This application claims priority to United States Provisional Application Nos.

62/656,949, filed April 12, 2018, 62/670,709, filed May 11, 2018, 62/715,684, filed August 07, 2018, 62/723,375, filed August 27, 2018, and 62/776,432, filed December 06, 2018, the entirety of each of which is incorporated herein by reference.

BACKGROUND

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

SUMMARY

[0003] Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugar, base, and/or internucleotidic linkages, and patterns thereof), and/or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotidic linkages), and/or patterns thereof), can have significant impact on oligonucleotide properties, e.g., activities, toxicities, e.g., as may be mediated by protein binding characteristics, stability, splicing-altering capabilities, etc. In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements, e.g., controlled chemical modification and/or controlled backbone stereochemistry patterns, provide unexpected properties, including but not limited to certain activities, toxicities, etc. In some embodiments, the present disclosure demonstrates that oligonucleotide properties, e.g., activities, toxicities, etc., can be modulated by chemical modifications (e.g., modifications of sugars, bases, internucleotidic linkages, etc.), chiral structures (e.g., stereochemistry of chiral internucleotidic linkages and patterns thereof, etc.), and/or combinations thereof.

[0004] In some embodiments, the present disclosure provides an oligonucleotide or an oligonucleotide composition. In some embodiments, an oligonucleotide or an oligonucleotide

composition is a DMD oligonucleotide or a DMD oligonucleotide composition. In some embodiments, a DMD oligonucleotide or a DMD oligonucleotide composition is an oligonucleotide or an oligonucleotide composition capable of modulating skipping of one or more exons of the target gene Dystrophin (DMD). In some embodiments, a DMD oligonucleotide or a DMD oligonucleotide composition is useful for treatment of muscular dystrophy. In some embodiments, an oligonucleotide or oligonucleotide composition is an oligonucleotide or oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide or oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage is capable of modulating the expression, level and/or activity of a gene target or a gene product thereof, including but not limited to, increasing or decreasing the expression, level and/or activity of a gene target or gene product thereof via any mechanism, including but not limited to: an RNase H-dependent mechanism, steric hindrance, RNA interference, modulation of skipping of one or more exon, etc. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage, in combination with any other structure or chemical moiety described herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide or DMD oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage.

[0005] In some embodiments, the present disclosure provides technologies related to an oligonucleotide or an oligonucleotide composition for reducing levels of a transcript and/or a protein encoded thereby. In some embodiments, as demonstrated by example data described herein, provided technologies are particularly useful for reducing levels of mRNA and/or proteins encoded thereby.

[0006] In some embodiments, the present disclosure provides technologies, e.g., oligonucleotides, compositions and methods, etc., for altering gene expression, levels and/or splicing of transcripts. In some embodiments, a transcript is Dystrophin (DMD). Splicing of a transcript, such as pre-mRNA, is an essential step for the transcript to perform its biological functions in many higher eukaryotes. In some embodiments, the present disclosure recognizes that targeting splicing, especially through compositions comprising oligonucleotides having base sequences and/or chemical modifications and/or stereochemistry patterns (and/or patterns thereof) described in this disclosure, can effectively correct disease-associated mutations and/or aberrant splicing, and/or introduce and/or enhance beneficial splicing that lead to desired products, e.g., mRNA, proteins, etc. which can repair, restore, or add new desired biological functions, e.g., one or more functions of Dystrophin.

[0007] In some embodiments, the present disclosure provides compositions and methods for altering splicing of DMD transcripts, wherein altered splicing deletes or compensates for an exon(s) comprising a disease-associated mutation.

[0008] For example, in some embodiments, a Dystrophin gene can comprise an exon comprising

one or more mutations associated with a disease, e.g., muscular dystrophy (including but not limited to Duchenne (Duchenne’s) muscular dystrophy (DMD) and Becker (Becker’s) muscular dystrophy (BMD)). In some embodiments, a disease-associated exon comprises a mutation (e.g., a missense mutation, a frame shift mutation, a nonsense mutation, a premature stop codon, etc.) in an exon. In some embodiments, the present disclosure provides compositions and methods for effectively skipping a disease-associated Dystrophin exon(s) and/or a different or an adjacent exon(s), while maintaining or restoring the reading frame so that a shorter (e.g., internally truncated) but partially functional dystrophin can be produced. A person having ordinary skill in the art appreciates that provided technologies (oligonucleotides, compositions, methods, etc.) can also be utilized for skipping of other exons, for example, those described in WO 2017/062862 and incorporated herein by reference, in accordance with the present disclosure to treat a disease and/or condition.

[0009] Among other things, the present disclosure demonstrates that chemical modifications and/or stereochemistry can be used to modulate transcript splicing by oligonucleotide compositions. In some embodiments, the present disclosure provides combinations of chemical modifications and stereochemistry to improve properties of oligonucleotides, e.g., their capabilities to alter splicing of transcripts. In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions that, when compared to a reference condition (e.g., absence of the composition, presence of a reference composition (e.g., a stereorandom composition of oligonucleotides having the same constitution (as understood by those skilled in the art, unless otherwise indicated constitution generally refers to the description of the identity and connectivity (and corresponding bond multiplicities) of the atoms in a molecular entity but omitting any distinction arising from their spatial arrangement), a different chirally controlled oligonucleotide composition, etc.), combinations thereof, etc.), provide altered splicing that can deliver one or more desired biological effects, for example, increase production of desired proteins, knockdown of a gene by producing mRNA with frameshift mutations and/or premature termination codons, knockdown of a gene expressing a mRNA with a frameshift mutation and/or premature termination codon, etc. In some embodiments, compared to a reference condition, provided chirally controlled oligonucleotide compositions are surprisingly effective. In some embodiments, desired biological effects (e.g., as measured by increased levels of desired mRNA, proteins, etc., decreased levels of undesired mRNA, proteins, etc. ) can be enhanced by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100 fold.

[0010] The present disclosure recognizes challenges of providing low toxicity oligonucleotide compositions and methods of use thereof. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced toxicity. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced immune responses. In some

embodiments, the present disclosure recognizes that various toxicities induced by oligonucleotides are related to cytokine and/or complement activation. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced cytokine and/or complement activation. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced complement activation via the alternative pathway. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced complement activation via the classical pathway. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced drug-induced vascular injury. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced injection site inflammation. In some embodiments, reduced toxicity can be evaluated through one or more assays widely known to and practiced by a person having ordinary skill in the art, e.g. , evaluation of levels of complete activation product, protein binding, etc.

