiliary in the DMD oligonucleotide 1 is capped, for example, by a capping reagent, during DMD oligonucleotide synthesis. 1may be purposefully capped to modulate DMD oligonucleotide properties. 1 -R 1 In some embodiments, R 1 In some embodiments, R' is an optionally substituted C(O)R'. 1~6 It is aliphatic. In some embodiments, R' is methyl.

[0433] In some embodiments, the chiral reagent for use according to the present disclosure is selected for its ability to be removed at a particular step in the cycle described above. For example, in some embodiments, it is desirable to remove the chiral reagent during the step of modifying the binding phosphorus. In some embodiments, it is desirable to remove the chiral reagent before the step of modifying the binding phosphorus. In some embodiments, it is desirable to remove the chiral reagent after the step of modifying the binding phosphorus. In some embodiments, it is desirable to remove the chiral reagent after the first coupling step has been performed but before the second coupling step has been performed so that no chiral reagent is present on the growing DMD oligonucleotide during the second coupling (and also for further subsequent coupling steps). In some embodiments, the chiral reagent is removed during a "deblocking" reaction that is performed after modification of the binding phosphorus but before the subsequent cycle begins. Exemplary methods and reagents for removal are described herein.

[0434] In some embodiments, the removal of chiral auxiliary is realized during the modification and / or deblocking step, as shown in scheme I. It may be beneficial to combine the removal of chiral auxiliary with other transformations, such as modification and deblocking. Those skilled in the art will understand that the reduction in steps / transformations may improve the overall synthesis efficiency, for example, in terms of yield and product purity, especially for longer DMD oligonucleotides. One example of the removal of chiral auxiliary during modification and / or deblocking is shown in scheme I.

[0435] In some embodiments, a chiral reagent for use in accordance with the methods of the present disclosure is characterized in that it is removable under certain conditions. For example, in some embodiments, a chiral reagent is selected for its ability to be removed under acidic conditions. In certain embodiments, a chiral reagent is selected for its ability to be removed under weakly acidic conditions. In certain embodiments, a chiral reagent is selected for its ability to be removed by an E1 elimination reaction (e.g., D due to the formation of a cationic intermediate on the chiral reagent under acidic conditions). (Removal occurs by causing cleavage of the chiral reagent from the MD oligonucleotide). In some embodiments, the chiral reagent is characterized by having a structure that recognizes it as being capable of accepting or promoting an E1 elimination reaction. One of ordinary skill in the relevant art will understand which structures may be envisioned as being susceptible to such an elimination reaction.

[0436] In some embodiments, the chiral reagent is selected for its ability to be removed with a nucleophile. In some embodiments, the chiral reagent is selected for its ability to be removed with an amine nucleophile. In some embodiments, the chiral reagent is selected for its ability to be removed with a nucleophile other than an amine.

[0437] In some embodiments, the chiral reagent is selected for its ability to be removed with a base. In some embodiments, the chiral reagent is selected for its ability to be removed with an amine. In some embodiments, the chiral reagent is selected for its ability to be removed with a base other than an amine.

[0438] In some embodiments, the chirally pure phosphoramidite containing the chiral auxiliary may be separated prior to use. In some embodiments, the chirally pure phosphoramidite containing the chiral auxiliary may be used without separation - in some embodiments, it may be used directly after formation.

[0439] activation As one of skill in the art will appreciate, DMD oligonucleotide preparations may utilize a variety of conditions, reagents, etc. to activate reaction components, for example, during phosphoramidite preparation, during one or more steps in the cycle, during post-cycle cleavage / deprotection, etc. Various activation techniques that may be utilized in the present disclosure include, but are not limited to, those described in U.S. Pat. No. 9,695,211, U.S. Pat. No. 9,605,019, U.S. Pat. No. 9,598,458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Patent Application Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 1 60741, WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 055951, WO 2019 / 200185 and / or WO 2019 / 217784 (each of which activation techniques are incorporated by reference). Specific activation techniques, such as reagents, conditions, methods, etc., are provided in the Examples.

[0440] Coupling In some embodiments, the cycle of the present disclosure includes a stereoselective condensation / coupling step to form chiral controlled internucleotide bonds. Condensation often uses an activating reagent such as 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT), benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole (NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole (BTT), 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate (CMPT), N-cyanomethylpiperidinium triflate, or N-cyanomethyldimethylammonium triflate. Suitable conditions and reagents include chiral phosphoramidites, and are described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006 ... Examples of suitable coupling techniques include those described in WO 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 160741, WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 055951, WO 2019 / 200185 and / or WO 2019 / 217784 (each of which condensation reagents, conditions and methods are incorporated by reference). Specific coupling techniques, such as reagents, conditions, methods, etc., are shown in the Examples.

