DMD antisense oligonucleotide-mediated exon skipping efficiency

Abstract

Provided herein are oligonucleotides, cell membrane-permeable peptides, and peptide-oligonucleotide conjugates. Also provided herein are methods for treating a muscular disease, viral infection, or bacterial infection in a subject in need thereof, the methods comprising administering to the subject the oligonucleotides, peptides, and peptide-oligonucleotide conjugates described herein. Provided herein are modified antisense oligonucleotides, which are 18 to 40 subunits in length and contain a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 408,505, filed September 21, 2022. The entire teachings of the above application are incorporated herein by reference in their entirety. [Background technology]

[0002] Antisense technology provides a means for regulating the expression of one or more specific gene products, including alternative splicing products, and is uniquely useful in many therapeutic, diagnostic, and research applications. The principle behind antisense technology is that antisense compounds, such as oligonucleotides that hybridize to target nucleic acids, regulate gene expression activities such as transcription, splicing, or translation through any one of a number of antisense mechanisms. The sequence specificity of antisense compounds makes them attractive as tools for target validation and gene function, as well as therapeutic agents that selectively regulate the expression of genes involved in disease.

[0003] Although significant advances have been made in the field of antisense technology, there remains a need for oligonucleotides and peptide-oligonucleotide-conjugates with improved antisense or antigene performance. Summary of the Invention [Means for solving the problem]

[0004] Provided herein are modified antisense oligonucleotides, which are 18 to 40 subunits in length and contain a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene. The modified antisense oligonucleotides may contain a non-natural chemical backbone selected from phosphoramidate or phosphorodiamidate morpholino oligomers (PMOs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), phosphorothioate oligomers, tricyclo-DNA oligomers, tricyclo-phosphorothioate oligomers, 2'O-Me-phosphorothioate oligomers, or any combination of the foregoing. In one embodiment, the antisense oligonucleotide is covalently linked to a cell membrane-permeable peptide. Each subunit of antisense oligonucleotide is combined in the order from the 5' end of antisense oligonucleotide to the 3' end of antisense oligonucleotide to form a targeting sequence, and the targeting region is in the exon of human dystrophin pre-mRNA, and the exon is flanked by the upstream 5' splice site of the exon by a slow intron, and the slow intron is the intron that is retained in dystrophin pre-mRNA for a longer period compared with the average retention time of the intron in the dystrophin pre-mRNA downstream of the slow intron.Antisense oligonucleotide is useful for treating neuromuscular disease.In one embodiment, neuromuscular disease is Duchenne muscular dystrophy.

[0005] In certain embodiments, the exon is selected from exon 10, exon 14, exon 17, exon 18, exon 21, exon 22, exon 42, exon 50, exon 53, and exon 70.

[0006] In one embodiment, the antisense oligonucleotide is an oligonucleotide conjugate of Formula I: [ka] (I) or a pharmaceutically acceptable salt thereof, and A', E', R 1, R 2 , and z are as defined herein.

[0007] In certain embodiments, the antisense oligonucleotide of Formula I is [ka] (Ia) and [ka] (Ib) is an oligonucleotide conjugate selected from In the formula, A', E', G, J, L, R 1 , R 2 , and z are as defined herein.

[0008] In certain embodiments, each R 1 is —N(CH). In other embodiments, each R 2 are independently selected from natural or unnatural nucleobases, and each R from 5' to 3' 2 The sequence formed by the combination of is the targeting sequence. In yet other embodiments, J is a cell membrane penetrating peptide.

[0009] In another aspect, provided herein is a pharmaceutical composition comprising an antisense oligonucleotide provided herein and a pharmaceutically acceptable carrier.

[0010] Also provided herein is a method for treating a disease associated with dysregulation of peripheral myelin protein 22, comprising administering to a subject in need thereof an antisense oligomer provided herein.

