Dystrophin exon skipping oligonucleotides

Abstract

Provided herein are antisense oligonucleotides (AONs), and pharma- ceutically acceptable derivatives thereof, that induce skipping of exon 51 of human dystrophin pre-mRNA. Pharmaceutical compositions containing the AONs, and methods of using the AONs and compositions to treat subjects with Duchenne muscular dystrophy, are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 362,189, filed March 30, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which is submitted herewith as an XML file entitled "035104WO_SL.xml", created on March 23, 2023, and 85,189 bytes in size, and is hereby incorporated by reference in its entirety.

[0003] Field Provided herein are antisense oligonucleotides for use in compositions and methods for dystrophin exon skipping and the treatment of Duchenne muscular dystrophy. [Background technology]

[0004] Antisense oligonucleotides (AONs) are in (pre)clinical development for many diseases and conditions, including cancer, inflammatory conditions, cardiovascular diseases, and neurodegenerative and neuromuscular disorders. Their mechanism of action is directed at various targets, e.g., RNaseH-mediated degradation of target RNA in the nucleus or cytoplasm, splice regulation (exon inclusion or skipping) in the nucleus, or translation inhibition by steric hindrance of ribosomal subunit binding in the cytoplasm. Splice regulation or splice switching AONs were first described to correct aberrant splicing of human β-globin pre-mRNA (Dominski and Kole PNAS, 1993, 90(18):8673-8677). They are currently being investigated for various genetic disorders.

[0005] AONs have been widely studied in the treatment of neuromuscular disorders Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which are the most common childhood forms of muscular dystrophy. DMD is a severe and fatal neuromuscular disorder that requires wheelchair assistance by age 12, and patients often die before age 30 from respiratory or cardiac failure. It is caused by frameshift deletions (about 67%) or duplications (about 7%) or point mutations (about 25%) of one or more exons in the 2.24 Mb dystrophin gene, resulting in the absence of functional dystrophin. BMD is also caused by mutations in the dystrophin gene that maintain the open reading frame and produce a semi-functional dystrophin protein, usually resulting in a milder phenotype and longer life span.

[0006] To date, four AONs (eteplirsen, golodirsen, casimersen, and viltolarsen) have been approved for the treatment of DMD, however, these agents have limited efficacy and are each approved for the treatment of only a small proportion of DMD patients.

[0007] Thus, there is a continuing need for exon-skipping AONs for use in compositions and methods for treating DMD. Summary of the Invention

[0008] Provided herein are AONs for use in compositions and methods for treating DMD. Provided herein are AONs that are reverse-complementary to a portion of exon 51 of human dystrophin pre-mRNA. In one embodiment, provided herein are AONs that contain at least one modification as defined herein. In another embodiment, provided herein are AONs that are 2'-O-methoxyethyl ("2'-MOE") RNA oligonucleotides with a phosphorothioate backbone. In another embodiment, provided herein are AONs that are 2'-O-methyl ("2'-OMe") RNA oligonucleotides with a phosphorothioate backbone. In another embodiment, provided herein are AONs that are 2'-MOE RNA oligonucleotides with a phosphorothioate backbone, in which all cytosines are replaced with 5-methylcytosines and all uracils are replaced with thymines. In another embodiment, provided herein are AONs that are 2'-OMe RNA oligonucleotides with a phosphorothioate backbone, in which all cytosines are replaced with 5-methylcytosines and all uracils are replaced with thymines.

[0009] Also provided herein are methods of treating DMD by administering an AON or composition provided herein. Also provided herein are methods of delaying the onset of DMD by administering an AON or composition provided herein. [Brief description of the drawings]

[0010] [Figure 1] 1 shows exon 51 skipping of the AONs provided herein. [Diagram 2] 1 shows an immunofluorescence analysis of dystrophin expression in the AONs provided herein. [Diagram 3] AB show in vivo exon skipping (A) and dystrophin levels (B) in the quadriceps and heart of hDMDdel52 / mdx mice of the AONs provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] I. Definition To facilitate understanding of the disclosure set forth herein, several terms are defined below.

[0012] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, the definitions in this section prevail unless expressly stated otherwise.

[0013] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0014] As used herein, a "subject" is an animal, such as a mammal, including a human patient.

[0015] As used herein, biological activity refers to the in vivo activity of a compound or the physiological response that occurs upon in vivo administration of a compound, composition, or other mixture. Thus, biological activity encompasses the therapeutic effects and pharmacokinetic behavior of such compounds, compositions, and mixtures. Biological activity can be observed in in vitro systems designed to test such activity.

[0016] As used herein, pharma- ceutically acceptable derivatives of compounds include, but are not limited to, their salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, clathrates, solvates, or hydrates. Such derivatives can be readily prepared by those skilled in the art using known methods for such derivatization. The compounds produced can be administered to animals or humans without substantial toxic effects and are pharma- ceutical active or are prodrugs. Pharmaceutically acceptable salts include amine salts (such as, but not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane); alkali metal salts (such as, but not limited to, lithium, potassium, and sodium). ); alkaline earth metal salts (e.g., but not limited to, barium, calcium, and magnesium); transition metal salts (e.g., but not limited to, zinc); and inorganic salts (e.g., but not limited to, sodium hydrogen phosphate and disodium phosphate), including mineral acid salts (e.g., but not limited to, hydrochloride and sulfate salts); and organic acid salts (e.g., but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, mesylate, and fumarate). Pharmaceutically acceptable esters include alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl esters of acidic groups (including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids, and boronic acids). Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of the formula C=C(OR), where R is alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl.Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of the formula C=C(OC(O)R), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl. Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or from 1 to about 100, or from 1 to about 10, or from 1 to about 2, 3, or 4 solvent or water molecules.

[0017] As used herein, treatment refers to any manner in which one or more of the symptoms of a disease or disorder are improved or otherwise beneficially altered. Treatment also includes any pharmaceutical use of the compositions herein (e.g., use in treating DMD).

[0018] As used herein, amelioration of symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any relief, whether permanent or temporary, persistent or transient, that can result from or be associated with administration of the compound or pharmaceutical composition.

[0019] As used herein, unless otherwise indicated, the terms "manage," "managing," and "management" encompass preventing the recurrence of a particular disease or disorder in a subject already suffering from a disease or disorder and / or extending the time that a subject suffering from a disease or disorder remains in remission. The terms include modulating the threshold, progression, and / or duration of a disease or disorder, or altering the way in which a subject responds to a disease or disorder.

[0020] Where moieties are specified in conventional chemical formulas written from left to right, they also encompass the chemically identical moieties that would result if the structure were written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0021] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, an alkyl group having the specified number of carbon atoms (i.e., C1-C 10means a straight-chain (i.e., unbranched) or branched-chain saturated hydrocarbon radical (which can include divalent and multivalent radicals) having from 1 to 10 carbons. Examples of alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0022] The term “alkenyl,” by itself or as part of another substituent, means, unless otherwise stated, an alkyl group having the specified number of carbon atoms (i.e., C1-C 10 means a straight-chain (i.e., unbranched) or branched-chain hydrocarbon radical having one or more carbon-carbon double bonds (which can include divalent and polyvalent radicals) having from 1 to 10 carbons. Examples of alkenyl groups include, but are not limited to, vinyl (i.e., ethenyl), 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and the higher homologs and isomers.

[0023] The term “alkynyl,” by itself or as part of another substituent, means, unless otherwise stated, an alkynyl group having the specified number of carbon atoms (i.e., C1-C 10 means a straight-chain (i.e., unbranched) or branched-chain hydrocarbon radical having one or more carbon-carbon triple bonds (which can include divalent and polyvalent radicals) having 1 to 10 carbons. Examples of alkynyl groups include, but are not limited to, ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

[0024] The term "alkylene," by itself or as part of another substituent, means a divalent radical derived from an alkyl, exemplified, but not limited to, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms (e.g., groups having 10 or fewer carbon atoms). A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having 6 or fewer carbon atoms.

[0025] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in the conventional sense to refer to an alkyl group attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0026] The term "heteroalkyl", alone or in combination with another term, means, unless otherwise stated, a straight or branched chain hydrocarbon radical consisting of heteroatoms selected from the group consisting of O, N, P, Si, and S, where the nitrogen and sulfur atoms may be optionally oxidized, the nitrogen atoms may have alkyl substituents to satisfy valence, and / or may be optionally quaternized. The heteroatom(s) O, N, P, Si, and S may be placed at any interior position of the heteroalkyl group. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, including, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-.

[0027] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively, including bicyclic, tricyclic, and bridged bicyclic groups. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornanyl, bicyclo[2.2.2]octanyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, 1- or 2-azabicyclo[2.2.2]octanyl, and the like.

[0028] The term "halo," by itself or as part of another substituent, means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" is meant to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0029] The term "aryl", unless otherwise specified, means a polyunsaturated aromatic hydrocarbon substituent, which may be a single ring or multiple rings (1-3 rings in one embodiment) fused together or covalently linked. The term "heteroaryl" refers to an aryl group containing 1-4 heteroatoms selected from N, O, and S in the ring(s), wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Heteroaryl groups may be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4- Examples of the heteroaryl ring system include thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. The substituent moieties for the aryl and heteroaryl ring systems can be selected from the group of acceptable substituent moieties described herein. The term "heteroarylium" refers to a heteroaryl group that is positively charged on one or more heteroatoms.

[0030] The term "oxo" as used herein means an oxygen atom that is double bonded to a carbon atom.

[0031] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl," and "heteroaryl") are meant to include both substituted and unsubstituted forms of the indicated radical. Non-limiting examples of substituent moieties for each type of radical are provided below.

[0032] In one embodiment, substituent portions of alkyl, heteroalkyl, alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups can be selected from deuterium, -OR', ═O, ═NR', ═N-OR', -NR'R", -SR', halo, -SiR'R"R"', -O, in a number ranging from zero to the number of hydrogen atoms in such radical. and -N. In one embodiment, the substituent moieties of the cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups include substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl. In one embodiment, R', R", R'", and R"" are each independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl groups. When a compound provided herein includes more than one R group, for example, each of the R groups is independently selected, as are each of the R', R", R"', and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.From the above discussion of substituent moieties, one of skill in the art will understand that the term "alkyl" is meant to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyls (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, and the like).

[0033] Substituent moieties for aryl and heteroaryl groups, in one embodiment, include deuterium, halo, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -COR', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', -S(O)2R' , -S(O)NR'R", -NRSOR', -CN and -NO, -R', -N, -CH(Ph), fluoro(C-C)alkoxy, and fluoro(C-C)alkyl, in a number ranging from 0 to the total number of hydrogens on the aromatic ring system; R', R", R'', and R"" are, in one embodiment, independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound provided herein includes more than one R group, for example, each of the R groups is independently selected, as are each of the R', R", R"', and R"" groups when more than one of these groups is present.