[0441] In some embodiments, the chiral phosphoramidite for coupling is [ka] wherein R 2sis -H, -F, or -OR, and each other variable is independently as described herein. 1 Or G 2 contains an electron withdrawing group as described in the present disclosure. In some embodiments, the chiral phosphoramidite for coupling is [ka] wherein each variable is independently as described in this disclosure. 1 is R' as described in this disclosure. In some embodiments, R 1 is R as described herein. In some embodiments, R is phenyl, optionally substituted as described herein. In some embodiments, R is phenyl. In some embodiments, R is C, optionally substituted as described herein. 1~6 In some embodiments, R is an optionally substituted C 1~6 It is an alkyl. For example, in some embodiments, R is methyl; in some embodiments, R is isopropyl; in some embodiments, R is t-butyl, etc. In some embodiments, R' is a 5' blocking group in oligonucleotide synthesis, such as DMTr. In some embodiments, BA is an optionally protected nucleobase as described herein. In some embodiments, BA is an optionally substituted A, T, G, C, U, or a tautomer thereof. In some embodiments, BA is a protected nucleobase. In some embodiments, BA is an optionally substituted protected A, T, G, C, U, or a tautomer thereof. In some embodiments, R' is a protecting group. In some embodiments, R' is DMTr. In some embodiments, R 2s is -H, -F or -OMe. 2sis -H. In some embodiments, R 2s is -F. In some embodiments, R 2s is -OMe.

[0442] In some embodiments, attached to the bound phosphorus: [ka] wherein each variable is independently as per the disclosure. is an internucleotide bond formed in a coupling step comprising:

[0443] In some embodiments, the coupling forms internucleotide bonds with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more stereoselectivity. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 90% or more. In some embodiments, the stereoselectivity is 91% or more. In some embodiments, the stereoselectivity is 92% or more. In some embodiments, the stereoselectivity is 93% or more. In some embodiments, the stereoselectivity is 94% or more. In some embodiments, the stereoselectivity is 95% or more. In some embodiments, the stereoselectivity is 96% or more. In some embodiments, the stereoselectivity is 97% or more. In some embodiments, the stereoselectivity is 98% or more. In some embodiments, the stereoselectivity is 99% or more.

[0444] Capping If the final nucleic acid is larger than a dimer, the unreacted -OH moieties are generally capped with a blocking / capping group. The chiral auxiliary in the oligonucleotide may also be capped with a blocking group to form a capped condensation intermediate. Suitable capping techniques (e.g., reagents, conditions, etc.) include those described in U.S. Pat. No. 9,695,211, U.S. Pat. No. 9,605,019, U.S. Pat. No. 9,598,458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Patent Application Publication No. 20170037399, WO 2017 / 015555, WO 2017 / 062862, WO 2017 / 160741, WO 2017 / 192664, WO 2017 / 192679, WO 2017 / 210647, WO 2017 / 220647, WO 2017 / 230647, WO 2017 / 240647, WO 2017 / 250647, WO 2017 / 260647, WO 2017 / 280647, WO 2017 / 29 ... Examples of capping techniques include those described in WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 055951, WO 2019 / 200185 and / or WO 2019 / 217784 (each of which is incorporated by reference). In some embodiments, the capping reagent is a carboxylic acid or a derivative thereof. In some embodiments, the capping reagent is R'COOH. In some embodiments, the capping step introduces R'COO- to the unreacted 5'OH group and / or amino group in the chiral auxiliary. In some embodiments, a cycle may include two or more capping steps. In some embodiments, a cycle includes a first capping (e.g., conversion of P(III) to P(V)) before modification of the coupling product and another capping after modification of the coupling product. In some embodiments, the first capping is carried out under amidation conditions, such as with an acylating reagent (e.g., (RC(O)) 2 Anhydrides having the structure O (e.g., Ac 2O)) and a base (e.g., 2,6-lutidine). In some embodiments, the first capping caps an amino group, e.g., the amino group of the chiral auxiliary in the internucleotide linkage. In some embodiments, the internucleotide formed in the capping step is [ka] wherein each variable is independently according to the present disclosure. 1 In some embodiments, R is CH 3 -. In some embodiments, each chiral controlled coupling (e.g., using a chiral auxiliary) is followed by a first capping. Typically, the cycle for the chiral non-controlled coupling that uses conventional phosphoramidite to construct a natural phosphate bond does not include a first capping. In some embodiments, the second capping is carried out, for example, under esterification conditions that cap free 5'-OH (e.g., under capping conditions of conventional phosphoramidite oligonucleotide synthesis).

[0445] Specific capping techniques, including reagents, conditions, methods, etc., are provided in the Examples.

[0446] qualification In some embodiments, an internucleotide bond whose bound phosphorus exists as P(III) is modified to form another modified internucleotide bond. In many embodiments, P(III) is modified by reaction with an electrophile. A variety of reactions suitable for P(III) may be utilized in the present disclosure. Suitable modification techniques (e.g., reagents (e.g., sulfurization reagents, oxidation reagents, etc.), conditions, etc.) include those described in U.S. Pat. No. 9,695,211, U.S. Pat. No. 9,605,019, U.S. Pat. No. 9,598,458, U.S. Patent Application Publication No. 2013 / 0178612, U.S. Patent Application Publication No. 20150211006, U.S. Pate...