[0011] In some embodiments, the present disclosure provides oligonucleotides with enhanced antagonism of hTLR9 activity. In some embodiments, certain diseases, e.g., DMD, are associated with inflammation in, e.g., muscle tissues. In some embodiments, provided technologies (e.g., oligonucleotides, compositions, methods, etc.) provides both enhanced activities (e.g., exon-skipping activities) and hTLR9 antagonist activities which can be beneficial to one or more conditions and/or diseases associated with inflammation. In some embodiments, provided oligonucleotides and/or compositions thereof provides both exon-skipping capabilities and decreased levels of toxicity and/or inflammation. In some embodiments, the present disclosure provides an oligonucleotide which comprises one or more non-negatively charged internucieotidic linkages, wherein the oligonucleotide agonizes TLR9 activity less than another oligonucleotide which does not comprise a non-negatively charged internucieotidic linkage or which comprises fewer non-negatively charged internucieotidic linkages and which is otherwise identical. In some embodiments, the present disclosure provides an oligonucleotide which comprises one or more non-negatively charged internucieotidic linkages, wherein the oligonucleotide agonizes TLR9 activity less than an otherwise identical oligonucleotide which does not comprise a non-negatively charged internucieotidic linkage or which comprises fewer non-negatively charged internucieotidic linkages. In some embodiments, the present disclosure pertains to an oligonucleotide comprising at least one non-negatively charged internucieotidic linkage. In some embodiments, the non-negatively charged internucieotidic is selected from: n001, n002, n003, n004, n005, n006, n007, n008, n009, or n010, or a chirally controlled stereoisomer of n001, n002, n003, n004, n005, n006, n007, n008, n009, or n010. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises at least two non-negatively charged internucieotidic linkages, wherein the linkages are different from each other. In some embodiments, the present disclosure pertains to an oligonucleotide comprising a CpG motif, wherein at least one internucleotidic linkage in the CpG (e.g., the p in CpG) or immediately upstream of the CpG (toward the 5’ end of the oligonucleotide) or immediately downstream of the CpG (toward the 3’ end of the oligonucleotide) is a non-negatively charged internucleotidic linkage. In some embodiments, TLR9 is a human TLR9. In some embodiments, TLR9 is a mouse TLR9.

[0012] In some embodiments, the present disclosure demonstrates that oligonucleotide properties, e.g., activities, toxicities, etc., can be modulated through chemical modifications. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which have a common base sequence, and comprise one or more modified internucleotidic linkages (or "non-natural internucleotidic linkages”, linkages that are not but can be utilized in place of a natural phosphate internucleotidic linkage (‒OP(O)(OH)O‒, which may exist as a salt form (‒OP(O)(O‒)O‒) at a physiological pH) found in natural DNA and RNA), one or more modified sugar moieties, and/or one or more natural phosphate linkages. In some embodiments, provided oligonucleotides may comprise two or more types of modified internucleotidic linkages. In some embodiments, a provided oligonucleotide comprises a non-negatively charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a neutral internucleotidic linkage comprises a triazole, alkyne, or guanidine (e.g., cyclic guanidine) moiety. Such moieties are optionally substituted. In some embodiments, a provided oligonucleotide comprises a neutral internucleotidic linkage and another internucleotidic linkage which is not a neutral backbone. In some embodiments, a provided oligonucleotide comprises a neutral internucleotidic linkage and a phosphorothioate internucleotidic linkage. In some embodiments, provided oligonucleotide compositions comprising a plurality of oligonucleotides are chirally controlled and level of the plurality of oligonucleotides in the composition is controlled or pre-detemrined, and oligonucleotides of the plurality share a common stereochemistry configuration at one or more chiral internucleotidic linkages. For example, in some embodiments, oligonucleotides of a plurality share a common stereochemistry configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral internucleotidic linkages, each of which is independently Rp or Sp; in some embodiments, oligonucleotides of a plurality share a common stereochemistry configuration at each chiral internucleotidic linkages. In some embodiments, a chiral internucleotidic linkage where a controlled level of oligonucleotides of a composition share a common stereochemistry configuration (independently in the Rp or Sp configuration) is referred to as a chirally controlled internucleotidic linkage.

[0013] In some embodiments, a modified internucleotidic linkage is a non-negatively charged

(neutral or cationic) internucleotidic linkage in that at a pH, (e.g., human physiological pH (~ 7.4), pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.), etc.), it largely (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc.; in some embodiments, at least 30%; in some embodiments, at least 40%; in some embodiments, at least 50%; in some embodiments, at least 60%; in some embodiments, at least 70%; in some embodiments, at least 80%; in some embodiments, at least 90%; in some embodiments, at least 99%; etc.;) exists as a neutral or cationic form (as compared to an anionic form (e.g., ‒O‒P(O)(O‒)‒O‒ (the anionic form of natural phosphate linkage), ‒O‒P(O)(S‒)‒O‒ (the anionic torn: of phosphorothioate linkage), etc.)), respectively. In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage in that at a pH, it largely exists as a neutral form. In some embodiments, a modified internucleotidic linkage is a cationic internucleotidic linkage in that at a pH, it largely exists as a cationic form. In some embodiments, a pH is human physiological pH (~ 7.4). In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage in that at pH 7,4 in a water solution, at least 90% of the internucleotidic linkage exists as its neutral form. In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage in that in a water solution of the oligonucleotide, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the internucleotidic linkage exists in its neutral form. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is at least 99%. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, when in its neutral form has no moiety with a pKa that is less than 8, 9, 10, 11.

12, 13, or 14. In some embodiments, pKa of an internucleotidic linkage in the present disclosure can be represented by pKa of CH3‒the internucleotidic linkage‒CH3 (i.e., replacing the two nucleoside units connected by the internucleotidic linkage with two -CH3 groups). Without wishing to be bound by any particular theory, in at least some cases, a neutral internucleotidic linkage in an oligonucleotide can provide improved properties and/or activities, e.g., improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape and/or improved nuclear uptake, etc,, compared to a comparable nucleic acid which does not comprises a neutral internucleotidic linkage.

[0014] In some embodiments, a non-negatively charged internucleotidic linkage has the structure of e.g., of formula I-n-1, I-n-2, 1-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc. In some embodiments, a non-negatively charged internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a guanidine moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a cyclic guanidine moiety. In some embodiments, a modified internucleotidic linkage comprising a cyclic

guanidine moiety has the structure of:
In some embodiments, a neutral internucleotidic linkage comprising a cyclic guanidine moiety is chirally controlled. In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage and at least one phosphorothioate internucleotidic linkage.