[0011] In another aspect, provided herein is a use of any of the antisense oligonucleotides provided herein for treating a neuromuscular disease. In one embodiment, the disease is Duchenne muscular dystrophy. [Brief explanation of the drawings]

[0012] [Figure 1A] Figure 1A shows a graphical representation of the splicing order of introns in the DMD gene (adapted from Gazzoli et al. 2016). Introns, indicated by lines between exons, are slowly spliced, while directly adjacent exons, indicated by "fast introns," are separated by "fast introns." Different exon classes based on adjacent introns are shown in orange, green, yellow, and pink, as shown on the right. The various shades of blue are visual aids unrelated to the hypothesis. The continuity of the exon reading frame is indicated by shape. [Figure 1B] Figure 1B shows the exon skipping efficiency of DMD transcripts carrying antisense oligonucleotides (AONs) for the various exons shown in Figure 1A. Each shape represents the average of two independent nucleofected samples, and each unique shape per bar corresponds to an AON targeting the same exon. Skipping efficiency was determined as the molar ratio of skipped product to total (full-length + skipped) product in RT-PCR analysis using appropriate primers for each exon. Exons belonging to the 5'Slow-3'Slow and 5'Slow-3'Fast classes show significantly higher exon skipping efficiency than those belonging to the 5'Fast-3'Slow and 5'Fast-3'Fast classes. Error bars: SEM. (*: P value < 0.05, **: P value < 0.01, ***: P value < 0.001, ****: P value < 0.0001; NS: not significant - one-way ANOVA). [Figure 1C] Figure 1C shows the data from Figure 1B reanalyzed and presented grouping distinct exons based on their upstream intron class, as indicated. 5'Slow exons show significantly higher skipping efficiency compared to 5'Fast exons. [Figure 1D] Figure 1D shows the data from Figure 1B reanalyzed and presented grouping distinct exons based on their downstream intron class, as indicated. 3' slow and 3' fast exons show no difference in skipping efficiency. [Figure 2A]Figure 2A shows the relative expression of various genes measured by RT-qPCR after nucleofection of exon 51-targeting PMO using the Lonza buffer system and Amaxa pulse program, as indicated. The DMD exon 50-52F_52R primer set measures the presence of the DMD gene when exon 51 is skipped, while the exon 49-50 primer set indicates the presence of both skipped and non-skipped DMD. MYOG and MYH3 are measured as indicators of myogenic proliferation. Data are normalized to the housekeeping genes GUSB and GAPDH. [Figure 2B] Figure 2B shows a comparison of the same set of RT-PCR samples measuring exon 51 skipping rate as the ratio of skipped to total (skipped + full-length) PCR products derived from a set of HC myotubes after nucleofection with a PMO targeting exon 51. Adjacent bars indicate the same PCR product measured with either system, as indicated. [Figure 3] Figure 3 shows the separated skipping data shown in Figures 1B-1D. Error bars indicate SD of two independent samples. [Figure 4A] Figure 4A shows a graphical representation of the splicing order of introns in the DMD gene (adapted from Gazzoli et al. 2016). Introns, indicated by lines between exons, are spliced ​​slowly, while directly adjacent exons, indicated by "fast introns," are separated by "fast introns." Different exon classes targeted for AON-mediated exon skipping based on adjacent introns are shown in green and yellow, as shown on the right. The various shades of blue are visual aids unrelated to the hypothesis. The continuity of the exon reading frames is indicated by their shapes. [Figure 4B]Figure 4B shows the exon skipping efficiency of DMD transcripts by AONs for various exons from the 5'Slow-3'Fast adjacent intron class, as shown in Figure 4A. Each point represents the average of two independently nucleofected samples. The x-axis represents the potential window for targeting 25-mer AONs within the exon sequence of the indicated exons, scaled from 0 to 100 for each exon to normalize for exon size. Lines corresponding to symbol colors represent the results of linear regression analyses of skipping efficiency as a function