[0034] Two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring are optionally represented by the formula -Q'-C(O)-(CRR') q-Q"-, where Q' and Q" are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring optionally form a ring of the formula A-(CH2) r A and B may be substituted with a substituent of the formula -B-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may be substituted with a substituent of the formula -(CRR') s -X'-(CR”R”') d - substituents, where s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. In one embodiment, the substituent moieties R, R', R", and R"' are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0035] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0036] As used herein, a prodrug is a compound that, when administered in vivo, is metabolized or otherwise undergoes chemical changes under physiological conditions by one or more steps or processes, or is otherwise converted into a biologically, pharmacologic, or therapeutically active form of the compound.In addition, a prodrug can be converted into a biologically, pharmacologic, or therapeutically active form of the compound by chemical or biochemical methods in an ex vivo environment.For example, when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent, a prodrug can be converted into a compound of the present invention.

[0037] Certain compounds provided herein may exist in unsolvated and solvated forms (including hydrated forms). In general, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds provided herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present disclosure.

[0038] Certain compounds provided herein possess asymmetric carbon atoms (optical centers) or double bonds, and the racemates, diastereomers, tautomers, geometric isomers, and individual isomers are encompassed within the scope of the disclosure. Compounds provided herein do not include those known in the art to be too unstable to synthesize and / or isolate.

[0039] The compounds provided herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain radioactive isotopes, such as tritium ( 3 H), Iodine-125( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds provided herein, whether radioactive or not, are encompassed within the scope of the disclosure.

[0040] II. ANTISENSE OLIGONUCLEOTIDES FOR USE IN COMPOSITIONS AND METHODS In one embodiment, provided herein is an AON that is reverse complementary to a portion of exon 51 of human dystrophin pre-mRNA. In one embodiment, the AON provided herein has or contains one of the sequences shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0041] In another embodiment, the AON provided herein has or contains one of the sequences provided herein, in which all nucleotides are RNA.In another embodiment, the AON provided herein has or contains one of the sequences provided herein, in which all nucleotides are DNA.In another embodiment, the AON provided herein has or contains one of the sequences provided herein, in which nucleotides are a mixture of RNA and DNA.

[0042] As known to those skilled in the art, natural oligonucleotides usually contain nucleotides that contain sugar and base moieties linked by a phosphodiester backbone. In one embodiment, the AONs provided herein contain a modification. The modification is a chemical modification of either the sugar or base moiety of one or more nucleotides in the AON, or a chemical modification of the phosphodiester backbone. Each modification can be independently selected. Thus, the AONs provided herein can have two or more different modifications.

[0043] In one embodiment, the modification is a chemical modification of the sugar moiety of one or more nucleotides in the AON, hi another embodiment, the modification is a chemical modification of the sugar moiety of one, two, three, four, or all of the nucleotides in the AON.

[0044] In one embodiment, modified sugar moieties used in the AONs provided herein are: 2'-O-modified RNAs (e.g., 2'-O-alkyl or 2'-O-(substituted)alkyl, such as 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-acetal esters (e.g., Biscans et al. Bioorg. Med. Chem. 2015, 23, 5360), 2'-O-allyl, 2'-O-(2S-methoxypropyl), 2'-O-(N-(aminoethyl)carbamoyl)methyl) (2'-AECM), 2'-O-(2-carboxyethyl), and carbamoyl derivatives (Yamada et al. al. Org. Biomol. Chem. 2014, 12, 6457), 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl), 2'-O-(haloalkoxy)methyl (Arai K. et al. Bioorg. Med. Chem. 2011, 21, 6285), such as 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME), 2'-O-[2-(methylthio)ethyl] (MCE ... )ethyl] (2'-MTE), 2'-(ω-O-serinol); 2'-halo, e.g., 2'-F, FANA (2'-F arabinosyl nucleic acid); 2',4'-difluoro-2'-deoxy; carba- and aza-sugar modifications; 3'-O-substituted (e.g., 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl); 4'-substituted (e.g., 4'-aminomethyl-2'-O-methyl or 4'-aminomethyl-2'-fluoro); 5'-substituted (e.g., 5'-methyl or CNA) (Ostergaard et al. ACS Chem. Biol. 2014, 22, 6227); and derivatives thereof.

[0045] In one embodiment, the 2'-substituted RNA is 2'-F, 2'-O-methyl, or 2'-O-(2-methoxyethyl) (i.e., 2'-MOE). In another embodiment, the 2'-substituted RNA is 2'-MOE. In another embodiment, the 2'-substituted RNA is 2'-OMe.

[0046] In one embodiment, the modified sugar moiety used in the AONs provided herein is a modification that increases the binding affinity to a target strand, and / or increases the melting temperature of the resulting duplex of said first and / or second oligonucleotide with its target, and / or decreases the immunostimulatory effect, and / or increases the biostability, and / or improves biodistribution and / or tissue distribution, and / or improves cellular uptake and transport.

[0047] In one embodiment, the modified sugar moiety used in the AONs provided herein is a bicyclic nucleic acid (BNA) monomer. In one embodiment, the BNA is a pentose-derived moiety that has been chemically modified to conformationally restrict the pentose ring. Non-limiting examples of BNAs used in the AONs provided herein are BNAs in which the first ring (e.g., pentose ring) forms a spirane with an additional cyclic moiety, where both rings share only one atom, BNAs in which the first ring (e.g., pentose ring) is fused with an additional cyclic moiety, where both rings share two adjacent atoms, and BNAs in which the first cycle (e.g., pentose ring) forms a bridged compound through a moiety that is attached to the first cyclic moiety at two non-adjacent atoms. Such adjacent atoms are called bridgehead atoms. A bridged compound contains multiple rings, each of which overlaps with at least three atoms. Instead, a compound that contains two rings, where the rings overlap with only two atoms, is a fused compound. In some bridged compounds, the smallest bond between the two bridgehead atoms is called the bridging moiety, or bridge moiety. In other bridged compounds, if a cycle is a characteristic cycle, such as the pentose cycle of a nucleotide, the part that does not make up the characteristic cycle is called the bridging moiety. Thus, the nomenclature of bridged bicyclic compounds is context-dependent. [ka]

[0048] Bicyclic compounds may contain additional rings. Bicyclic compounds contain at least two rings, which constitute a spirane, a fused system, or a bridged system, or a combination thereof. In one embodiment, the bicyclic compound is a fused and bridged compound. In some embodiments, the BNA is a bridged nucleic acid monomer.

[0049] In one embodiment, each occurrence of a BNA in an AON is independently a conformationally restricted nucleotide (CRN) monomer, a locked nucleic acid (LNA) monomer, a xylo-LNA monomer, an α-LNA monomer, an α-L-LNA monomer, a β-D-LNA monomer, a 2'-amino-LNA monomer, a 2'-(alkylamino)-LNA monomer, a 2'-(acylamino)-LNA monomer, a 2'-N-substituted-2'-amino-LNA monomer, a 2'-thio-LNA monomer, a (2'-O,4'-C) restricted ethyl (cEt) BNA monomer, a (2'-O,4'-C) restricted methoxyethyl (cMOE) BNA monomer, a 2',4'-BNANC(NH) monomer, a 2',4'-BNANC(N-Me) monomer, a 2',4'-BNANC(N- Bn) monomers, ethylene-bridged nucleic acid (ENA) monomers, carba LNA (cLNA) monomers, 3,4-dihydro-2H-pyran nucleic acid (DpNA) monomers, 2'-C-bridged bicyclic nucleotide (CBBN) monomers, heterocyclic bridged BNA monomers, amide bridged BNA monomers, urea bridged BNA monomers, sulfonamide bridged BNA monomers, bicyclic carbocyclic nucleotide monomers, TriNA monomers, α-L-TriNA monomers, bicyclic DNA (bcDNA) monomers, F-bcDNA monomers, tricyclic DNA (tcDNA) monomers, F-tcDNA monomers, oxetane nucleotide monomers, locked PMO monomers (derived from 2'-amino-LNA), and derivatives thereof are provided. In one embodiment, multiple different scaffold BNA modifications can be used in the oligonucleotide.In some embodiments, each occurrence of the BNA is independently selected from the group consisting of a conformationally constrained nucleotide (CRN) monomer, a locked nucleic acid (LNA) monomer, a xylo-LNA monomer, an α-L-LNA monomer, a β-D-LNA monomer, a 2'-amino-LNA monomer, a 2'-(alkylamino)-LNA monomer, a 2'-(acylamino)-LNA monomer, a 2'-N-substituted-2'-amino-LNA monomer, a (2'-O,4'-C) constrained ethyl (cEt) LNA monomer, a (2'-O,4'-C) constrained methoxyethyl (cMOE) BNA monomer, a 2',4'-BNANC(NH) monomer, a 2',4'-BNANC(N-Me) monomer, an ethylene-bridged nucleic acid (ENA) monomer, a 2'-C-bridged bicyclic nucleotide (CBBN) monomer, and derivatives thereof.

[0050] In one embodiment, each occurrence of the BNA is a locked nucleic acid (LNA) monomer. In another embodiment, the AONs provided herein contain an LNA and a 2'-MOE monomer. In another embodiment, the AONs provided herein contain an LNA and a 2'-MeO monomer. In another embodiment, the AONs provided herein contain one to four LNA monomers. In another embodiment, the AONs provided herein contain one to three LNA monomers. In another embodiment, the AONs provided herein contain one or two LNA monomers. In another embodiment, the AONs provided herein contain one, two, three, or four LNA monomers. In another embodiment, the AONs provided herein contain one LNA monomer. In another embodiment, the AONs provided herein contain two LNA monomers. In another embodiment, the AONs provided herein contain three LNA monomers. In another embodiment, the AONs provided herein contain four LNA monomers. In another embodiment, the AON provided herein contains two LNA monomers at the two 5'-terminal nucleotide positions of the AON and two LNA monomers at the two 3'-terminal nucleotide positions of the AON.