[0015] In some embodiments, a non-negatively charged internucleotidic linkage is n001, n002, n003, n004, n005, n006, n007, or n008. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled, e.g., n001R, n002R, n0G3R, n004R, n005R, n006R, n007R, n008R, n009R, n001S, n002S, n003S, n004S, n005S, n006S, n007S, n008S, n009S, etc.

[0016] In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage and at least one phosphorothioate internucleotidic linkage, wherein the phosphorothioate internucleotidic linkage is a chirally controlled internucleotidic linkage in the Sp configuration.

[0017] In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage and at least one phosphorothioate internucleotidic linkage, wherein the phosphorothioate internucleotidic linkage is a chirally controlled internucleotidic linkage in the Rp configuration.

[0018] In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage selected from a neutral internucleotidic linkage comprising an optionally substituted triazolyl group, a neutral internucleotidic linkage comprising an optionally substituted alkynyl group, and a neutral internucleotidic linkage

comprising a moiety
and at least one phosphorothioate internucleotidic linkage. In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage selected from a neutral internucleotidic linkage comprising an optionally substituted triazolyl group, a neutral internucleotidic linkage comprising an optionally

substituted alkynyl group, and a neutral internucleotidic linkage comprising a Tmg group
and at least one phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one non-negatively charged internucleotidic linkage and at least one phosphorothioate internucieotidic linkage. In some embodiments, the non-negatively charged internucieotidic linkage is n001. In some embodiments, the non-negatively charged internucieotidic linkage and the phosphorothioate internucieotidic linkage are independently chirally controlled. In some embodiments, each of the non-negatively charged internucieotidic linkage and the phosphorothioate internucieotidic linkages are independently chirally controlled.

[0019] In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucieotidic linkage selected from a neutral internucieotidic linkage comprising an optionally substituted triazolyl group, a neutral internucieotidic linkage comprising an optionally substituted alkynyl group, and a neutral internucieotidic linkage comprising a Tmg group, and at least one phosphorothioate, wherein the phosphorothioate is a chirally controlled internucieotidic linkage in the Sp configuration.

[0020] In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage selected from a neutral internucieotidic linkage comprising an optionally substituted triazolyl group, a neutral internucieotidic linkage comprising an optionally substituted alkynyl group, and a neutral internucieotidic linkage comprising a Tmg group, and at least one phosphorothioate, wherein the phosphorothioate is a chirally controlled internucieotidic linkage in the Rp configuration.

[0021] Various types of internucleotidic linkages differ in properties. Without wishing to be bound by any theory, the present disclosure notes that a natural phosphate linkage (phosphodiester internucieotidic linkage) is anionic and may be unstable when used by itself without other chemical modifications in vivo; a phosphorothioate internucieotidic linkage is anionic, generally more stable in vivo than a natural phosphate linkage, and generally more hydrophobic; a neutral internucieotidic linkage such as one exemplified in the present disclosure comprising a cyclic guanidine moiety is neutral at physiological pH, can be more stable in vivo than a natural phosphate linkage, and more hydrophobic.

[0022] In some embodiments, an internucieotidic linkage (e.g., a non-negatively charged internucleotidic linkage, a chirally controlled non-negatively charged internucleotidic linkage, etc.) is neutral at physiological pH, chirally controlled, stable in vivo, hydrophobic, and may increase endosomal escape.

[0023] In some embodiments, an oligonucleotide or oligonucleotide composition is: a DMD oligonucleotide or oligonucleotide composition; an oligonucleotide or oligonucleotide composition comprising a non-negatively charged internucieotidic linkage; or a DMD oligonucleotide comprising a non-negatively charged internucieotidic linkage.

[0024] In some embodiments, an oligonucleotide has, as non-limiting examples, a wing-core-wing, wing-core, core-wing, wing-wing-core-wing-wing, wing-wing-core-wing, or wing-core-wing-wing

structure (in some embodiments, a wing-wing comprises or consists of a first wing and a second wing, wherein the first wing is different than the second wing, and the first and second wings are different than the core). A wing or core can be defined by any structural elements and/or patterns and/or combinations thereof. In some embodiments, a wing and core is defined by nucleoside modifications, sugar modifications, and/or internucleotidic linkages, wherein a wing comprises a nucleoside modification, sugar modification and/or internucleotidic linkage and/or pattern and/or combination thereof, that the core region does not have, or vice versa. In some embodiments, oligonucleotides of the present disclosure comprise or consist of a 5’-end region, a middle region, and a 3’-end region. In some embodiments, a 5’-end region is a 5’-wing region. In some embodiments, a 5’-wing region is a 5’-end region. In some embodiments, a 3’-end region is a 3’-wing region. In some embodiments, a 3’-wing region is a 3’-end region. In some embodiments, a core region is a middle region.

[0025] In some embodiments, each wing region (or each of the 5’-end and 3’-end regions) independently comprises one or more modified phosphate linkages and no natural phosphate linkages, and the core region (the middle region) comprises one or more modified internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, each wing region (or each of the 5’-end and 3’-end regions) independently comprises one or more natural phosphate linkages and optionally one or more modified internucleotidic linkages, and the core (or the middle region) comprises one or more modified internucleotidic linkages and optionally one or more natural phosphate linkages. In some embodiments, a wing (or a 5’-end or 3’-end region) comprises modified sugar moieties. In some embodiments, a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.

[0026] Among other things, the present disclosure encompasses the recognition that stereorandom oligonucleotide preparations contain a plurality of distinct chemical entities that differ from one another, e.g., in the stereochemical structure of individual backbone chiral centers within the oligonucleotide chain. Without control of stereochemistry of backbone chiral centers, stereorandom oligonucleotide preparations provide uncontrolled (or stereorandom) compositions comprising undetermined levels of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and/or chemical modifications, they are different chemical entities at least due to their different backbone stereochemistry, and they can have, as demonstrated herein, different properties, e.g., activities, toxicides, distribution etc. Among other things, the present disclosure provides chirally controlled compositions that are or contain particular stereoisomers of oligonucleotides of interest; in contrast to chirally uncontrolled compositions, chirally controlled compositions comprise controlled levels of particular stereoisomers of oligonucleotides. In some embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its pattern of backbone linkages, its pattern of backbone chiral centers, and pattern of backbone phosphorus modifications, etc. As is understood in the art, in some embodiments, base sequence may refer solely to the sequence of bases and/or to the identity and/or modification status of nucleoside residues (e.g., of sugar and/or base components, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) in an oligonucleotide and/or to the hybridization character (i.e., the ability to hybridize with particular complementary residues) of such residues. In some embodiments, the present disclosure demonstrates that property improvements (e.g., improved activities, lower toxicities, etc.) achieved through inclusion and/or location of particular chiral structures within an oligonucleotide can be comparable to, or even better than those achieved through use of chemical modifications, e.g., particular backbone linkages, residue modifications, etc. (e.g., through use of certain types of modified phosphates [e.g., phosphorothioate, substituted phosphorothioate, etc.], sugar modifications [e.g., 2'-modifications, etc.], and/or base modifications [e.g., inethylation, etc.]). In some embodiments, the present disclosure demonstrates that chirally controlled oligonucleotide compositions of oligonucleotides comprising certain chemical modifications (e.g., 2'-F, 2'-OMe, phosphorothioate internucleotidic linkages, lipid conjugation, etc.) demonstrate unexpectedly high exon-skipping efficiency.