of targeting position. The individual slopes of each regression analysis are shown below the plots. [Figure 4C] Figure 4C shows the exon skipping efficiency of DMD transcripts by AONs for various exons from the 5'Fast-3'Slow flanking intron class as shown in Figure 4A. The plots, axes, and analytical descriptions are the same as those in Figure 4B. [Figure 4D] Figure 4D shows a plot reanalyzing and summarizing the DMD exon skipping efficiency of all AONs used in Figures 1B, 4B, and 4C as a function of their position within the exon, as indicated on the x-axis. Circles represent data from Figure 1B, and triangles represent data from Figures 4B-4C. The linear regression analysis (red line) shows a negative slope, indicating that, in general, AON targeting closer to the 5' end of the exon is more efficient at skipping the targeted exon than AONs targeted more distally. [Figure 5A] Figure 5A shows the separated skipping data shown in Figure 4B. Error bars indicate the SD of two independent samples. [Figure 5B] Figure 5B shows the separated skipping data shown in Figure 4C. Error bars indicate SD of two independent samples. [Figure 5C] FIG. 5C shows the data from FIGS. 4B and 4C reanalyzed and presented grouped into separate exons based on their upstream and downstream intron classes, as indicated. [Figure 5D]Figure 5D shows the data from Figures 4B and 4C reanalyzed and presented grouped into separate exons based on whether efficient skipping of AONs results in in-frame or out-of-frame transcripts, as indicated. [Figure 6] Figure 6 shows the separated skipping data shown in Figure 4. Error bars indicate the SD of two independent samples. [Figure 7A] Figure 7A shows the exon skipping efficiency of exon 51 of the DMD gene in HC myotubes after treatment with AON targeting exon 51. Skipping of exon 51 results in an out-of-frame transcript. [Figure 7B] Figure 7B shows the exon skipping efficiency of exon 53 of the DMD gene in HC myotubes after treatment with AON targeting exon 53. Skipping of exon 53 results in an out-of-frame transcript. [Figure 7C] Figure 7C shows the exon skipping efficiency of exon 51 of the DMD gene in myotubes from a DMD patient (ΔExon 48-50) after treatment with exon 51-targeting AON. Skipping of exon 51 results in an in-frame transcript. [Figure 7D] Figure 7D shows the exon skipping efficiency of exon 53 of the DMD gene in myotubes from a DMD patient (Δexon 45-52) after treatment with exon 53-targeting AON. Skipping of exon 53 results in an in-frame transcript. [Figure 8A] Figures 8A-8D show RT-qPCR expression of DMD levels for the samples presented in Figures 7A-7D. Measurements of DMD transcripts are shown using primer sets upstream (Ex38-39) and downstream (Ex55-56) of the skipped exons. MYH3 expression is used as an indicator of myogenic proliferation in the samples. Data are normalized to the housekeeping genes GUSB and GAPDH. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 9A] Figure 9A shows the exon skipping efficiency of HC and DMD cells nucleofected with no PMO (WT) or a mixture of 12 exon 65 targeting PMOs and treated with DMSO or CHX as indicated. [Figure 9B] Figure 9B shows RT-qPCR expression of DMD levels for the samples presented in Figure 9A. Measurements of DMD transcripts spanning exons 38-39 and exons 55-56 are shown. MYH3 expression is used as an indicator of myogenic proliferation in the samples. Data are normalized to the housekeeping genes GUSB and GAPDH. [Figure 9C] Figure 9C shows the exon skipping efficiency of exon 51 of the DMD gene in HC myotubes after treatment with exon 51 targeting AON and CHX as indicated. [Figure 9D] Figure 9D shows the exon skipping efficiency of exon 53 of the DMD gene in HC myotubes after treatment with exon 53 targeting AON and CHX as indicated. [Figure 9E] Figure 9E shows the exon skipping efficiency (Δexon 48–50) of exon 51 of the DMD gene in myotubes from DMD patients after treatment with exon 51 targeting AON and CHX as indicated. [Figure 9F] Figure 9F shows the exon skipping efficiency (Δexon 45-52) of exon 53 of the DMD gene in myotubes from DMD patients after treatment with exon 53 targeting AON and CHX as indicated. DETAILED DESCRIPTION OF THE INVENTION