[0051] Examples of the structures of these BNAs are shown below: where B is a nucleotide base (e.g., A, G, T, C, 5-methylcytosine, etc.), X is a variable and represents O, S, or NR, R is H or alkyl, X2 is a hydroxyl moiety or another 2'-substitution as defined herein, and L is a backbone bond as described herein. As known to those skilled in the art, the naming of such modifications in the literature is often arbitrary and does not follow a uniform convention. In this application, the names provided below are intended to refer to the structures provided below. For comparison, the cyclic scaffold of a conventional RNA monomer is shown first. In the structures shown below, the monomer is typically depicted as the 3'-end monomer. When chirality is not indicated, each enantiomer is referred to individually. The heteroatoms contained in the cyclic moiety can be replaced with other heteroatoms (e.g., N, O, or S). [ka] [ka] [ka]

[0052] In another embodiment, the BNAs used in the present invention include: cEt (2'-O,4'-C constrained ethyl) LNA (doi:10.1021 / ja710342q), cMOE (2'-O,4'-C constrained methoxyethyl) LNA (Seth et al., J. Org. Chem. 2010, 75, 1569-1581), 2',4'-BNANC(NH), 2',4'-BNANC(N-Me), ethylene bridged nucleic acid (ENA) (doi:10.1093 / nass / 1.1.241), carba LNA (cLNA) (doi:10.1021 / jo100170g), DpNA (Osawa et al., J. Org. Chem., 2015, 80(21), pp 10474-10481), 2'-C-bridged bicyclic nucleotides (CBBN, e.g., WO2014 / 145356 (MiRagen Therapeutics)), heterocycle-bridged LNA (e.g., WO2014 / 126229 (Mitsuoka Y et al.)), amide-bridged LNA (e.g., Yamamoto et al. Org. Biomol. Chem. 2015, 13, 3757), urea-bridged LNA (e.g., Nishida et al. Chem. Commun. 2010, 46, 5283), sulfonamide-bridged LNA (e.g., WO2014 / 112463 (Obika S et al.)), bicyclic carbocyclic nucleosides (e.g., WO2015 / 142910 (Ionis Pharmaceuticals)), TriNA (Hanessian et al. al., J. Org. Chem., 2013, 78(18), pp 9064-9075), α-L-TriNA, bicycloDNA (bcDNA) (Bolli et al., Chem Biol. 1996 Mar;3(3):197-206), F-bcDNA (DOI:10.1021 / jo402690j), tricycloDNA (tcDNA) (Murray et al., Nucl. Acids Res., 2012, Vol. 40, No. 13 6135-6143), F-tcDNA (doi:10.1021 / acs.joc.5b00184), or oxetane nucleotide monomers (Nucleic Acids Res. 2004, 32, 5791-5799).In other embodiments, BNAs as used herein include those disclosed in WO2011 / 097641 (ISIS / Ionis Pharmaceuticals) and WO2016 / 017422 (Osaka University).

[0053] In another embodiment, the chemical modification of the sugar moiety of one or more nucleotides in the AONs provided herein comprises the replacement of the sugar with another chemical moiety. In these embodiments, the sugar moiety is replaced with, for example, morpholine (PMO, PPMO, PMO-X), peptide derivatives (PNAs), boron cluster modified PNAs, pyrrolidine-based oxypeptide nucleic acids (POPNAs), glycol or glycerol-based nucleic acids (GNAs), threose-based nucleic acids (TNAs), acyclic threoninol-based nucleic acids (aTNAs), cationic morpholino-based oligomers (PMOPlus), oligonucleotides with integrated bases and backbones (ONIBs); pyrrolidine amide oligonucleotides (POMs), and derivatives thereof.

[0054] In one embodiment, the modification is a chemical modification of the base moiety of one or more nucleotides in the AON, hi another embodiment, the modification is a chemical modification of the base moiety of one, two, three, four, or all of the nucleotides in the AON.

[0055] In one embodiment, the AONs provided herein contain at least one modified base moiety. In another embodiment, the AONs provided herein have all base moieties modified. In another embodiment, the AONs provided herein have or contain one of the sequences provided herein in which at least one thymine base is uracil. In another embodiment, the AONs provided herein have or contain one of the sequences provided herein in which all thymine bases are uracil. In another embodiment, the AONs provided herein have or contain one of the sequences provided herein in which at least one cytosine base is 5-methylcytosine. In another embodiment, the AONs provided herein have or contain one of the sequences provided herein in which all cytosine bases are 5-methylcytosine. In another embodiment, the AONs provided herein have or contain one of the sequences provided herein in which all thymine bases are uracil and all cytosine bases are 5-methylcytosine.

[0056] In one embodiment, the modification is a chemical modification of the backbone of the AON. In another embodiment, the modification is a chemical modification of the backbone of the AON, where one, two, three, four, or all of the phosphodiester bonds in the AON are modified. In another embodiment, the AONs provided herein have at least one phosphorothioate backbone linkage. In another embodiment, the AONs provided herein have a complete phosphorothioate backbone linkage. In another embodiment, the AONs provided herein have a mixture of phosphorothioate backbone linkages and phosphate backbone linkages. In another embodiment, the AONs provided herein have at least one phosphorodiamidate backbone linkage. In another embodiment, the AONs provided herein have a complete phosphorodiamidate backbone linkage. In another embodiment, the AONs provided herein have a mixture of phosphorodiamidate backbone linkages and phosphate backbone linkages.

[0057] In another embodiment, the backbone of the AON provided herein is a chirally pure phosphorothioate, phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methyl phosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methyl boranophosphonothioate, and derivatives thereof. Alternative modifications include phosphoramidites, phosphoramidates, N3'→P5' phosphoramidates, phosphodiamidates, phosphorothiodiamidates, sulfamates, dimethylenesulfoxides, sulfonates, triazoles, oxalyl, carbamates, methyleneimino (MMI), 3'-S-phosphorothiolates, and thioacetamide nucleic acid (TANA), and derivatives thereof.

[0058] In one embodiment, the AONs provided herein contain hydroxyalkoxy groups. The hydroxyalkoxy groups used in the AONs provided herein comprise or consist of ethylene glycol monomers, ethylene glycol oligomers, or ethylene glycol polymers (also known as polyethylene glycol (PEG)).

[0059] In another embodiment, the hydroxyalkoxy group, or at least one of the hydroxyalkoxy groups, or all of the hydroxyalkoxy groups used in the AONs provided herein, comprises or consists of an ethylene glycol monomer, ethylene glycol oligomer, or ethylene glycol polymer (also known as polyethylene glycol (PEG)). In one embodiment, the bond between the AON and the hydroxyalkoxy group is a covalent bond.

[0060] PEGylation, i.e. the attachment of (chemically activated) hydroxyalkoxy groups containing ethylene glycol monomers or chains, is well known to those skilled in the art. PEGylation can be carried out at the -OH group of the 5'- and / or 3'-terminal monomers of the nucleic acid. This can be done directly or via a spacer (e.g., an aminoalkylhydroxyalkoxy group), for example by click chemistry, as known by those skilled in the art.

[0061] Also included is the use of modified PEGylation, where the (poly)ethylene glycol is chemically modified and / or contains moieties attached thereto. In this way, the hydroxyalkoxy group acquires additional properties known in the art. For example, the hydroxyalkoxy group can be cleavable or fluorescent. An example of modified PEGylation includes, but is not limited to, cleavable PEGylation, where the bond is a degradable (cleavable) bond. Examples include, for example, bonds that respond to pH, light, temperature, reducing or oxidizing environments, nucleophiles, synthesis reagents, enzymes, proteases, cathepsins, click release reactions, or (other) external stimuli.

[0062] In one embodiment, the hydroxyalkoxy group is an unmodified ethylene glycol monomer, ethylene glycol oligomer, or ethylene glycol polymer. In another embodiment, the hydroxyalkoxy group is a modified ethylene glycol monomer, ethylene glycol oligomer, or ethylene glycol polymer (e.g., a modified PEG).

[0063] In one embodiment, the hydroxyalkoxy group used in the AONs provided herein comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol monomers. In some embodiments, the hydroxyalkoxy group comprises or consists of 1-20, 1-16, 1-12, 1-8, 1-6, 2-16, 2-12, 2-8, 2-6, 3-12, 3-8, or 3-6 ethylene glycol monomers. In some embodiments, the hydroxyalkoxy group comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ethylene glycol monomers. In some embodiments, the hydroxyalkoxy group comprises or consists of 3, 4, 5, or 6 ethylene glycol monomers. In some embodiments, the hydroxyalkoxy group comprises or consists of 3 or 6 ethylene glycol monomers. In some embodiments, the hydroxyalkoxy group comprises or consists of three ethylene glycol monomers.

[0064] In one embodiment, the hydroxyalkoxy group is a diethylene glycol, triethylene glycol (TEG), tetraethylene glycol, pentaethylene glycol, or hexaethylene glycol (HEG) group. In some embodiments, the hydroxyalkoxy group is TEG or HEG. In some embodiments, the hydroxyalkoxy group is TEG. In some embodiments, the hydroxyalkoxy group is HEG.

[0065] In the AONs provided herein, the hydroxyalkoxy group can be attached to the AON using methods well known to those skilled in the art. For example, the 5'- and / or 3'-terminal OH of the AON can be derivatized as a phosphoramidite, chloroformate, chloramidate, or thiophosphoramidite, which is then reacted with a hydroxyalkoxy group (e.g., diethylene glycol, triethylene glycol (TEG), tetraethylene glycol, pentaethylene glycol, or hexaethylene glycol (HEG)) under standard coupling conditions to obtain a hydroxyalkoxylated AON. Alternatively, the OH of a hydroxyalkoxy group (e.g., diethylene glycol, triethylene glycol (TEG), tetraethylene glycol, pentaethylene glycol, or hexaethylene glycol (HEG)) can be derivatized as a phosphoramidite, chloroformate, chloramidate, or thiophosphoramidite, which is then reacted with the 5'- and / or 3'-terminal OH of the AON under standard coupling conditions to obtain a hydroxyalkoxylated AON.

[0066] In one embodiment, the hydroxyalkoxy group is attached to the AON via a phosphate linker (PO). In another embodiment, the hydroxyalkoxy group is a TEG group and the hydroxyalkoxylated AON is a TEG-PO-AON (i.e., HO(CH2CHO)3-P(O)(OH)-AON), with the TEG-PO attached to the 5'-OH of the AON. In another embodiment, the hydroxyalkoxy group is a TEG group and the hydroxyalkoxylated AON is a TEG-PO-AON, with the TEG-PO attached to the 3'-OH of the AON. In another embodiment, two TEG groups are attached to the AON, one to the 3'-OH and the other to the 5'-OH, with the hydroxyalkoxylated AON being a (TEG-PO)2-AON.

[0067] In another embodiment, the hydroxyalkoxy group is attached to the AON via a phosphorothioate linker (PS). In another embodiment, the hydroxyalkoxy group is a TEG group and the hydroxyalkoxylated AON is a TEG-PS-AON (i.e., HO(CH2CHO)3-P(S)(OH)-AON), where the TEG-PS is attached to the 5'-OH of the AON. In another embodiment, the hydroxyalkoxy group is a TEG group and the hydroxyalkoxylated AON is a TEG-PS-AON, where the TEG-PS is attached to the 3'-OH of the AON. In another embodiment, two TEG groups are attached to the AON, one to the 3'-OH and the other to the 5'-OH, where the hydroxyalkoxylated AON is a (TEG-PS)2-AON.