[0027] In some embodiments, provided oligonucleotides are blockmers. In some embodiments, a blockmer is an oligonucleotide comprising one or more blocks.

[0028] In some embodiments, a block is a portion of an oligonucleotide. In some embodiments, a block is a wing or a core. In some embodiments, a blockmer comprises one or more blocks. In some embodiments, a 5’-block is a 5’-end region or 5’-wing. In some embodiments, a 3’-block is a 3’-end region or 3’-wing.

[0029] In some embodiments, provided oligonucleotide are altmers. In some embodiments, provided oligonucleotides are altmers comprising alternating blocks. In some embodiments, a blockmer or an altmer can be defined by chemical modifications (including presence or absence), e.g., base modifications, sugar modification, internucleotidic linkage modifications, stereochemistry, etc.

[0030] In some embodiments, provided oligonucleotides comprise blocks comprising different internucleotidic linkages. In some embodiments, provided oligonucleotides comprise blocks comprising modified internucleotidic linkages and/or natural phosphate linkages.

[0031] In some embodiments, provided oligonucleotides comprise blocks comprising sugar modifications. In some embodiments, provided oligonucleotides comprise one or more blocks comprising one or more 2'-F modifications (2'-F blocks). In some embodiments, provided oligonucleotides comprise blocks comprising consecutive 2'-F modifications. In some embodiments, a block comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive 2'-F modifications.

[0032] In some embodiments, provided oligonucleotides comprises one or more blocks

comprising one or more 2’-OR1 modifications (2’-OR1 blocks), wherein R1 is independently as defined and described herein and below. In some embodiments, provided oligonucleotides comprise both 2'-F and 2'-OR1 blocks. In some embodiments, provided oligonucleotides comprise alternating 2'-F and 2'-OR1 blocks. In some embodiments, provided oligonucleotides comprise a first 2'-F block at the 5’-end, and a second 2'-F block at the 3’-end, each of which independently comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive 2'-F modifications.

[0033] In some embodiments, provided oligonucleotides comprise a 5’-block wherein each sugar moiety of the 5’-block comprises a 2'-F modification. In some embodiments, provided oligonucleotides comprise a 3’-block wherein each sugar moiety of the 3’-block comprises a 2'-F modification. In some embodiments, such provided oligonucleotides comprise one or more 2'-OR1 blocks, and optionally one or more 2'-F blocks, between the 5’ and 3’ 2'-F blocks. In some embodiments, such provided oligonucleotides comprise one or more 2'-OR1 blocks, and one or more 2'-F blocks, between the 5’ and 3’ 2'-F blocks (e.g., WV-3047, WV-3048, etc).

[0034] In some embodiments, a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5’-block is an Rp block. In some embodiments, a 3’-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5’-block is an Sp block. In some embodiments, a 3’-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks.

[0035] In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.

[0036] In some embodiments, a 5’-block is an Sp block wherein each sugar moiety comprises a, 2'-F modification. In some embodiments, a 5’-block is an Sp block wherein each internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, a 5’-block is an Sp block wherein each internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, a 5’-block comprises 4 or more nucleoside units.

[0037] In some embodiments, a 3’-block is an Sp block wherein each sugar moiety comprises a 2'-F modification. In some embodiments, a 3’-block is an Sp block wherein each internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, a 3’-block is an Sp block wherein each internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, a 3’-block

comprises 4 or more nucleoside units.

[0038] In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties and/or unmodified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties and unmodified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties, wherein the modified sugar moieties comprise different 2'-modifications. For example, in some embodiments, provided oligonucleotide comprises alternating blocks comprising 2'-OMe and 2'-F, respectively.

[0039] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which:

1) have a common base sequence complementary to a target sequence in a transcript; and

2) comprise one or more modified sugar moieties and modified internucleotidic linkages.

[0040] In some embodiments, a provided oligonucleotide composition is characterized in that, when it is contacted with the transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

[0041] In some embodiments, a reference condition is absence of the composition. In some embodiments, a reference condition is presence of a reference composition. Example reference compositions comprising a reference plurality of oligonucleotides are extensively described in this disclosure. In some embodiments, oligonucleotides of the reference plurality have a different structural elements (chemical modifications, stereochemistry, etc.) compared with oligonucleotides of the plurality in a provided composition. In some embodiments, a reference composition is a stereorandom preparation of oligonucleotides having the same chemical modifications. In some embodiments, a reference composition is a mixture of stereoisomers while a provided composition is a chirally controlled oligonucleotide composition of one stereoisomer. In some embodiments, oligonucleotides of the reference plurality have the same base sequence, same sugar modifications, same base modifications, same internucleotidic linkage modifications, and/or same stereochemistry as oligonucleotide of the plurality in a provided composition but different chemical modifications, e.g., base modification, sugar modification, internucleotidic linkage modifications, etc.

[0042] Example splicing systems are widely known in the art. In some embodiments, a splicing system is an in vivo or in vitro system including components sufficient to achieve splicing of a relevant target transcript. In some embodiments, a splicing system is or comprises a spliceosome (e.g., protein and/or RNA components thereof). In some embodiments, a splicing system is or comprises an organellar

membrane (e.g., a nuclear membrane) and/or an organelle (e.g., a nucleus). In some embodiments, a splicing system is or comprises a cell or population thereof. In some embodiments, a splicing system is or comprises a tissue. In some embodiments, a splicing system is or comprises an organism, e.g., an animal, e.g., a mammal such as a mouse, rat, monkey, dog, human, etc.

[0043] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which:

1) have a common base sequence complementary to a target sequence in a transcript; and

2) comprise one or more modified sugar moieties and modified internucleotidic linkages, the oligonucleotide composition being characterized in that, when it is contacted with the transcript in a transcript splicing system, spticmg of the transcript is altered relative to that observed under reference conditions selected from the group consi sting of absence of the composition, presence of a reference composition, and combinations thereof.