[0013] Provided herein are modified antisense oligonucleotides, the modified antisense oligonucleotides being 18 to 40 subunits in length and comprising a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene;

[0014] The modified antisense oligonucleotide comprises a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2'O-Me-phosphorothioate oligomer, or any combination of the foregoing;

[0015] each subunit together from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence;

[0016] The target region is located in the exon of human dystrophin pre-mRNA, and the exon is flanked by the upstream 5' splice site of the exon by a slow intron, and the slow intron is the intron that is retained in dystrophin pre-mRNA for a longer period than the average retention time of the intron in the dystrophin pre-mRNA downstream of the slow intron.In one embodiment, antisense oligonucleotide is covalently linked to cell membrane-permeable peptide.Antisense oligonucleotide is useful for treating various diseases in subjects who need it, including but not limited to Duchenne muscular dystrophy.

[0017] Many modified antisense oligonucleotide analogues have been developed, in which the phosphodiester bond of natural DNA is replaced by other bonds that are resistant to nuclease degradation.For example, see Barawkar and Bruice (1998) Proc Natl Acad Sci USA 95(1): 11047-52; Linkletter et al. (2001) Nucleic Acids Res 29(11): 2370-6 and Micklefield (2001) Curr Med Chem 8(10): 1157-79.Antisense oligonucleotides with various backbone modifications other than internucleoside bond have also been prepared (Crooke (2001) Antisense Drug Technology: Principles, Strategies, and Applications. New York, Marcel Dekker; Micklefield (2001)).In addition, oligonucleotides have been modified by peptide conjugation to enhance cellular uptake. See, for example, Moulton et al. (2004) Bioconjug Chem 15(2):290-9, and Nelson et al. (2005) Bioconjug Chem 16(4):959-66.

[0018] Morpholino-based oligomers (including antisense oligonucleotides) are described, for example, in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, 5,506,337, and PCT Publication Nos. WO / 2009 / 064471, WO / 2012 / 043730, WO 2008 / 036127, and Turton et al. 1997, Antisense and Nucleic Acid Drug Development, 7, 187-195, all of which are incorporated herein by reference in their entireties.

[0019] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this disclosure, preferred methods and materials are described. For purposes of this disclosure, the following terms are defined below.

[0020] The term "about" will be understood by those of skill in the art and will vary to some extent in the context in which it is used. As used herein, when referring to a measurable value, such as an amount, duration, etc., the term "about" is meant to encompass a variation of ±10%, including ±5%, ±1%, and ±0.1%, as such variations are appropriate for practicing the disclosed methods.

[0021] The term "alkyl" refers, in certain embodiments, to a saturated, straight-chain, or branched-chain hydrocarbon moiety containing 1 to 6, or 1 to 8, carbon atoms. 1-6 Examples of -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, and n-hexyl moieties; 1-8 Examples of -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

[0022] The number of carbon atoms in an alkyl substituent is expressed as "C x-y " where x is the minimum and y is the maximum number of carbon atoms in the substituent. x Chain means an alkyl chain containing x carbon atoms.

[0023] The term "heteroalkyl," by itself or in combination with another term, unless otherwise stated, refers to a stable straight- or branched-chain alkyl group containing the specified number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatom may be placed at any position of the heteroalkyl group, including between the remainder of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O-CH-CH-CH, -CH-CH-CH-OH, -CH-CH-NH-CH, -CH-S-CH-CH, and -CH-CH-S(=O)-CH. 3。 Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-SS-CH3.

[0024] The term "aryl," when used alone or in combination with other terms, unless otherwise specified, refers to a carbocyclic aromatic system containing one or more rings (generally one, two, or three rings), which may be attached in a pendant fashion, such as biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. In various embodiments, example aryl groups are phenyl (e.g., C-aryl) and biphenyl (e.g., C 12 In some embodiments, the aryl group has 6 to 16 carbon atoms. In some embodiments, the aryl group has 6 to 12 carbon atoms (e.g., C 6-12 -aryl). In some embodiments, the aryl group has 6 carbon atoms (e.g., C6-aryl).

[0025] As used herein, the terms "heteroaryl" or "heteroaromatic" refer to heterocycles having aromatic character. Heteroaryl substituents include, for example, C1-9 -Heteroaryl may be defined by the number of carbon atoms, such that it indicates the number of carbon atoms contained in the heteroaryl group, not including the number of heteroatoms. For example, C 1-9 -Heteroaryl will contain 1 to 4 additional heteroatoms. Polycyclic heteroaryls can contain one or more partially saturated rings. Non-limiting examples of heteroaryls include pyridyl, pyrazinyl, pyrimidinyl (including, for example, 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, for example, 2-pyrrolyl), imidazolyl, thiazolyl, pyrazolyl (including, for example, 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0026] Non-limiting examples of polycyclic heterocycles and heteroaryls include indolyl (e.g., 3-, 4-, 5-, 6-, and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (e.g., including 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (e.g., including 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (e.g., 3-, 4-, 5-, 6-, and 7-indolyl), benzo ... and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (e.g., 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (e.g., 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (e.g., including 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbonyl, acridinyl, pyrrolidinyl, and quinolizidinyl.