[0068] In another embodiment, two TEG groups are attached to the AON, one to the 3'-OH and the other to the 5'-OH, and the hydroxyalkoxylated AON is TEG-PO-AON-PS-TEG; TEG-PO is at the 5' and TEG-PS is at the 3'; or TEG-PO is at the 3' and TEG-PS is at the 5'.

[0069] In another embodiment, the hydroxyalkoxy group is a HEG group and the hydroxyalkoxylated AON is a HEG-PO-AON (i.e., HO(CH2CHO)6-P(O)(OH)-AON), where the HEG-PO is attached to the 5'-OH of the AON. In another embodiment, the hydroxyalkoxy group is a HEG group and the hydroxyalkoxylated AON is a HEG-PO-AON, where the HEG-PO is attached to the 3'-OH of the AON. In another embodiment, two HEG groups are attached to the AON, one to the 3'-OH and the other to the 5'-OH, where the hydroxyalkoxylated AON is a (HEG-PO)2-AON.

[0070] In another embodiment, the hydroxyalkoxy group is a HEG group and the hydroxyalkoxylated AON is a HEG-PS-AON (i.e., HO(CH2CHO)6-P(S)(OH)-AON), and the HEG-PS is attached to the 5'-OH of the AON. In another embodiment, the hydroxyalkoxy group is a HEG group and the hydroxyalkoxylated AON is a HEG-PS-AON, and the HEG-PS is attached to the 3'-OH of the AON. In another embodiment, two HEG groups are attached to the AON, one to the 3'-OH and the other to the 5'-OH, and the hydroxyalkoxylated AON is a (HEG-PS)2-AON.

[0071] In another embodiment, two HEG groups are attached to the AON, one to the 3'-OH and the other to the 5'-OH, and the hydroxyalkoxylated AON is HEG-PO-AON-PS-HEG; HEG-PO at 5' and HEG-PS at 3'; or HEG-PO at 3' and HEG-PS at 5'. In another embodiment, one TEG group and one HEG group are attached to the AON, one to the 3'-OH and the other to the 5'-OH, and the hydroxyalkoxylated AON is TEG-PO-AON-PS-HEG; TEG-PO at 5' and HEG-PS at 3', or TEG-PO at 3' and HEG-PS at 5'. In another embodiment, one TEG group and one HEG group are attached to the AON, one to the 3'-OH and the other to the 5'-OH, and the hydroxyalkoxylated AON is HEG-PO-AON-PS-TEG; either HEG-PO is at the 5' and TEG-PS is at the 3', or HEG-PO is at the 3' and TEG-PS is at the 5'.

[0072] In another embodiment, the AONs provided herein have or contain the sequence of AON No. 33, 34, 35, 36, 37, 38, or 39. In another embodiment, the AONs provided herein have or contain the sequence of AON No. 33, 34, 35, 36, 37, 38, or 39, in which all thymine bases are replaced with uracil. In another embodiment, the AONs provided herein have or contain the sequence of AON No. 33, 38, or 39. In another embodiment, the AONs provided herein have or contain the sequence of AON No. 33, 38, or 39, in which all thymine bases are replaced with uracil.

[0073] In one embodiment, the AON provided herein has one of the sequences provided herein, with one or two nucleotides deleted from the 5' end of the AON, or one or two nucleotides deleted from the 3' end of the AON, or one nucleotide deleted from the 5' end and one nucleotide deleted from the 3' end of the AON. Thus, in this embodiment, the AON provided herein is a 16-mer or a 17-mer.

[0074] In another embodiment, the AONs provided herein contain one of the sequences provided herein or contain a 16-mer or 17-mer as described above and are 16-30 nucleotides in length. In such an embodiment, one of skill in the art would be able to readily determine which nucleotides, and in what order, to add to the 5' and / or 3' ends of the sequences provided herein to obtain an AON that is greater than 18 nucleotides in length and up to 30 nucleotides in length that is up to 90%, 95%, 96%, 97%, 98%, 99%, or 100% reverse complementary to a portion of human dystrophin exon 51 based on the known sequence of human dystrophin exon 51. Such longer AONs are within the scope of the present disclosure. In another embodiment, the AONs provided herein are 16-25 nucleotides in length. In another embodiment, the AONs provided herein are 16-20 nucleotides in length. In another embodiment, the AONs provided herein are 16, 17, 18, 19, or 20 nucleotides in length. In another embodiment, the AONs provided herein are 18 nucleotides in length.

[0075] In another embodiment, provided herein is an AON having one of the sequences provided herein and which is fully 2'-MOE RNA modified, where all cytosines are replaced with 5-methylcytosines and the backbone is a fully phosphorothioate backbone.

[0076] In another embodiment, provided herein are AONs having one of the sequences provided herein and that are fully 2'-OMe RNA modified, where all cytosines are replaced with 5-methylcytosines and the backbone is a fully phosphorothioate backbone.

[0077] In another embodiment, provided herein is an AON having one of the sequences provided herein, wherein all cytosines are replaced with 5-methylcytosines, the two 5' terminal nucleotides of the AON are LNA, the two 3' terminal nucleotides of the AON are LNA, the remaining nucleotides are 2'-MOE RNA modified, and the backbone is a fully phosphorothioate backbone.

[0078] In another embodiment, provided herein is an AON having one of the sequences provided herein, where all cytosines are replaced with 5-methylcytosines, the two 5' terminal nucleotides of the AON are LNA, the two 3' terminal nucleotides of the AON are LNA, the remaining nucleotides are 2'-OMe RNA modified, and the backbone is a fully phosphorothioate backbone.

[0079] The AONs provided herein can be synthesized using standard solid-phase oligonucleotide synthesis techniques. For example, AONs can be synthesized using either an OP-10 synthesizer (GE / AKTA Oligopilot) or a MerMade12 synthesizer (BioAutomation) using standard phosphoramidite protocols. AONs can be cleaved and deprotected in a two-step sequence (e.g., DEA, followed by concentrated NH4OH treatment), purified by anion exchange chromatography, desalted by size exclusion chromatography, and lyophilized.

[0080] III. Pharmaceutical Compositions Pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more AONs provided herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0081] The AONs can be formulated into suitable pharmaceutical preparations (e.g., solutions, suspensions, powders, sterile solutions or suspensions for ophthalmic or parenteral administration, and transdermal patch preparations). In general, the AONs described herein are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999).

[0082] In the compositions, an effective concentration of one or more compounds or pharma- ceutically acceptable salts are mixed with a suitable pharmaceutical carrier or vehicle. In certain embodiments, the concentration of AON in the composition is effective to deliver an amount that, upon administration, treats, prevents, or ameliorates one or more of the symptoms and / or progression of a disease or disorder disclosed herein.

[0083] Usually, the composition is formulated for single administration.To formulate the composition, the weight fraction of the compound is dissolved, suspended, dispersed or otherwise mixed in the selected vehicle at effective concentration so as to relieve or improve the treated condition.The pharmaceutical carrier or vehicle suitable for administration of the AON provided herein includes any such carrier known by those skilled in the art to be suitable for specific administration mode.

[0084] Furthermore, AONs may be formulated as the only pharma- ceutically active ingredient in a composition, or may be formulated in combination with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharma- ceutically acceptable carriers. They may be prepared according to methods known to those skilled in the art. For example, liposomal formulations may be prepared as known in the art. Briefly, liposomes, such as multivesicles (MLVs), may be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) in a flask. A solution of a compound provided herein in phosphate-buffered saline (PBS) without divalent cations is added, and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation, and then resuspended in PBS.

[0085] The active compound is included in a pharma- ceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without undesirable side effects in the subject being treated. Therapeutically effective concentrations can be empirically determined by testing the AON in the in vitro and in vivo systems described herein, and then extrapolated to human dosages. In some embodiments, the AON is administered in a manner that results in a therapeutically effective concentration of the drug. In some embodiments, a companion diagnostic (e.g., Olsen D and Jorgensen JT, Front. Oncol., 2014 May 16, 4:105, doi:10.3389 / fonC.2014.00105) is used to determine the therapeutic concentration and safety profile of the active compound in a particular subject or population of subjects.

[0086] The concentration of the AON in the pharmaceutical composition will depend on the absorption of the active compound, tissue distribution, inactivation and excretion rate of the compound, the physicochemical properties of the compound, the dosing schedule, and the amount administered, as well as other factors known to those skilled in the art, for example, the amount delivered will be sufficient to ameliorate one or more of the symptoms of a disease or disorder disclosed herein.

[0087] In certain embodiments, a therapeutically effective dosage should produce a serum concentration of the active ingredient of about 0.1 ng / mL to about 50-100 μg / mL. In one embodiment, the pharmaceutical composition provides a dosage of about 0.001 mg to about 2000 mg of compound per kilogram of body weight per day. Pharmaceutical unit dosage forms are prepared to obtain about 1 mg to about 1000 mg, and in certain embodiments, about 10 to about 500 mg of the essential active ingredient or combination of essential ingredients per dosage unit form.

[0088] AON may be administered at once or divided into several small doses at intervals. It is understood that the exact dosage and duration of treatment depend on the disease being treated and can be empirically determined using known test protocols or by extrapolating from in vivo or in vitro test data. It is also noted that concentration and dosage values ​​may vary as the severity of the condition is alleviated. It should also be understood that for any particular subject, a particular dosage regimen must be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0089] Thus, an effective concentration or amount of one or more of the AONs described herein, or a pharma- ceutically acceptable salt thereof, is mixed with a pharmaceutical carrier or vehicle suitable for systemic, local, or topical administration to form a pharmaceutical composition. The AON is included in an amount effective to ameliorate or treat, slow the progression of, or prevent one or more symptoms of a disease or disorder. The concentration of the active compound in the composition will depend on the absorption, tissue distribution, inactivation, excretion rate of the active compound, administration schedule, dosage, particular formulation, and other factors known to those skilled in the art.

[0090] The compositions are intended to be administered by a suitable route, including, but not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, dermal, transdermal, or buccal.

[0091] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain any of the following components: a sterile diluent (e.g., water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, dimethylacetamide, or other synthetic solvents); antibacterial agents (e.g., benzyl alcohol and methylparabens); antioxidants (e.g., ascorbic acid and sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetates, citrates, and phosphates); and agents for adjusting isotonicity (e.g., sodium chloride or dextrose). Parenteral preparations can be enclosed in ampoules, pens, disposable syringes, or single- or multi-dose vials made of glass, plastic, or other suitable material.