[0044] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides of a particular 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.

[0045] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides of a particular 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,

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

the oligonucleotide composition being characterized in that, when it is contacted with the transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

[0046] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

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

[0047] In some embodiments, each region (e.g., a block, wing, core, 5’-end, 3’-end, or middle region, etc.) of an oligonucleotide independently comprises 3, 4, 5, 6, 7, 8, 9, 10 or more bases. In some embodiments, each region independently comprises 3 or more bases. In some embodiments, each region independently comprises 4 or more bases. In some embodiments, each region independently comprises 5 or more bases. In some embodiments, each region independently comprises 6 or more bases. In some embodiments, each sugar moiety in a region is modified. In some embodiments, a modification is a 2'-modification. In some embodiments, each modification is a 2'-modification. In some embodiments, a modification is 2'-F. In some embodiments, each modification is 2'-F. In some embodiments, a modification is 2'-OR1. In some embodiments, each modification is 2'-OR1. In some embodiments, a modification is 2’-OR1. In some embodiments, each modification is 2'-OMe. In some embodiments, each modification is 2'-OMe. In some embodiments, each modification is 2'-MOE. In some embodiments, each modification is 2’-MOE. In some embodiments, a modification is an LNA sugar modification. In some embodiments, each modification is an LNA sugar modification. In some embodiments, each internucleotidic linkage in a region is a chiral internucleotidic linkage. In some embodiments, each internucleotidic linkage in a wing, or 5’-end or 3’-end region, is an Sp chiral internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a core or middle region comprises one or more natural phosphate linkages and one or more modified internucleotidic linkages. In some embodiments, a core or middle region comprises one or more natural phosphate linkages and one or more chiral internucleotidic linkages. In some embodiments, a core region comprises one or more natural phosphate linkages and one or more Sp chiral internucleotidic linkages. In some embodiments, a core or middle region comprises one or more natural phosphate linkages and one or more Sp phosphorothioate linkages.

[0048] In some embodiments, a region (e.g., a block, wing, core, 5’-end, 3’-end, middle region, etc.) of an oligonucleotide comprises a non-negatively charged internucleotidic linkage, e.g., of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc. In some embodiments, a region comprises a neutral internucleotidic linkage. In some embodiments, a region comprises an internucleotidic linkage which comprises a triazole or alkyne moiety. In some embodiments, a region comprises an internucleotidic linkage which comprises a cyclic guanidine guanidine. In some embodiments, a region comprises an internucleotidic linkage which comprises a cyclic guanidine moiety. In some embodiments, a region comprises an internucleotidic linkage having the structure of

In some embodiments, such internucleotidic linkages are chirally controlled.

[0049] In some embodiments, the base sequence of an oligonucleotide, e.g., the base sequence of a plurality of oligonucleotides of a particular oligonucleotide type, is or comprises a base sequence disclosed herein (e.g., a base sequence of an example oligonucleotide (e.g., those listed in the tables, examples, etc.), a target sequence, etc.) (or a portion thereof which is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases long). In some embodiments, a provided oligonucleotide has a base sequence comprising the base sequence of any example oligonucleotides or another base sequence disclosed herein, and a length of up to 30 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising the base sequence of any example oligonucleotides or another base sequence disclosed herein, and a length of up to 40 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising the base sequence of any example oligonucleotides or another base sequence disclosed herein, and a length of up to 50 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising at least 15 contiguous bases of the base sequence of an oligonucleotide example or another sequence disclosed herein, and a length of up to 30 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising at least 15 contiguous bases of the base sequence of an oligonucleotide example or another sequence disclosed herein, and a length of up to 40 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising at least 15 contiguous bases of the base sequence of an oligonucleotide example or another sequence disclosed herein, and a length of up to 50 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising a sequence having no more than 5 mismatches from the base sequence of an example oligonucleotide or another sequence disclosed herein, and a length of up to 30 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising a sequence having no more than 5 mismatches from the base sequence of an example oligonucleotide or another sequence disclosed herein, and a length of up to 40 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising a sequence having no more than 5 mismatches from the base sequence of an example oligonucleotide or another sequence disclosed herein, and a length of up to 50 bases.

[0050] In some embodiments, the base sequence of a provided oligonucleotide is the base sequence of an example oligonucleotide or another sequence disclosed herein, and a pattern of backbone

chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage. In some embodiments, the base sequence of a provided oligonucleotide is the base sequence of an example oligonucleotide or another sequence disclosed herein, the oligonucleotide has a length of up to 30 bases, and a pattern of backbone chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage. In some embodiments, the base sequence of a provided oligonucleotide is the base sequence of an example oligonucleotide or another sequence di sclosed herein, the oligonucleotide has a length of up to 40 bases, and a pattern of backbone chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage. In some embodiments, the base sequence of a provided oligonucleotide comprises at least 15 contiguous bases of any example oligonucleotides or another sequence disclosed herein, the oligonucleotide has a length of up to 30, 40, or 50 bases, and a pattern of backbone chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage.

[0051] In some embodiments, a mismatch is a difference between the base sequence or length when two sequences are maximally aligned and compared. As a non-limiting example, a mismatch is counted if a difference exists between the base at a particular location in one sequence and the base at the corresponding position in another sequence. Thus, a mismatch is counted, for example, if a position in one sequence has a particular base (e.g., A), and the corresponding position on the other sequence has a different base (e.g., G, C or U). A mismatch is also counted, e.g., if a position in one sequence has a base (e.g., A), and the corresponding position on the other sequence has no base (e.g., that position is an abasic nucleotide which comprises a phosphate-sugar backbone but no base) or that position is skipped. A single-stranded nick in either sequence (or in the sense or antisense strand) may not be counted as mismatch, for example, no mismatch would be counted if one sequence comprises the sequence 5’-AG-3’, but the other sequence comprises the sequence 5’-AG-3’ with a single-stranded nick between the A and the G. A base modification is generally not considered a mismatch, for example, if one sequence comprises a C, and the other sequence comprises a modified C (e.g., with a 2'-modification) at the same position, no mismatch may be counted.

[0052] In some embodiments, oligonucleotides of a particular type are chemically identical in that they have the same base sequence (including length), the same pattern of chemical modifications to sugar and base moieties, the same pattern of backbone linkages (e.g., pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, non-negatively charged linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., pattern of stereochemistry (Rp/Sp) of chiral internucleotidic linkages), and the same pattern of backbone phosphorus modifications (e.g., pattern of modifications on the internucleotidic phosphorus atom, such as ‒S‒, and ‒L‒R1 of

formula I).