[0027] The term "protecting group" or "chemical protecting group" refers to a chemical moiety that blocks some or all reactive moieties in a compound, preventing them from participating in a chemical reaction until the protecting group is removed. For example, such moieties are listed and described in T.W. Greene, P.G.M. Buts, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons (1999). When different protecting groups are used, it can be advantageous for each (different) protecting group to be removable by a different means. Protecting groups that are cleaved under completely different reaction conditions allow for differential removal of such protecting groups. For example, protecting groups can be removed by acid, base, and hydrogenolysis. Groups such as trityl, monomethoxytrityl, dimethoxytrityl, acetal, and tert-butyldimethylsilyl are acid labile and can be used to protect carboxy and hydroxy reactive moieties in the presence of hydrogenolysis-removable Cbz groups and amino groups protected with the base-labile Fmoc group. Carboxylic acid moieties may be blocked with base-labile groups such as, but not limited to, methyl or ethyl, and hydroxy reactive moieties may be blocked with base-labile moieties such as acetyl in the presence of acid-labile groups such as tert-butyl carbamate, or amines blocked with both acid- and base-stable but hydrolytically removable carbamates.

[0028] Carboxylic acid and hydroxyl reactive moieties may also be blocked with hydrolytically removable protecting groups such as benzyl groups, while amine groups may be blocked with base-labile groups such as Fmoc. A particularly useful amine protecting group for the synthesis of compounds of Formula I is trifluoroacetamide. Carboxylic acid reactive moieties may be blocked with oxidatively removable protecting groups such as 2,4-dimethoxybenzyl, while existing amino groups may be blocked with fluoride-labile silyl carbamates.

[0029] Allyl-blocking groups are useful in the presence of acid- and base-protecting groups because the former are stable and can be subsequently removed by metal or pi-acid catalysts. For example, allyl-blocked carboxylic acids can be deprotected with a palladium(0)-catalyzed reaction in the presence of acid-labile t-butyl carbamate or base-labile acetate amine protecting groups. Another form of protecting group is a resin to which a compound or intermediate can be attached. As long as the residue is attached to the resin, the functional group is blocked and cannot react. Once released from the resin, the functional group becomes reactive.

[0030] The terms "nucleobase," "base pairing moiety," "nucleobase-pairing moiety," or "base" refer to the heterocyclic portion of a nucleoside, nucleotide, and / or morpholino subunit. The nucleobase can be naturally occurring or can be a modified or analogous version of these naturally occurring nucleobases, e.g., one or more nitrogen atoms of a nucleobase can be independently replaced by carbon in each occurrence. Exemplary analogs include hypoxanthine (the base building block of the nucleoside inosine), 2,6-diaminopurine; 5-methylcytosine; C5-propynyl-modified pyrimidines, 10-(9-(aminoethoxy)phenoxazinyl) (G-clamp), and the like.

[0031] Further examples of base-pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine and pyrimidine analogs such as acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purines, xanthines, or hypoxanthines (the latter two being natural degradation products). Also contemplated are modified nucleobases disclosed in Chiu and Rana (2003) RNA 9:1034-1048, Limbach et al. (1994) Nucleic Acids Res. 22:2183-2196, and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, the contents of which are incorporated herein by reference.

[0032] Further examples of base pairing moiety include, but are not limited to, the expanded nucleobase that one or more benzene rings are added to.The nucleobase substitutions described in Glen Research Catalog (www.glenresearch.com), Krueger AT et al. (2007) Acc. Chem. Res.40:141-150, Kool ET (2002) Acc. Chem. Res.35:936-943, Benner SA et al. (2005) Nat. Rev. Genet. 6:553-543, Romesberg FE et al. (2003) Curr. Opin. Chem. Biol.7:723-733, Hirao, I (2006) Curr. Opin. Chem. Biol.10:622-627 (these contents are incorporated herein by reference) are contemplated to be useful for synthesizing the oligomer described herein. Examples of extended size nucleobases are: [ka]

[0033] The term "oligonucleotide" or "oligomer" refers to a compound comprising a plurality of linked nucleosides, nucleotides, or a combination of both nucleosides and nucleotides. In certain embodiments provided herein, the oligonucleotide is a morpholino oligonucleotide.