[0092] If the AON exhibits insufficient solubility, methods for dissolving the AON may be used. Such methods are known to those skilled in the art and include, but are not limited to, using a co-solvent (e.g., dimethyl sulfoxide (DMSO)), using a surfactant (e.g., TWEEN®), or dissolving in aqueous sodium bicarbonate.

[0093] Upon mixing or addition of the AON(s), the resulting mixture may be a solution, suspension, emulsion, etc. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the AON in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder, or condition being treated and may be empirically determined.

[0094] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage form (e.g., powders, granules, sterile parenteral solutions, or suspensions and oil-water emulsions containing an appropriate amount of the AON or a pharma- ceutically acceptable salt thereof). The pharma- ceutically therapeutically active AON and its salts are formulated and administered in unit dosage form or multiple dosage form. Unit dosage form, as used herein, refers to physically discrete units suitable for human and animal subjects and individually packaged, as known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes, individually packaged tablets or capsules. A single dosage form may be administered in portions or multiples thereof. A multiple dosage form is a plurality of identical single dosage forms packaged in a single container to be administered in divided single dosage form. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons. Thus, a multiple dose form is an undivided multiple of a unit dose when packaged.

[0095] Sustained release formulations can also be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the compounds provided herein, which matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include iontophoretic patches, polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON). DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time. If encapsulated compounds remain in the body for long periods of time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in structure. Depending on the mechanism of action involved, rational strategies for stabilization can be devised. For example, if the mechanism of aggregation is found to be intermolecular S--S bond formation via thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acidic solution, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0096] Dosage forms or compositions containing active ingredients in the range of 0.005%-100%, with the remainder consisting of non-toxic carriers, may be prepared. Such compositions include, but are not limited to, solutions, suspensions, powders, and sustained release formulations (including but not limited to implants and microencapsulated delivery systems), as well as biodegradable, biocompatible polymers (e.g., collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like). Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain about 0.001%-100% active ingredients, in certain embodiments, about 0.1-85% or about 75-95%.

[0097] The active AON or pharma- ceutically acceptable salt may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coatings.

[0098] The composition may contain other active AONs to obtain a desired combination of properties. As described herein, the AONs or pharma- ceutically acceptable salts thereof provided herein may also be advantageously administered for therapeutic or prophylactic purposes with another pharmacologically active substance known in the art to be of value in treating one or more of the above-mentioned diseases or conditions (e.g., diseases associated with oxidative stress). It should be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.

[0099] A. Injections, solutions, and emulsions Parenteral administration, generally characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquids or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. In some embodiments, the suspension is a suspension of microparticles or nanoparticles. In some embodiments, the emulsion is an emulsion of microparticles or nanoparticles. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins. It is also contemplated herein to implant a sustained-release or sustained-release system such that a constant level of dosage is maintained.Briefly, the AONs provided herein are dispersed in a solid inner matrix (e.g., polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers (e.g., acrylate and methacrylate hydrogels), collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate) that is insoluble in body fluids, which solid inner matrix is ​​dispersed in an outer polymeric membrane (e.g., polyethylene, polypropylene, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers (e.g., acrylate and methacrylate hydrogels), collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate) that is insoluble in body fluids. The AON is surrounded by a polymeric membrane (e.g., propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride copolymer with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomeric polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer, and ethylene / vinyloxyethanol copolymer). The AON diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active AON contained in such parenteral compositions is highly dependent on its specific nature, as well as the activity of the compound and the needs of the subject.

[0100] Parenteral administration of the composition includes intravenous, subcutaneous, and intramuscular administration. Preparations for parenteral administration include sterile solutions for injection, sterile dry soluble products (e.g., lyophilized powders) ready to mix with a solution immediately before use (e.g., subcutaneous tablets), sterile suspensions for injection, sterile dry insoluble products ready to mix with a vehicle immediately before use, and sterile emulsions. The solutions can be either aqueous or non-aqueous.

[0101] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0102] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances.

[0103] Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations should be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0104] The concentration of the pharma- ceutically active AON is adjusted to obtain an effective amount to produce the desired pharmacological effect upon injection, with the exact dose depending on the age, weight, and condition of the subject or animal as known in the art.

[0105] Single-dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

[0106] Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active AON is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the active AON injected as needed to produce the desired pharmacological effect.

[0107] The injectables are designed for local and systemic administration. Typically, a therapeutically effective dosage is formulated to contain at least about 0.1% w / w and at most about 90% w / w or more (e.g., more than 1% w / w) of active AON in the tissue(s) to be treated. The active AON may be administered once or divided into several smaller doses spaced apart. It is understood that the exact dosage and duration of treatment will depend on the tissue to be treated and may be empirically determined using known testing protocols or by extrapolating from in vivo or in vitro test data. It is also noted that concentration and dosage values ​​may vary with the age of the individual to be treated. Furthermore, it should be understood that for any particular subject, a particular dosing regimen must be tailored over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the formulation, and that the concentration ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed formulations.

[0108] The AONs may be suspended in micronized or other suitable form, or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture will depend on a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration will be sufficient to ameliorate the symptoms of the condition and can be empirically determined.

[0109] B. Freeze-dried powder Also provided herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

[0110] Sterile lyophilized powders are prepared by dissolving the AONs or pharma- ceutically acceptable salts thereof provided herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solutions prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer such as citrate, sodium or potassium phosphate, or other buffers as known to those of skill in the art, in one embodiment, a buffer with a pH of about neutral. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, will yield the desired formulation. Typically, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dose (including but not limited to 10-1000 mg or 100-500 mg) or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions (eg, at about 4° C. to room temperature).

[0111] A solution of the lyophilized powder and water for injection provides a formulation for parenteral administration. For reconstitution, about 1-50 mg, about 5-35 mg, or about 9-30 mg of lyophilized powder is added per mL of sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amounts can be empirically determined.

[0112] C. Topical Administration Topical mixtures are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and is formulated as a cream, gel, ointment, emulsion, liquid, elixir, lotion, suspension, tincture, paste, foam, aerosol, wash, spray, suppository, bandage, skin patch, or other formulation suitable for topical administration.

[0113] The compound can be formulated for topical application or local application (e.g., topical application to the skin and mucous membranes), for example, in the form of gels, creams, and lotions, as well as for application to the eye, or for intracisternal or intrathecal application, for example, in the eye. Topical administration is contemplated for transdermal delivery, as well as for administration to the eye or mucous membranes, or for inhalation therapy. Nasal solutions of the active compound alone or in combination with other pharma- ceutically acceptable excipients can also be administered.

[0114] These solutions, particularly those intended for ophthalmic use, may be formulated as 0.01%-10% isotonic solutions (pH about 5-7) containing appropriate salts.

[0115] D. Sustained Release Compositions The AONs provided herein can be administered by controlled release means or delivery devices known to those skilled in the art.Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and U.S. Patent Nos. 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,639,480, 5,733,566, 5,733,567, 5,733,568, 5,733,569, 5,733,570, 5,733,571, 5,733,572, 5,733,573, 5,733,574, 5,733,575, 5,733,576, 5,733,577, 5,733,578, 5,733,579 ... 39,108, 5,891,474, 5,922,356, 5,972,891, 5,980,945, 5,993,855, 6,045,830, 6,087,324, 6,113,943, 6,197,350, 6,248,363, 6,264,970, 6,267,981, 6,376,461, 6,419,961, 6,589,548, 6,613,358, 6,699,500, and 6,700,000. No. 6,740,634, each of which is incorporated herein by reference. For example, hydroxypropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres, or combinations thereof are used to provide sustained or controlled release of one or more active ingredients, and such dosage forms can be used to obtain desired release profiles at various rates. Suitable controlled release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the active ingredients provided herein.

[0116] All controlled release pharmaceutical products have a common goal of improving drug therapy beyond that achieved by their non-controlled counterparts. In one embodiment, the use of optimally designed controlled release preparations in treatment is characterized by the minimum amount of drug substance used to cure or control the pathology in the minimum amount of time. In certain embodiments, the advantages of controlled release formulations include extended drug activity, reduced dosing frequency, and improved subject compliance. In addition, controlled release formulations can be used to affect other characteristics such as the time of onset of action or blood concentration of the drug, and therefore can affect the occurrence of side (e.g., adverse) effects.

[0117] Many controlled release formulations are designed to initially release an amount of drug (active ingredient) that quickly produces the desired therapeutic effect, and then slowly and continuously release another amount of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time.To maintain a constant drug level in the body, the drug must be released from the dosage form at a rate that compensates for the amount of drug metabolized and excreted from the body.The controlled release of AONs can be stimulated by a variety of conditions, including but not limited to pH, temperature, enzymes, water, or other physiological conditions or compounds.

[0118] In certain embodiments, AONs can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, pumps can be used (see Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used. In yet another embodiment, the controlled release system can be placed near the therapeutic target, i.e., so that only a fraction of the systemic dose is required (see, for example, Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).

[0119] Other controlled release systems are described in the review by Langer (Science 249:1527-1533 (1990)). AONs may be dispersed in a solid inner matrix (e.g., polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers (e.g., acrylate and methacrylate hydrogels), collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate) that is insoluble in body fluids. The AON is surrounded by a polymeric membrane (e.g., ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride copolymer with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomeric polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer, and ethylene / vinyloxyethanol copolymer). The AON then diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active ingredient contained in such parenteral compositions is highly dependent on their specific nature and the needs of the subject.

[0120] E. Targeted Formulations The AONs or pharma- ceutically acceptable salts thereof provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of the subject to be treated. This includes liposome delivery systems, resealed red blood cell delivery systems, and antibody-based delivery systems. Many such targeting methods are well known to those skilled in the art. All such targeting methods for use in the present compositions are contemplated herein. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, and 6,071,495. Nos. 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

[0121] In one embodiment, the antibody-based delivery system is an antibody drug conjugate ("ADC"), e.g., as described in: Hamilton GS, Biologicals, 2015 September, 43(5):318-32; Kim EG and Kim KM, Biomol. Ther. (Seoul), 2015 November, 23(6):493-509; and Peters C and Brown S, Biosci. Rep., 2015 Jun. 12, 35(4) pii:e00225, each of which is incorporated herein by reference.

[0122] In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharma-ceutically acceptable carriers. They may be prepared according to methods known to those skilled in the art. For example, liposomal formulations may be prepared as described in U.S. Pat. No. 4,522,811. Briefly, liposomes, such as multivesicles (MLVs), may be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) in a flask. A solution of AONs provided herein in phosphate buffered saline (PBS) without divalent cations is added, and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compounds, pelleted by centrifugation, and then resuspended in PBS.