[0053] In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions of oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotidic linkages, and particularly for oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotidic linkages, wherein the oligonucleotides comprise at least one, and in some embodiments, more than 5, 6, 7, 8, 9, or 10 chirally controlled internucleotidic linkages. In some embodiments, in a chirally controlled composition of oligonucleotides each chiral internucleotidic linkage of the oligonucleotides is independently a chirally controlled internucleotidic linkage. In some embodiments, in a stereorandom or racemic composition of oligonucleotides, each chiral internucleotidic linkage is formed with less than 90: 10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, in a stereoselective or chirally controlled composition of oligonucleotides, each chirally controlled internucleotidic linkage of the oligonucleotides independently has a diastereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus (either Rp or Sp). Among other things, the present disclosure pro vides technologies to prepare oligonucleotides of high diastereopurity. In some embodiments, diastereopurity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5’-nucleoside of the dimer is the same as the nucleoside to the 5’-end of the chiral internucleotidic linkage, and the 3’-nucleoside of the dimer is the same as the nucleoside to the 3’-end of the chiral internucleotidic linkage.

[0054] As described herein, provided compositions and methods are capable of altering splicing of transcripts. In some embodiments, provided compositions and methods provide improved splicing patterns of transcripts compared to reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof. An improvement can be an improvement of any desired biological functions. In some embodiments, for example, in DMD, an improvement is production of an mRNA from which a dystrophin protein with improved biological activities is produced.

[0055] In some embodiments, the present disclosure provides a method for altering splicing of a target transcript, comprising administering a provided composition, wherein the splicing of the target transcript is altered relative to reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

[0056] In some embodiments, the present disclosure provides a method of generating a set of spliced products from a target transcript, the method comprising steps of:

contacting a splicing system containing the target transcript with an oligonucleotide composition

comprising a plurality of oligonucleotides (e.g., a provided chirally controlled oligonucleotide composition), in an amount, for a time, and under conditions sufficient for a set of spliced products to be generated that is different from a set generated under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

[0057] In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising administering to a subject an oligonucleotide composition described herein.

[0058] In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising administering to a subject an oligonucleotide composition comprising a plurality of oligonucleotides, which:

1) have a common base sequence complementary to a target sequence in a transcript: and

2) comprise one or more modified sugar moieties and modified internucleotidic linkages, the oligonucleotide composition being characterized in that, when it is contacted with the transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

CLAIMS

1. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular 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,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

oligonucleotides of the plurality comprise 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 internucleotidic linkages.

2. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular 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,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

the oligonucleotide composition being characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

3. The oligonucleotide of claim 2, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.

4. The oligonucleotide composition of claim 1, wherein when the oligonucleotide composition is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

5. The oligonucleotide of any one of claims 1-4, wherein one or more non-negatively charged internucleotidic linkage are independently chirally controlled.

6. The composition of claim 5, wherein a non-negatively charged internucleotidic linkage has the

structure of formula I:

or a salt form thereof, wherein:

PL is P( =W ), P, or P→ B(R')3;

W is O, N(‒L‒R5), S or Se;

each of R1 and R5 is independently ‒H, -L‒R', halogen, ‒CN, -NO2, ‒L‒Si(R')3, ‒OR', ‒SR', or ‒N(R')2;

X is -N(‒L‒R5)- ;

each of Y and Z is independently ‒O‒, ‒S‒, ‒N(‒L‒R5)‒, or L;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C1_6 alkylene, C1_6 alkenylene, -C≡C‒, a bivalent C1-C6 heteroaliphatic group having 1-5 heteroatoms, ‒C(R')2‒, ‒Cy‒, ‒O‒, -S-, -S-S-, ‒N(R')‒, -C(O)-, -C(S)- , ‒C(NR')‒, ‒C(O)N(R')‒, ‒N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)- , ‒S(O)2‒, ‒S(O)2N(R') , ‒C(O)S-, ‒C(O)O‒, ‒P(O)(OR')‒, ‒P(O)(SR')‒, ‒P(O)(R')‒ ‒P(O)(NR')-, -P(S)(OR')‒, -P(S)(SR')‒, ‒P(S)(R')‒, ‒P(S)(NR')‒, ‒P(R')- , -P(OR')-, ‒P(SR')‒, ‒P(NR')‒, ‒P(OR')[B(R')3]‒, ‒OP(O)(OR')O‒, ‒OP(O)(SR')O‒, ‒OP(O)(R')O‒ ‒OP(O)(NR')O‒, -OP(OR')O‒, ‒OP(SR')O‒, ‒OP(NR')O‒, ‒OP(R')O‒, or ‒OP(OR')[B(R')3]O‒, and one or more CH or carbon atoms are optionally and independently replaced with CyL ;

each ‒Cy‒ is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each CyL is independently an optionally substituted trivalent or tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each R' is independently ‒R, ‒C(O)R, ‒C(O)OR, or ‒S(O)2R;

each R is independently ‒H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylahphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or

two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyciic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyciic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

7. The composition of claim 5, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3:

or a salt form thereof, wherein:

PL is P( =W), P, or P→ B(R')3;

W is O, N(‒L‒R5), S or Se;

each of R1 and R5 is independently ‒H, ‒L‒R', halogen, ‒CN, ‒NO2, ‒L‒Si(R')3, ‒OR', ‒SR', or ‒N(R')2;

each of Y and Z is independently ‒O‒, ‒S‒, ‒N(‒L‒R5)‒, or L;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C1_6 alkylene, C1_6 alkenylene, -C≡C‒, a bivalent C1_6 heteroaliphatic group having 1-5 heteroatoms, ‒C(R')2-, ‒Cy‒, ‒O‒, ‒S‒, ‒S‒S‒, ‒N(R')‒, ‒C(O)‒, ‒C(S)‒, ‒C(NR')‒, ‒C(O)N(R')‒, ‒N(R')C(O)N(R')‒, ‒N(R’)C(O)O‒, -S(O)- , ‒S(O)2‒, ‒S(O)2N(R') , ‒C(O)S-, ‒C(O)O‒, ‒P(O)(OR')‒, ‒P(O)(SR')‒, ‒P(O)(R')‒ ‒P(O)(NR')-, -P(S)(OR')‒, -P(S)(SR')‒, ‒P(S)(R')‒, ‒P(S)(NR')‒, ‒P(R')- , -P(OR')-, ‒P(SR')‒, ‒P(NR')‒, ‒P(OR')[B(R')3]‒, ‒OP(O)(OR')O‒, ‒OP(O)(SR')O‒, ‒OP(O)(R')O‒ ‒OP(O)(NR')O‒, -OP(OR')O‒, ‒OP(SR')O‒, ‒OP(NR')O‒, ‒OP(R')O‒, or ‒OP(OR')[B(R')3]O‒, and one or more CH or carbon atoms are optionally and independently replaced with CyL;

each ‒Cy‒ is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each CyL is independently an optionally substituted bivalent or tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each R' is independently -R, ‒C(O)R, ‒C(O)OR, or ‒S(O)2R;

each R is independently ‒H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroahphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1- 10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicydic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

8. The composition of claim 5, wherein a non-negatively charged internucleotidic linkage has the

structure of

9. The composition of claim 8, wherein the non-negatively charged internucleotidic linkage

is chirally controlled and is Rp.