[0034] As used herein, the terms "antisense oligomer," "antisense compound," and "antisense oligonucleotide" are used interchangeably and refer to a sequence of subunits, each having bases carried on a backbone subunit composed of a ribose or other pentose sugar or a morpholino group, joined by intersubunit linkages that allow the bases in the compound to hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence. The oligomer may have exact or near-exact sequence complementarity to the target sequence. Such an antisense oligonucleotide is designed to block or inhibit translation of mRNA containing the target sequence and can be said to be "directed" to the sequence to which it hybridizes.

[0035] Also contemplated herein as types of "antisense oligomer," "antisense compound," or "antisense oligonucleotide" are phosphoramidate or phosphorodiamidate morpholino oligomers (PMO), phosphorothioate modified oligomers, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), 2'-fluoro modified oligomers, 2'-O,4'-C-ethylene bridged nucleic acids (ENAs), tricyclo-DNA, tricyclo-DNA phosphorothioate modified oligomers, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified oligomers, 2'-O-methyl phosphorothioate modified oligomers, 2'-O-methoxyethyl (2'-O-MOE) modified oligomers, and 2'-O-methyl oligonucleotides, or combinations thereof, as well as other antisense agents known in the art.

[0036] The term "morpholino oligonucleotide" or "PMO" refers to a modified oligonucleotide having morpholino subunits joined by phosphoramidate or phosphorodiamidate linkages linking the morpholino nitrogen of one subunit to the 5'-exocyclic carbon of an adjacent subunit. Each morpholino subunit contains a nucleobase pairing moiety effective to bind to a target nucleobase by nucleobase-specific hydrogen bonding.

[0037] An antisense oligonucleotide "specifically hybridizes" to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions at a Tm greater than 37°C, greater than 45°C, preferably at least 50°C, and typically 60°C to 80°C or higher. The "Tm" of an oligomer is the temperature at which 50% hybridizes to a complementary polynucleotide. Tm is determined under standard conditions in saline, for example, as described in Miyada et al. (1987) Methods Enzymol. 154:94-107. Such hybridization can occur through "near" or "substantial" complementarity of the antisense oligonucleotide to the target sequence, as well as complete complementarity.

[0038] The terms "complementary" and "complementarity" refer to oligonucleotides (i.e., nucleotide sequences) related by the base-pairing rules. For example, the sequence "TGA(5'-3')" is complementary to the sequence "TCA(5'-3')." Complementarity can be "partial," in which only some of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete," "total," or "perfect" (100%) complementarity between nucleic acids. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desirable, some embodiments can include one or more mismatches to the target RNA, preferably 6, 5, 4, 3, 2, or 1 mismatch. Such hybridization can occur with "near" or "substantial" complementarity of the antisense oligomer to the target sequence, as well as complete complementarity. In some embodiments, the oligomer can hybridize to the target sequence with about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementarity. Variation at any position within the oligomer is included. In certain embodiments, sequence variation near the ends of the oligomer is generally preferred over variation within the interior, and, if present, is typically about 6, 5, 4, 3, 2, or 1 nucleotide at the 5' end, 3' end, or both ends.

[0039] Naturally occurring nucleotide bases include adenine, guanine, cytosine, thymine, and uracil, which have the symbols A, G, C, T, and U, respectively. Nucleotide bases can also include analogs of the naturally occurring nucleotide bases. Base pairing typically occurs between purine A and pyrimidine T or U, and between purine G and pyrimidine C.

[0040] Oligonucleotides may also contain nucleobase (simply referred to in the art as "base") modifications or substitutions. Oligonucleotides containing modified or substituted bases include oligonucleotides in which one or more of the purine or pyrimidine bases most commonly found in nucleic acids are replaced with less common or unnatural bases. In some embodiments, the nucleobase is covalently linked to the morpholine ring of the nucleoside at the N atom of a purine base or the N atom of a pyrimidine base.

[0041] Purine bases contain a pyrimidine ring fused to an imidazole ring, as described by the general formula: [ka]

[0042] Adenine and guanine are the two most commonly found purine nucleobases in nucleic acids, which can be substituted with other naturally occurring purines, including but not limited to, N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0043] Pyrimidine bases contain a six-membered pyrimidine ring as described by the general formula: [ka]

[0044] Cytosine, uracil, and thymine are the most commonly found pyrimidine bases in nucleic acids. They can be substituted with other naturally occurring pyrimidines, including, but not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain thymine bases instead of uracil.