[0123] F.Manufactured products The AON or pharma- ceutically acceptable salt can be packaged as an article of manufacture containing packaging material, an AON provided herein, or a pharma- ceutically acceptable salt thereof (used for the treatment, prevention, or amelioration of one or more symptoms or progression of a disease or disorder disclosed herein), and a label indicating that the compound or a pharma- ceutically acceptable salt thereof is used for the treatment, prevention, or amelioration of one or more symptoms or progression of a disease or disorder disclosed herein.

[0124] The manufactured articles provided herein contain packaging materials. The packaging materials used in packaging pharmaceutical products are well known to those skilled in the art. For example, see U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, pens, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment. A wide range of formulations of the AONs and compositions provided herein are contemplated.

[0125] In certain embodiments, kits are also provided herein that, when used by a medical practitioner, can simplify administration of an appropriate amount of an AON to a subject. In certain embodiments, the kits provided herein include a container and a dosage form of an AON provided herein or a pharma- ceutically acceptable salt, solvate, or prodrug thereof.

[0126] In certain embodiments, the kit comprises a container containing a dosage form of an AON or a pharma- ceutically acceptable salt, solvate, or prodrug thereof provided herein within a container that contains one or more other therapeutic agent(s) described herein.

[0127] The kits provided herein may further include a device used to administer the AONs. Examples of such devices include, but are not limited to, syringes, needleless injectors, drip bags, patches, and inhalers.

[0128] The kits provided herein may further include a pharma- ceutically acceptable vehicle that can be used to administer one or more active ingredients. For example, if the active ingredient is provided in a solid form that needs to be prepared into a solution for parenteral administration, the kit may include a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particulate-free sterile solution suitable for parenteral administration. Examples of pharma- ceutically acceptable vehicles include, but are not limited to, aqueous vehicles (e.g., but are not limited to, water for injection (USP), sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection); water-miscible vehicles (e.g., but are not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol); and non-aqueous vehicles (e.g., but are not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate).

[0129] IV. Administration The AONs and pharmaceutical compositions provided herein may be administered in specific therapeutically or prophylactically effective amounts, at specific time intervals, in specific dosage forms, and in specific dosage administration methods, as described below.

[0130] In certain embodiments, a therapeutically or prophylactically effective amount of the AON is about 0.005 to about 1,000 mg per day, about 0.01 to about 500 mg per day, about 0.01 to about 250 mg per day, about 0.01 to about 100 mg per day, about 0.1 to about 100 mg per day, about 0.5 to about 100 mg per day, about 1 to about 100 mg per day, about 0.01 to about 50 mg per day, about 0.1 to about 50 mg per day, about 0.5 to about 50 mg per day, about 1 to about 50 mg per day, about 0.02 to about 25 mg per day, about 0.05 to about 10 mg per day, about 0.05 to about 5 mg per day, about 0.1 to about 5 mg per day, or about 0.5 to about 5 mg per day.

[0131] In certain embodiments, the therapeutically or prophylactically effective amount is about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, or about 150 mg per day.

[0132] In one embodiment, the recommended daily dose range of the AON or derivative thereof provided herein for the conditions described herein is in the range of about 0.5 mg to about 50 mg per day, in one embodiment administered as a single dose once a day or in divided doses throughout the day. In some embodiments, dosages range from about 1 mg to about 50 mg per day. In other embodiments, dosages range from about 0.5 to about 5 mg per day. Specific daily doses include 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg / day.

[0133] In certain embodiments, the recommended starting dosage may be 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, or 50 mg / day. In another embodiment, the recommended starting dosage may be 0.5, 1, 2, 3, 4, or 5 mg / day. The dose may be increased to 15, 20, 25, 30, 35, 40, 45, and 50 mg / day. In certain embodiments, the AON may be administered in an amount of about 25 mg / day. In certain embodiments, the AON may be administered in an amount of about 10 mg / day. In certain embodiments, the AON may be administered in an amount of about 5 mg / day. In certain embodiments, the AON may be administered in an amount of about 4 mg / day. In certain embodiments, the AON may be administered in an amount of about 3 mg / day.

[0134] In certain embodiments, the therapeutically or prophylactically effective amount is about 0.001 to about 100 mg / kg / day, about 0.01 to about 50 mg / kg / day, about 0.01 to about 25 mg / kg / day, about 0.01 to about 10 mg / kg / day, about 0.01 to about 9 mg / kg / day, 0.01 to about 8 mg / kg / day, about 0.01 to about 7 mg / kg / day, about 0.01 to about 6 mg / kg / day, about 0.01 to about 5 mg / kg / day, about 0.01 to about 4 mg / kg / day, about 0.01 to about 3 mg / kg / day, about 0.01 to about 2 mg / kg / day, about 0.01 to about 1 mg / kg / day, or about 0.01 to about 0.05 mg / kg / day.

[0135] Dosages administered can be expressed in units other than mg / kg / day. For example, parenteral doses can be expressed in mg / m 2 The dosage can be expressed as mg / kg / day to mg / m2 for either the height or weight of the subject, or both. 2 It will be readily apparent how to convert this to a daily dose (see www.fda.gov / cder / cancer / animalframe.htm). For example, for a human weighing 65 kg, a dose of 1 mg / kg / day is approximately 38 mg / m 2 / day.

[0136] In certain embodiments, the amount of AON administered is sufficient to provide a steady-state plasma concentration of the compound in the range of about 0.001 to about 500 μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, about 1 to about 50 μM, about 0.02 to about 25 μM, about 0.05 to about 20 μM, about 0.1 to about 20 μM, about 0.5 to about 20 μM, or about 1 to about 20 μM.

[0137] In other embodiments, the amount of AON administered is sufficient to provide a steady state plasma concentration of the compound in the range of about 5 to about 100 nM, about 5 to about 50 nM, about 10 to about 100 nM, about 10 to about 50 nM, or about 50 to about 100 nM.

[0138] As used herein, the term "steady state plasma concentration" refers to the concentration reached after a period of administration of a compound provided herein or a derivative thereof. When steady state is reached, there are small peaks and troughs in the time-dependent curve of the plasma concentration of the compound.

[0139] In certain embodiments, the amount of AON administered is sufficient to provide a maximum plasma concentration (peak concentration) of the compound in the range of about 0.001 to about 50 μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, about 1 to about 50 μM, about 0.02 to about 25 μM, about 0.05 to about 20 μM, about 0.1 to about 20 μM, about 0.5 to about 20 μM, or about 1 to about 20 μM.

[0140] In certain embodiments, the amount of AON administered is sufficient to provide a minimum plasma concentration (trough concentration) of the compound in the range of about 0.001 to about 500 μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, about 1 to about 50 μM, about 0.01 to about 25 μM, about 0.01 to about 20 μM, about 0.02 to about 20 μM, about 0.02 to about 20 μM, or about 0.01 to about 20 μM.

[0141] In certain embodiments, the amount of AON administered is sufficient to provide an area under the curve (AUC) of the compound in the range of about 100 to about 100,000 ng×hr / mL, about 1,000 to about 50,000 ng×hr / mL, about 5,000 to about 25,000 ng×hr / mL, or about 5,000 to about 10,000 ng×hr / mL.

[0142] The methods provided herein encompass treating patients regardless of the subject's age, although some diseases or disorders are more prevalent in certain age groups.

[0143] Depending on the disease to be treated and the condition of the subject, the AONs or derivatives thereof provided herein may be administered parenterally (e.g., intramuscularly, intraperitoneally, intravenously, CIV, intracisternal injection or infusion, subcutaneous injection, or implant) or topically (e.g., transdermally or topically) via administration routes. The AONs or derivatives thereof provided herein may be formulated, alone or in combination, in suitable dosage units with pharma- ceutically acceptable excipients, carriers, adjuvants, and vehicles appropriate for each administration route.

[0144] In another embodiment, the AONs or derivatives thereof provided herein are administered parenterally. In yet another embodiment, the AONs or derivatives thereof provided herein are administered intravenously.

[0145] The AONs or derivatives thereof provided herein can be delivered as a single dose (e.g., a single bolus injection) or over an extended period of time (e.g., continuous infusion over an extended period of time or divided bolus doses over an extended period of time). The AONs can be administered repeatedly as needed, for example, until the subject experiences stable or regression of disease, or until the subject experiences disease progression or unacceptable toxicity.

[0146] The AONs or derivatives thereof provided herein can be administered once a day (QD) or divided into multiple daily doses (e.g., twice a day (BID), three times a day (TID), and four times a day (QID)). Furthermore, administration can be continuous (i.e., daily or every day), intermittent (e.g., cyclical (i.e., with days, weeks, or months of drug-free rest)). As used herein, the term "daily" is intended to mean that a therapeutic compound, such as a compound provided herein or a derivative thereof, is administered, for example, one or more times each day for a period of time. The term "continuous" is intended to mean that a therapeutic compound, such as a compound provided herein or a derivative thereof, is administered daily for an uninterrupted period of at least 10 days to 52 weeks. As used herein, the term "intermittent" or "intermittently" is intended to mean stopping and starting at regular or irregular intervals. For example, intermittent administration of the AONs or derivatives thereof provided herein can be administration for 1-6 days per week, cyclic administration (e.g., once daily for 2-8 consecutive weeks, followed by a rest period of up to 1 week without administration), or administration every other day. As used herein, the term "cycling" is intended to mean that a therapeutic compound, such as a compound provided herein or a derivative thereof, is administered daily or continuously (with a rest period). In some such embodiments, administration is once daily for 2-6 days, followed by a rest period of 5-7 days without administration.

[0147] In some embodiments, the frequency of administration ranges from about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. In one embodiment, the compound provided herein or a derivative thereof is administered once a day. In another embodiment, the AON or a derivative thereof provided herein is administered twice a day. In yet another embodiment, the AON or a derivative thereof provided herein is administered three times a day. In yet another embodiment, the AON or a derivative thereof provided herein is administered four times a day.

[0148] In certain embodiments, the AONs or derivatives thereof provided herein are administered once a day for 1 day to 6 months, 1 week to 3 months, 1 week to 4 weeks, 1 week to 3 weeks, or 1 week to 2 weeks. In certain embodiments, the AONs or derivatives thereof provided herein are administered once a day for 1 week, 2 weeks, 3 weeks, or 4 weeks. In one embodiment, the AONs or derivatives thereof provided herein are administered once a day for 4 days. In one embodiment, the AONs or derivatives thereof provided herein are administered once a day for 5 days. In one embodiment, the AONs or derivatives thereof provided herein are administered once a day for 6 days. In one embodiment, the AONs or derivatives thereof provided herein are administered once a day for 1 week. In another embodiment, the AONs or derivatives thereof provided herein are administered once a day for 2 weeks. In yet another embodiment, the AONs or derivatives thereof provided herein are administered once a day for 3 weeks. In yet another embodiment, the AONs or derivatives thereof provided herein are administered once a day for 4 weeks.