10. The composition of claim 8, wherein the transcript is a Dystrophin transcript.

11. The composition of claim 10, wherein splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.

12. The composition of claim 8, wherein each chiral internucleotidic linkage of the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.

13. The composition of claim 8, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.

14. The composition of claim 11, wherein the oligonucleotide type comprises any of: cholesterol; L- camitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex- antennary, alpha and beta).

15. The composition of claim 11, wherein each non-negatively charged internucleotidic linkage is

independently an internucleotidic linkage at least 50% of which exists in its non-negatively charged form at pH 7.4.

16. The composition of claim 11, wherein the oligonucleotides of the plurality each comprise one or more sugar modifications.

17. The composition of claim 16, wherein one or more sugar modifications are 2'-F modifications.

18. The composition of any one of the preceding claims, wherein each heteroatom is independently boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.

19. A pharmaceutical composition comprising an oligonucleotide composition of any one of the preceding claims and a pharmaceutically acceptable carrier.

20. A method for altering splicing of a target transcript, comprising administering an oligonucleotide composition of any one of the preceding claims.

21. The method of claim 20, wherein the target transcript is pre-mRNA of dystrophin.

22. The method of claim 21, wherein exon 45 of dystrophin is skipped at an increased level relative to absence of the composition.

23. The method of claim 21, wherein exon 51 of dystrophin is skipped at an increased level relative to absence of the composition.

24. The method of claim 21, wherein exon 53 of dystrophin is skipped at an increased level relative to absence of the composition.

25. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition of any one of the preceding claims.

26. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, wherein the oligonucleotide comprises one or more non-negatively charged internucleotidic linkages, comprising providing a phosphoramidite compound having the structure of:

wherein:

R5s is independently R' or ‒OR';

each BA is independently an optionally substituted group selected from C3-30 cycloaliphatic, C6-30 aryl, C5-30 heteroaryl having 1-10 heteroatoms, C3-30 heterocyclyl having 1-10 heteroatoms, a natural nucleobase moiety, and a modified nueleobase moiety;

each R5 is independently -H, halogen, ‒CN, ‒N3, -NO, -NO2, -L-R', ‒L‒Si(R)3, -L-OR', ‒L‒SR', ‒L‒N(R')2, ‒O‒L‒R', ‒O‒L‒Si(R)3, ‒O‒L‒OR', ‒O‒L‒SR', or ‒O‒L‒N(R')2;

each s is independently 0-20;

each Ls is independently ‒C(R5s)2‒, or L;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C1-6 alkylene, C1-6 alkenylene, -C≡C-, a bivalent C1 -C6 heteroaliphatic group having 1-5 heteroatoms, ‒C(R')2‒, ‒Cy‒, ‒O‒, ‒S‒, ‒S‒S‒, ‒N(R')‒, ‒C(O)‒, ‒C(S)‒, ‒C(NR')‒, ‒C(O)N(R')‒, ‒N(R')C(O)N(R')‒, ‒N(R’)C(O)O‒, -S(O)- , ‒S(O)2‒, ‒S(O)2N(R') , ‒C(O)S-, ‒C(O)O‒, ‒P(O)(OR')‒, ‒P(O)(SR')‒, ‒P(O)(R')‒, ‒P(O)(NR')‒ ‒P(S)(OR')‒ ‒P(S)(SR')‒ ‒P(S)(R')‒, ‒P(S)(NR')‒, ‒P(R')‒, ‒P(OR')‒, ‒P(SR')‒, ‒P(NR')‒, ‒P(OR')[B(R')3]‒, ‒OP(O)(OR')O‒, ‒OP(O)(SR')O‒, ‒OP(O)(R')O‒ ‒OP(O)(NR')O‒, -OP(OR')O‒, ‒OP(SR')O‒, ‒OP(NR')O‒, ‒OP(R')O‒, or ‒OP(OR')[B(R')3]O‒, and one or more CH or carbon atoms are optionally and independently replaced with CyL ;

each — Cy— is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3- 20 membered heterocyclyl ring having 1-10 heteroatoms;

each CyL is independently an optionally substituted trivalent or tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heteroeyclyl ring having 1-10 heteroatoms;

each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each of G1, G2, G3, G4, G5, and G8is independently R1;

each R1 is independently ‒H, ‒L‒R', halogen, ‒CN, ‒NO2, ‒L‒Si(R')3, ‒OR', ‒SR', or ‒N(R')2; each R' is independently ‒R, ‒C(O)R, C(O)OR. or S(O)2 R;

each R is independently -H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heteroeyclyi having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms; and

wherein G2 comprises an electron-withdrawing group.

27. The method of claim 26, wherein G5 and one of G3 and G4 are taken together to form an optionally substituted 3-8 membered saturated ring having 0-3 heteroatoms in addition to the nitrogen of ‒NG5‒

28. The method of claim 26, wherein the oligonucleotide comprises an interaucleotidic linkage

having the structure of

29. The method of any one of claims 26-28, wherein G2 comprises an electron-withdrawing group.

30. The method of claim 29, wherein G2 is ‒L'‒S(O)2R', wherein IT is optionally substituted ‒CH2‒.

31. The method of claim 30, wherein R' is optionally substituted C1-6 aliphatic.

32. The method of claim 30, wherein R' is t-butyl.

33. The method of claim 30, wherein R' is optionally substituted phenyl.

34. The method of claim 30, wherein R' is phenyl.

35. The method of claim 29, comprising one or more cycles, each of which independently comprises or consisting of:

1) deblocking;

2) coupling;

3) optionally a first capping;

4) modifying; and

5) optionally a second capping.