[0045] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamps and their derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaaden ... These include degenerate or universal bases such as -aza-7-deaza-2,6-diaminopurine, super-G, super-A, and N4-ethylcytosine, or their derivatives; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP); pseudouracil or its derivatives; and abasic bases such as 2,6-difluorotoluene or abasic moieties (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen is replaced by nitrogen (azaribose)). Pseudouracil is a naturally occurring isomerized version of uracil, with a C-glycosidic rather than the usual N-glycosidic form in uridine.

[0046] Certain modified or substituted nucleobases are particularly useful for improving the binding affinity of the antisense oligonucleotides of the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcitrine. In various embodiments, the nucleobases may contain 5-methylcytosine substituents, which have been shown to improve the stability of the double helix structure of nucleic acids by 0.6-1.2°C.

[0047] In some embodiments, modified or substituted nucleobases are useful for facilitating the purification of antisense oligonucleotides. For example, in certain embodiments, antisense oligonucleotides may contain three or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases. In certain antisense oligonucleotides, a string of three or more consecutive guanine bases may cause the oligonucleotide to aggregate, complicating purification. In such antisense oligonucleotides, one or more consecutive guanines may be substituted with hypoxanthine. Substituting hypoxanthine for one or more guanines in a string of three or more consecutive guanine bases may reduce the aggregation of antisense oligonucleotides, thereby facilitating purification.

[0048] The oligonucleotides provided herein are synthetic and do not include antisense compositions of biological origin. The molecules of the present disclosure may be mixed, encapsulated, conjugated, or otherwise associated with molecules, molecular structures, or mixtures of compounds, or combinations thereof, such as liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations, for example, to aid in uptake, distribution, or absorption.

[0049] As used herein, "nucleic acid analog" refers to a non-naturally occurring nucleic acid molecule. Nucleic acids are polymers of nucleotide subunits linked together in a linear structure. Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar, which is attached to a phosphate group. Successive phosphate groups are linked together through phosphodiester bonds to form the polymer. Two common forms of naturally occurring nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). One end of the chain bears a free phosphate group attached to the 5'-carbon atom of the sugar moiety, referred to as the 5'-end of the molecule. The other end has a free hydroxyl (-OH) group at the 3'-carbon of the sugar moiety, referred to as the 3'-end of the molecule. Nucleic acid analogs may contain one or more non-naturally occurring nucleobases, sugars, and / or internucleotide linkages, such as phosphorodiamidate morpholino oligomers (PMOs). As disclosed herein, in certain embodiments, a "nucleic acid analog" is a PMO, and in certain embodiments, a "nucleic acid analog" is a positively charged cationic PMO.

[0050] "Morpholino oligomer" or "PMO" refers to a polymeric molecule having a backbone supporting bases capable of hydrogen bonding to a typical polynucleotide, where the polymer lacks a pentose sugar backbone moiety and, more specifically, a ribose backbone linked by phosphodiester bonds typical of nucleotides and nucleosides, but instead contains ring nitrogens linked through the ring nitrogens. Exemplary "morpholino" oligomers include morpholino subunit structures linked together by phosphoramidate or phosphorodiamidate linkages, with the morpholino nitrogen of one subunit attached to the 5' exocyclic carbon of an adjacent subunit, and each subunit containing a purine or pyrimidine base-pairing moiety effective to bind to a base in a polynucleotide by base-specific hydrogen bonding. Morpholino oligomers (including antisense oligomers) are described in detail in, for example, U.S. Patent Nos. 5,034,506, 5,142,047, 5,166,315, 5,185,444, 5,217,866, 5,506,337, 5,521,063, 5,698,685, 8,076,476, and 8,299,206, and PCT Publication No. 2009 / 064471, all of which are incorporated herein by reference in their entireties.

[0051] Preferred morpholino oligomers are phosphorodiamidate-linked morpholino oligomers, referred to herein as PMOs. Such oligomers are composed of morpholino subunit structures such as those shown below: [ka] [ka] wherein X is NH2, NHR, or NR2 (R is lower alkyl, preferably methyl), Y1 is O, Z is O, and P i and P jis a purine or pyrimidine base-pairing moiety effective to bind to bases in a polynucleotide by base-specific hydrogen bonding. Also preferred are structures having alternating phosphorodiamidate linkages, where X is a lower alkoxy such as methoxy or ethoxy, Y is NH or NR, R is a lower alkyl, and Z is O.