[0149] V. Treatment Method In another embodiment, a method for treating a subject with Duchenne muscular dystrophy (DMD) is provided. In one embodiment, the method comprises administering to a subject an AON or a composition as provided herein. In another embodiment, a method is provided for delaying the onset of DMD by administering an AON or a composition as provided herein. In another embodiment, the method alleviates one or more symptoms (or symptoms) of DMD.

[0150] The alleviation of one or more symptoms of DMD in an individual using the AONs provided herein may be assessed by any of the following assays: prolongation of time to loss of ambulation, improvement in muscle strength, improvement in ability to lift weights, improvement in time to rise from floor, improvement in time to walk 9 meters, improvement in time to climb 4 flights of stairs, improvement in leg function grade, improvement in lung function, improvement in cardiac function, improvement in quality of life. Each of these assays is known to those skilled in the art. In each of these assays, upon finding a detectable improvement or prolongation of the parameter measured in the assay, it would preferably mean that one or more symptoms of DMD have been alleviated in an individual using the AONs provided herein. In one embodiment, the detectable improvement or prolongation is a statistically significant improvement or prolongation as described in Neuromuscular Disorders 2006;16:591-602. Alternatively, the alleviation of one or more symptoms of DMD may be assessed by measuring improvements in muscle fiber function, integrity, and / or survival. In another embodiment, one or more symptom(s) of a DMD patient is alleviated and / or one or more characteristic(s) of one or more muscle cells from a DMD patient are improved. Such symptoms or characteristics may be assessed at the cellular or tissue level, or in the patient themselves.

[0151] Alleviation of one or more characteristics of muscle cells from a patient may be assessed by any of the following tests on myoblasts or muscle cells from a patient: decreased calcium uptake by muscle cells, decreased collagen synthesis, changes in morphology, changes in lipid biosynthesis, reduced oxidative stress, and / or improved muscle fiber function, integrity, and / or survival. These parameters are typically assessed using immunofluorescence and / or histochemical analysis of cross-sections of muscle biopsies.

[0152] Improved muscle fiber function, integrity and / or survival may be assessed using at least one of the following tests: a detectable decrease in creatine kinase in the blood, a detectable decrease in muscle fiber necrosis in a biopsy cross-section of a muscle suspected to be dystrophic, and / or a detectable increase in uniformity of muscle fiber diameter in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to those of skill in the art.

[0153] Creatine kinase can be detected in the blood as described in Hodgetts et al. (2006). A detectable decrease in creatine kinase can mean a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater decrease compared to the creatine kinase concentration of the same DMD patient before treatment.

[0154] The detectable reduction in muscle fiber necrosis is preferably assessed by muscle biopsy, more preferably using a biopsy cross-section as described in Hodgetts et al. (2006). The detectable reduction in necrosis may be a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in the area of ​​confirmed necrosis using a biopsy cross-section. The reduction is measured relative to necrosis assessed in the same DMD patient before treatment.

[0155] A detectable increase in muscle fiber diameter uniformity is preferably assessed in muscle biopsy cross sections, more preferably as described in Hodgetts et al., supra. The increase is measured relative to the muscle fiber diameter uniformity of the same DMD patient prior to treatment.

[0156] In one embodiment, the AON provided herein is capable of providing a functional or semi-functional dystrophin protein to the individual and at least partially reducing the production of abnormal dystrophin protein in the individual.

[0157] In one embodiment, providing a functional or semi-functional dystrophin protein to an individual refers to increased production of functional or semi-functional dystrophin protein. Increased production of functional or semi-functional dystrophin mRNA or functional or semi-functional dystrophin protein means a detectable increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200% or more compared to the initial amount of functional or semi-functional mRNA or functional or semi-functional dystrophin protein (as determined by RT-digital droplet PCR (mRNA) (Verheul et al., PLoS ONE 2016, 11(9):e0162467) or immunofluorescence (Beekman et al., PLoS ONE 2014;9(9):e107494), Western blot, or capillary Western immunoassay (Beekman et al., PLoS ONE 2018;13(4):e0195850) analysis (protein). In another embodiment, the initial amount is the amount of functional or semi-functional mRNA or functional or semi-functional dystrophin protein at the time when a compound of the invention is used to initiate induction of exon skipping of dystrophin pre-mRNA in a cell, organ, tissue, and / or individual.

[0158] Reducing the production of abnormal dystrophin mRNA or abnormal dystrophin protein means 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of abnormal dystrophin mRNA or abnormal dystrophin protein, which is still detectable by RT digital droplet PCR (mRNA) or immunofluorescence, Western blot, or capillary Western immunoassay (Wes) analysis (protein). In one embodiment, the initial amount is the amount of abnormal dystrophin mRNA or abnormal dystrophin protein at the time when the AON of the present invention is used to start inducing exon skipping of dystrophin pre-mRNA in cells, organs, tissues, and / or individuals. Abnormal dystrophin mRNA or protein is also referred to herein as less functional or non-functional dystrophin mRNA or protein (compared to wild-type functional dystrophin protein). Non-functional dystrophin protein is a dystrophin protein that cannot bind to actin and / or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does not usually have or encode a dystrophin protein with an intact C-terminus of the protein. Detection of functional or semi-functional dystrophin mRNA or protein can be performed on abnormal dystrophin mRNA or protein.

[0159] When DMD patients are provided with functional or semi-functional dystrophin protein, at least part of the cause of DMD is eliminated.Therefore, it is expected that DMD symptoms will then be at least partially alleviated, or the rate at which symptoms worsen will decrease, resulting in a slowdown in debilitation.Increasing the frequency of skipping also increases the level of functional or semi-functional dystrophin protein produced in muscle cells of DMD individuals.

[0160] VI. Combination Therapy with a Second Active Agent The AONs or derivatives thereof provided herein can also be combined or used in combination with other therapeutic agents useful in the treatment and / or prevention of DMD.

[0161] In one embodiment, provided herein is a method for treating, preventing, or managing DMD, the method comprising administering to a subject an AON or derivative thereof provided herein in combination with one or more second active agents.

[0162] As used herein, the term "combination" includes the use of more than one therapies (e.g., one or more prophylactic and / or therapeutic agents). However, the use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject with a disease or disorder. The first therapy (e.g., a prophylactic or therapeutic agent, such as an AON provided herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second therapy (e.g., a prophylactic or therapeutic agent) to the subject. Triple therapy is also contemplated herein.

[0163] The administration of the AON or derivative thereof provided herein and one or more second active agents to a subject can be performed simultaneously or sequentially by the same or different routes of administration. The suitability of a particular route of administration for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without being degraded before entering the bloodstream) and the disease or disorder being treated.

[0164] The route of administration of the AON or derivative thereof provided herein is independent of the route of administration of the second therapy. In another embodiment, the AON or derivative thereof provided herein is administered intravenously. Thus, according to these embodiments, the AON or derivative thereof provided herein is administered intravenously, and the second therapy can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, buccally, intranasally, intraliposomally, by inhalation, intravaginally, intraocularly, locally delivered by catheter or stent, subcutaneously, intraadipose, intraarticularly, intrathecally, or in sustained release form. In one embodiment, the AON or derivative thereof provided herein and the second therapy are administered by the same mode of administration (e.g., IV). In another embodiment, the AON or derivative thereof provided herein is administered by one mode of administration (e.g., IV), while the second agent is administered by another mode of administration (e.g., orally).

[0165] In one embodiment, the second active agent is administered intravenously or subcutaneously once or twice daily in an amount of about 1 to about 1000 mg, about 5 to about 500 mg, about 10 to about 350 mg, or about 50 to about 200 mg. The specific amount of the second active agent will depend on the particular agent used, the type of disease being treated or managed, the severity and stage of the disease, and the amount of the AON or derivative thereof provided herein and any additional active agents being administered concomitantly to the subject.

[0166] In the methods and compositions provided herein, one or more second active ingredients or agents can be used in conjunction with the AONs or derivatives thereof provided herein. The second active agent can be a large molecule (e.g., a protein) or a small molecule (e.g., a synthetic inorganic molecule, an organometallic molecule, or an organic molecule).

[0167] Examples of macromolecular active agents include, but are not limited to, hematopoietic growth factors, cytokines, AONs, and monoclonal and polyclonal antibodies.Representative macromolecular active agents are biological molecules (e.g., natural or synthetic or recombinant proteins).Specific examples used herein include eteplirsen, casimersen, golodirsen, viltolarsen, and SRP-5051 (Sarepta Therapeutics).

[0168] Examples of small molecules include corticosteroids (eg, deflazacort).

[0169] Other therapies that may be combined with the AONs provided herein include gene therapy (e.g., SRP-9001, GALGT2, or GNT 0004 (Sarepta Therapeutics)), gene editing (e.g., CRISPR / CAS9 (Sarepta Therapeutics)), and cell therapy (e.g., CAP-1002 (Capricor Therapeutics / Nippon Shinyaku Co. Ltd.)). EXAMPLES

[0170] The following examples are intended to illustrate certain embodiments provided herein, and are not intended to limit the scope of the disclosure.

[0171] Example 1 The model system was the KM571 human immortalized myoblast cell line with a deletion of exon 52. KM571 cells were previously immortalized using hTERT and CDK4 (PMID: 22040608). On day 1, 138,000 cells per well were nucleofected (Lonza, set DS-150, SF solution) in the presence of 1.4 micromolar of individual AONs (2'-MOE (2'-O-methoxyethyl RNA), 5-methylcytosine, thymine (no uracil), and phosphorothioate backbone), then diluted 10-fold in skeletal muscle growth medium (PromoCell [catalog number: C-23060], final volume 12.5% ​​HI-FBS and 100 units / mL Pen / Strep) and transferred to a 96-well tissue culture plate. On day 4, cells were harvested and 10 microliters of RNA was reverse transcribed using TaqMan Gene Expression Cells-to-CT (Thermo Fisher Scientific, final lysis volume 55 microliters), and the resulting cDNA (4 microliters) was then assessed by qPCR (skip forward primer 5'-AAAGCAGCCTGACCTAGCT-3' (SEQ ID NO: 94), skip probe 5'-ACCACTATTGGAGCCTTTGAAAGA-3' (SEQ ID NO: 95), and skip reverse primer 5'-CTTGTACTTCATCCCACTGATTCTGA-3' (SEQ ID NO: 96)). Double-stranded DNA fragments of the skipped amplicon (IDT, exon 50 spliced ​​to exon 53) were used to validate the assay and to quantify copy numbers of unknown samples (reported as KM571-skip copies e50-53). Blank cells in the table were undetermined background levels. In the case of KM571 cells, DMD message was undergoing nonsense-mediated decay without treatment. Successful skipping of exon 51 in these cells restored the reading frame, extended the half-life of the mRNA, and allowed more complete translation.