Claims

1. fA*SfG*SfUn001RfU*SfU*SfCn001RmCfU*SfU*SmA*SfG*SmUmA*SfA*SfC*SfC*SfAn001RfC*SfA*SfG (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemical 1】 where phosphorus is in the Rp configuration. An oligonucleotide having the structure: or a pharmaceutically acceptable salt thereof.

2. fG*SfU*SfAn001RfC*SfC*SfUn001RfC*SfC*SmAfA*SmC*SfA*SmUfC*SfA*SfA*SfGn001RfG*SfA*SfA (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 2】 where phosphorus is in the Rp configuration. An oligonucleotide having the structure: or a pharmaceutically acceptable salt thereof.

3. fU*SfG*SfGn001RfC*SfA*SfUn001RfU*SfU*SmCfU*SmA*SfG*SmUfU*SfU*SfG*SfGn001RfA*SfG*SfA (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 3】 where phosphorus is in the Rp configuration. An oligonucleotide having the structure: or a pharmaceutically acceptable salt thereof.

4. fG*SfG*SfCn001RfA*SfU*SfUn001RmUfC*SfU*SmA*SfG*SmUmU*SfU*SfG*SfG*SfAn001RfG*SfA*SfU (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 4】 where phosphorus is in the Rp configuration. An oligonucleotide having the structure: or a pharmaceutically acceptable salt thereof.

5. fU*SfG*SfGn001RfC*SfA*SfGn001RfU*SfU*SmUfC*SmC*SfU*SmUfA*SfG*SfU*SfAn001RfA*SfC*SfC (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 5】 where phosphorus is in the Rp configuration. An oligonucleotide having the structure: or a pharmaceutically acceptable salt thereof.

6. The oligonucleotide according to any one of claims 1 to 5, which is a pharmaceutically acceptable salt.

7. The oligonucleotide of any one of claims 1 to 5, having a diastereomeric purity of about 50% or more.

8. A composition, wherein a level of every oligonucleotide in said composition is independently fA*SfG*SfUn001RfU*SfU*SfCn001RmCfU*SfU*SmA*SfG*SmUmA*SfA*SfC*SfC*SfAn001RfC*SfA*SfG (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 6】 where phosphorus is in the Rp configuration. or a pharmaceutically acceptable salt thereof.

9. A composition, wherein a level of every oligonucleotide in said composition is independently fG*SfU*SfAn001RfC*SfC*SfUn001RfC*SfC*SmAfA*SmC*SfA*SmUfC*SfA*SfA*SfGn001RfG*SfA*SfA (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 7】 where phosphorus is in the Rp configuration. or a pharmaceutically acceptable salt thereof.

10. A composition, wherein a level of every oligonucleotide in said composition is independently fU*SfG*SfGn001RfC*SfA*SfUn001RfU*SfU*SmCfU*SmA*SfG*SmUfU*SfU*SfG*SfGn001RfA*SfG*SfA (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 8】 where phosphorus is in the Rp configuration. or a pharmaceutically acceptable salt thereof.

11. A composition, wherein a level of every oligonucleotide in said composition is independently fG*SfG*SfCn001RfA*SfU*SfUn001RmUfC*SfU*SmA*SfG*SmUmU*SfU*SfG*SfG*SfAn001RfG*SfA*SfU (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 9】 where phosphorus is in the Rp configuration. or a pharmaceutically acceptable salt thereof.

12. A composition, wherein a level of every oligonucleotide in said composition is independently fU*SfG*SfGn001RfC*SfA*SfGn001RfU*SfU*SmUfC*SmC*SfU*SmUfA*SfG*SfU*SfAn001RfA*SfC*SfC (In the formula, f represents a 2'-F modified nucleoside; *S represents Sp phosphorothioate; m represents a 2'-OMe modified nucleoside; and n001R is, 【Chemistry 10】 where phosphorus is in the Rp configuration. or a pharmaceutically acceptable salt thereof.

13. The composition of any one of claims 8 to 12, which is a liquid composition and wherein the oligonucleotide is one or more salts dissolved in the composition.

14. The composition according to any one of claims 8 to 12, wherein the oligonucleotides are each independently a pharmaceutically acceptable salt.

15. The composition of any one of claims 8 to 12, wherein the level is about 50% or greater.

16. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

17. 1. An oligonucleotide composition comprising a plurality of oligonucleotides, said plurality of oligonucleotides comprising: 1) a common base sequence, and 2) independently, the same bond phosphorus stereochemistry at one or more chiral internucleotide linkages Share An oligonucleotide composition, wherein the composition is enriched for the plurality of oligonucleotides compared to a substantially racemic preparation of the plurality of oligonucleotides, or at least 10% of the oligonucleotides in the composition that share the common base sequence are the plurality of oligonucleotides.

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