36. An oligonucleotide, comprising an internucleotidic linkage having the structure of fonnula III:

wherein:

, or

Q- is an anion;

e each of R1 and R5 is independently -H, ‒L‒R', halogen, ‒CN, ‒NO2, ‒L‒Si(R')3, ‒OR', ‒SR', or ‒N(R')2;

each of Y and Z is independently -O-, -S-, ‒N(‒L‒R5)‒, or L;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C1-6 alkylene, C1-6 alkenylene, -C≡C-, a bivalent C1-C6 heteroaliphatic group having 1-5 heteroatoms, ‒C(R')2‒, -Cy-, ‒O‒, ‒S‒, ‒S‒S‒, ‒N(R')‒, ‒C(O)‒, ‒C(S)‒, ‒C(NR')‒, ‒C(O)N(R')‒, ‒N(R')C(O)N(R')‒, ‒N(R’)C(O)O‒, -S(O)- , ‒S(O)2‒, ‒S(O)2N(R') , ‒C(O)S-, ‒C(O)O‒, ‒P(O)(OR')‒, ‒P(O)(SR')‒, ‒P(O)(R')‒ ‒P(O)(NR')-, -P(S)(OR')‒, -P(S)(SR')‒, ‒P(S)(R')‒, ‒P(S)(NR')‒, ‒P(R')- , -P(OR')-, ‒P(SR')‒, ‒P(NR')‒, ‒P(OR')[B(R')3]‒, ‒OP(O)(OR')O‒, ‒OP(O)(SR')O‒, ‒OP(O)(R')O‒ ‒OP(O)(NR')O‒, -OP(OR')O‒, ‒OP(SR')O‒, ‒OP(NR')O‒, ‒OP(R')O‒, or ‒OP(OR')[B(R')3]O‒, and

one or more CH or carbon atoms are optionally and independently replaced with CyL;

each ‒Cy‒ is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each CyL is independently an optionally substituted trivalent or tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each R' is independently ‒R, ‒C(O)R, ‒C(O)OR, or ‒S(O)2R;

each R is independently -H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms; and

-X- IS

wherein G' comprises an electron -

withdrawing group.

37. The oligonucleotide of claim 36, wherein G2 is ‒L'‒S(O)2R', wherein L' is optionally substituted ‒CH2‒.

38. The oligonucleotide of claim 37, wherein R' is optionally substituted C1-6 aliphatic.

39. The oligonucleotide of claim 38, wherein R' is t-butyl.

40. The oligonucleotide of claim 37, wherein R' is optionally substituted phenyl.

41. The oligonucleotide of claim 40, wherein R' is phenyl.

42. The oligonucleotide of any one of claims 36-41, wherein R1 is ‒C(O)R'.

43. The oligonucleotide of claim 42, wherein R' is ‒CH3.

44. The oligonucleotide of any one of claims 36-41, wherein Q- is F-, Cl- , Br-, BF4-, PF6-, TfO- , Tf2N- , AsF6-, ClO4-, or SbF6-,

45. The oligonucleotide of any one of claims 36-44, wherein the oligonucleotide is atached to a solid support.

46. The oligonucleotide of claim 45, wherein the solid support is CPG.

47. A method for preparing an oligonucleotide, comprising contacting an oligonucleotide of any one of claims 36-46 with a base.

48. The method of claim 47, wherein the contact is performed substantially absent of water.

49. The method of claim 47 or 48, wherein the contact is after the oligonucleotide length is achieved before deprotection and cleavage of oligonucleotide .

50. The method of any one of claims 47-49, wherein the base is an amine base having the structure of NR3.

51. The method of claim 50, wherein the base is N,N-diethylamine .

52. The oligonucleotide, compound or method of any one of Example Embodiments 1-420.

53. An oligonucleotide, wherein the oligonucleotide is, WV-20104, WV-20103, WV-20102, WV-20101, WV-20100, WV-20099, WV-20098, WV-20097, WV-20096, WV-20095, WV-20094, WV-20106, WV-20119, WV-20118, WV-13739, WV-13740, WV-9079, WV-9082, WV-9100, WV-9096, WV-9097, WV-9106, WV-9133, WV-9148, WV-9154, WV-9898, WV-9899, WV-9900, WV-9906, WV-9907, WV-9908, WV-9909, WV-9756, WV-9757, WV-9517, WV-9714, WV-9715, WV-9519, WV-9521, WV-9747, WV-9748, WV-9749, WV-9897, WV-9898, WV-9900, WV-9899, WV-9906, WV-9912, WV-9524, WV-9912, WV-9906, WV-9900, WV-9899, WV-9899, WV-9898, WV-9898, WV-9898, WV-9898, WV-9898, WV-9897, WV-9897, WV-9897, WV-9897, WV-9897, WV-9747, WV-9714, WV-9699, WV-9517, WV-9517, WV-13409, WV-13408, WV-12887, WV-12882, WV-12881, WV-12880, WV-12880, WV-WV12880, WV-12878, WV-12877, WV-12877, WV-12876, WV-12873, WV-12872, WV-12559, WV-12559, WV-12558, WV-12558, WV-12557, WV-12556, WV-12556, WV- 12555, WV-12555, WV-12554, WV-12553, WV-12129, WV-12127, WV-12125. WV-12123, WV- 11342, WV-11342, WV-11341, WV-11341, WV-11340, WV-10672, WV-10671, WV-10670, WV- 10461, WV-10455, WV-9897, WV-9898, WV-13826, WV-13827, WV-13835, WV-12880, WV-14344,

WV-13864, WV-13835, WV-14791, WV-14344, WV-13754, WV-13766, WV-11086, WV-11089, WV-17859, WV-17860, WV-20070, WV-20073, WV-20076, WV-20052, WV-20099, WV-20049, WV- 20085, WV -20087, WV-20034, WV -20046, WV-20052, WV-20061, WV-20064, WV-20067, WV- 20092, WV-20091 , WV-20093, WV-20084, WV-9738, WV-9739, WV-9740, WV-9741, WV-15860, WV-15862, WV-11084, WV-11086, WV-11088, WV-11089, WV-14522, WV-14523, WV-17861 , WV-

17862, WV-13815, WV-13816, WV-13817, WV-13780, WV-17862, WV-17863, WV-17864, WV-17865, WV-17866, WV-20082, WV-20081, WV-20080, WV-20079, WV-20076, WV-20075, WV-20074, WV-20073, WV-20072, WV-20071, WV -20064, WV-20059, WV-20058, WV-20057, WV-20056, WV-20053, WV-20052, WV-20051, WV-20050, WV-20049, WV-20094, WV-20095, or a salt form thereof.