[0172] result The results are shown in Table 2. [Table 2-1] [Table 2-2]

[0173] Example 2 In vitro screening of AONs in myotube cultures from DMD patients (del 48-50) A series of overlapping AONs were screened at 800 nM in myotube cultures of DMD patients (del 48-50). As shown in Table 3 below, the base sequence of AON No. 79 and AON No. 79A with 2'-OMe phosphorothioate modification with 5-methylcytosine and 5' and 3' LNAs were optimized for LNA content and position to implement one or two 5' terminal guanine-LNAs, one 3' terminal cytosine-LNA, and / or one or two additional internal guanine-LNAs.

[0174] [Table 3]

[0175] Comparative testing of these AONs at 800 nM identified three AONs with the highest exon 51 skipping efficiency (AON#79B, AON#79C, and AON#79F) (Figure 1). Of these AONs with slightly different LNA profiles, AON#79C was the most efficient (7.3%). Subsequent in vitro comparative screening assays revealed that AON#79C was 10-fold more effective than drisapersen.

[0176] Example 3 Immunofluorescence analysis of dystrophin expression in sarcolemma Frozen sections (8 μm) of quadriceps muscles were mounted on Superfrost Ultra Plus microscope slides (Fisher Scientific) and incubated for 2 h with the primary antibody rabbit polyclonal anti-dystrophin (Ab15277, Abcam, dilution 1 / 200). Slides were rinsed twice for 5 min with PBS and then incubated for 1 h with the secondary antibody goat anti-rabbit AlexaFluor488 (ThermoFisher) at a dilution of 1 / 250. Imaging of slides was performed on the same day with a Zeiss LSM710 confocal microscope using a 25× objective and a laser intensity of 6%. Images of three different mice per VEH (vehicle) or AON group are shown in Figure 2. Top panel: control sections of non-dystrophic hDMD mice (positive = dystrophin primary antibody Ab15277, negative = rabbit IgG isotype primary antibody Ab27478).

[0177] Example 4 hDMDdel52 / mdx mice were analyzed after 13 weeks of tail vein injections of 18 mg / kg AON #79C or AON #79G. At the doses tested in the hDMDdel52 / mdx mouse study, AON #79C and AON #79G appeared to be similarly well tolerated. There was no effect on survival, body weight, or survival routine clinical chemistry or hematology. In general, there were no or minimal histopathological changes in the kidney, liver, spleen, lymph nodes, skeletal muscle, and heart. In the liver, hepatocyte single-cell necrosis was observed in 6 of 11 mice receiving AON #79G, but the severity was minimal in most cases. Additionally, minimal hepatocyte single-cell necrosis was observed in 1 of 10 mice receiving AON #79C, but not in any vehicle control mice of either sex. In the heart, myocardial fibrosis / fibroplasia was observed at a slightly higher incidence and / or severity in the treatment group than in the control group. The relationship between these histopathological changes and AON treatment was unclear but could not be excluded. Other histopathological findings in the heart were observed at similar incidence / severity in the control and treatment groups and therefore were considered unlikely to be related to AON treatment. In skeletal muscle, fiber necrosis, muscle atrophy, basophilia / regeneration, inflammation, calcification, and fatty changes were observed as part of the expected morphological appearance of skeletal muscle in this DMD mouse model (see Table 4 below). After treatment with each of the AONs (AON #79C or AON #79G), the incidence and / or severity of muscle fiber atrophy, necrosis, and inflammation was reduced compared to vehicle-treated mice. This may be related to the restoration of dystrophin expression.

[0178] [Table 4]

[0179] Example 5 hDMDdel52 / mdx mice were analyzed after 13 weeks of tail vein injection with 18 mg / kg AON number 79C or an equivalent AON with a 2'-MOE glycomodification instead of the 2'-OMe glycomodification and a 5'-TEG group. Mice were killed 14 days after the last injection. All mice survived the study. Exon skipping and dystrophin levels were similar in the quadriceps and heart of both groups. The results are shown in Figure 3A-B.

[0180] Example 6 The AONs provided herein were tested for dystrophin pre-mRNA exon 51 skipping using the assay described in Example 1. The data reported in Tables 5 and 6 were compared using additional qPCR assays against UBC housekeeping genes to determine the dystrophin pre-mRNA exon 51 skipping rate. -ΔΔCT The results are expressed as fold change relative to AON number 79C by applying the method. Briefly, the average potency was calculated as relative fold based on potency results 1–4. Delta ct: skip ct - housekeeping gene ct Double Delta CT: Delta CT - Average Delta CT without treatment Multiple: 2^(double delta ct) Efficiency: Multiples of AON No. 79C

[0181] result The average potency of the AONs provided herein in inducing dystrophin pre-mRNA exon 51 skipping is shown in the following table. In Table 5, the AONs tested are fully phosphorothioate backbones, all cytosines are substituted with 5-methylcytosines, and all nucleosides are 2'-MOE RNA nucleosides. In Table 6, the AONs tested are fully phosphorothioate backbones, all cytosines are substituted with 5-methylcytosines, nucleotides 1, 2, 17, and 18 are LNA, and nucleotides 3-16 are 2'-MOE RNA nucleosides.

[0182] [Table 5] [Table 6]

[0183] The present disclosure is not limited in scope by the embodiments disclosed in the examples, which are intended as single descriptions of individual aspects, and all equivalents are within the scope of the present disclosure. Various modifications in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0184] Various references, such as patents, patent applications, and publications, are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. An antisense oligonucleotide (AON) comprising, or consisting of, one of the base sequences of sequence numbers 1 to 93, including at least one modification.

2. The AON according to claim 1, wherein one of the at least one modifications is a chemical modification of the sugar portion of all nucleotides in the AON.

3. The AON according to claim 2, wherein the chemical modification of the sugar portion of all nucleotides in the AON includes a 2'-O-methoxyethyl (2'-MOE) modification.

4. The AON according to claim 3, wherein the AON comprises one, two, three or four locked nucleic acids (LNAs), and the remaining nucleotides that are not LNAs are 2'-MOE modified.

5. The AON according to claim 4, wherein two 5' terminal nucleotides and two 3' terminal nucleotides of the AON each contain LNA, and the remaining nucleotide that is not LNA is 2'-MOE modified.

6. The AON according to claim 2, wherein the chemical modification of the sugar portion of all nucleotides in the AON includes a 2'-O-methyl (2'-OMe) modification.

7. The AON according to claim 6, wherein the AON comprises one, two, three or four locked nucleic acids (LNAs), and the remaining nucleotides that are not LNAs are 2'-OMe modified.

8. The AON according to claim 7, wherein two 5' terminal nucleotides and two 3' terminal nucleotides of the AON each contain LNA, and the remaining nucleotide that is not LNA is 2'-OMe modified.

9. The AON according to claim 1, wherein the sugar portion of one or more nucleotides in the AON comprises a modification selected from the group consisting of morpholine (PMO, PPMO, PMO-X), peptide derivatives (PNA), boron cluster modified PNA, pyrrolidine-based oxypeptide nucleic acid (POPNA), glycol or glycerol-based nucleic acid (GNA), threose-based nucleic acid (TNA), acyclic threoninol-based nucleic acid (aTNA), cationic morpholino-based oligomer (PMOPlus), oligonucleotides having an integrated base and backbone (ONIB), and pyrrolidineamide oligonucleotide (POM), or derivatives thereof.

10. The AON according to claim 5, wherein the AON comprises at least one chemical modification of the base portion of nucleotide 1, 2, 3, 4 or all of the nucleotides in the AON.

11. The AON according to claim 10, wherein all cytosine bases are 5-methylcytosine bases.

12. The AON according to claim 10, wherein all thymine bases are uracil bases.

13. The AON according to claim 10, wherein the main chain is a complete phosphorothioate main chain bond.

14. The AON according to claim 1, wherein the AON contains a hydroxyalkoxy group at the 5' end of the AON, at the 3' end of the AON, or at each of the 5' and 3' ends of the AON.

15. The AON according to claim 14, wherein the hydroxyalkoxy group comprises or consists of an ethylene glycol monomer, an ethylene glycol oligomer, or an ethylene glycol polymer.

16. The AON according to claim 15, wherein the hydroxyalkoxy group is a triethylene glycol (TEG) group or a hexaethylene glycol (HEG) group.

17. The AON according to claim 1, comprising or consisting of the sequence of sequence numbers 33, 34, 35, 36, 37, 38, or 39.

18. The AON according to claim 17, comprising or consisting of the sequence of sequence numbers 33, 38, or 39.

19. The AON according to claim 16, wherein the AON has a length of 16 to 30 nucleotides.

20. The AON according to claim 19, wherein the AON has a length of 16, 17, 18, 19, or 20 nucleotides.

21. The AON according to claim 20, wherein the AON has a length of 18 nucleotides.

22. The AON according to claim 1, wherein the AON is completely 2'-MOE RNA modified, all cytosine bases are 5-methylcytosine bases, and the AON has a completely phosphorothioate-modified main chain.

23. The AON according to claim 1, wherein all cytosine bases are 5-methylcytosine bases, two 5' terminal nucleotides of the AON are LNA, two 3' terminal nucleotides of the AON are LNA, the remaining nucleotides in the AON that are not LNA are 2'-MOE RNA modified, and the AON has a fully phosphorothioate-modified main chain.

24. The AON according to claim 1, wherein the AON is completely 2'-OMe RNA modified, all cytosine bases are 5-methylcytosine bases, and the AON has a completely phosphorothioate-modified main chain.

25. The AON according to claim 1, wherein all cytosine bases are 5-methylcytosine bases, two 5' terminal nucleotides of the AON are LNA, two 3' terminal nucleotides of the AON are LNA, the remaining nucleotides in the AON that are not LNA are 2'-OMe RNA modified, and the AON has a fully phosphorothioate-modified main chain.

26. A pharmaceutical composition comprising the AON described in any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

27. ​​The pharmaceutical composition according to claim 26 for treating a subject suffering from Duchenne muscular dystrophy (DMD).

28. The pharmaceutical composition according to claim 26 for delaying the onset of Duchenne muscular dystrophy (DMD) in a subject.

29. The pharmaceutical composition according to claim 26 for inducing the skipping of exon 51 of human dystrophin pre-mRNA.

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