[0052] Representative PMOs include those in which the intersubunit linkage is linkage (A1). See Table 1. [Table 1]

[0053] A "phosphoramidate" group contains a phosphorus with three attached oxygen atoms and one attached nitrogen atom, while a "phosphorodiamidate" group contains a phosphorus with two attached oxygen atoms and two attached nitrogen atoms. Representative examples of phosphorodiamidates are shown below: [ka] Each P i is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein the nucleobase is independently selected at each occurrence from pyridine, pyrimidine, triazinane, purine, and deazapurine. 3-6 It contains a heterocycle, and n is an integer of 6 to 38.

[0054] In the uncharged or modified intersubunit linkages of the oligomers described herein, one nitrogen is always pendant to the backbone. The second nitrogen in the phosphorodiamidate linkage is typically a ring nitrogen in a morpholino ring structure.

[0055] PMOs are water-soluble, uncharged, or substantially uncharged antisense molecules that inhibit gene expression by preventing the binding or progression of splicing or translation machinery components. PMOs have also been shown to inhibit or block viral replication (Stein, Skilling et al. 2001; McCaffrey, Meuse et al. 2003). They are highly resistant to enzymatic digestion (Hudziak, Barofsky et al. 1996). PMOs have demonstrated high antisense specificity and efficacy in vitro in cell-free and cell culture models (Stein, Foster et al. 1997; Summerton and Weller 1997), and in vivo in zebrafish, frog, and sea urchin embryos (Heasman, Kofron et al. 2000; Nasevicius and Ekker 2000), as well as in adult animal models such as rats, mice, rabbits, dogs, and pigs (see, e.g., Arora and Iversen 2000; Qin, Taylor et al. 2000; Iversen 2001; Kipshidze, Keane et al. 2001; Devi 2002; Devi, Oldenkamp et al. 2002; Kipshidze, Kim et al. 2002; Ricker, Mata et al. 2002).

[0056] Antisense PMO oligomers have been shown to be taken up into cells with fewer non-specific effects and to be more consistently effective in vivo than other widely used antisense oligonucleotides (see, for example, P. Iversen, "Phosphoramidite Morpholino Oligomers," in Antisense Drug Technology, S.T. Crooke, ed., Marcel Dekker, Inc., New York, 2001). Conjugation of PMOs to arginine-rich peptides has been shown to increase their cellular uptake (see, for example, U.S. Patent No. 7,468,418, the entire contents of which are incorporated herein by reference).

[0057] As used herein, "charged," "uncharged," "cationic," and "anionic" refer to the prevailing state of a chemical moiety at about neutral pH, e.g., about 6 to 8. For example, the terms can refer to the prevailing state of a chemical moiety at physiological pH, i.e., about 7.4.

[0058] "Cationic PMO" or "PMO+" refers to a phosphorodiamidate morpholino oligomer containing any number of (1-piperazino)phosphinylideneoxy, (1-(4-(-guanidino-alkanoyl))-piperazino)phosphinylideneoxy linkages (A2 and A3, see Table 1) previously described (see, e.g., PCT Publication No. 2008 / 036127, incorporated herein by reference in its entirety).

[0059] The "backbone" of an oligonucleotide analog (e.g., an uncharged oligonucleotide analog) refers to the structure supporting the base-pairing moieties; for example, for the morpholino oligomers described herein, the "backbone" comprises morpholino ring structures connected by intersubunit linkages (e.g., phosphorus-containing linkages). A "substantially uncharged backbone" refers to the backbone of an oligonucleotide analog in which less than 50% of the intersubunit linkages are charged at about neutral pH. For example, a substantially uncharged backbone can contain less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or even less than 0% of the intersubunit linkages that are charged at about neutral pH. In some embodiments, a substantially uncharged backbone contains at most one charged (at physiological pH) intersubunit linkage for every four uncharged (at physiological pH), at most one uncharged linkage for every eight uncharged linkages, or at most one uncharged linkage for every 16 uncharged linkages. In some embodiments, the nucleic acid analogs described herein are completely uncharged.

[0060] The term "targeting base sequence," or simply "targeting sequence," refers to a sequence in a nucleic acid analog that is complementary (and thus substantially complementary) to a target sequence, e.g., a target sequence in the RNA genome of the human Duchenne muscular dystrophy (DMD) gene. The entire sequence of the analog compound, or only a portion thereof, may be complementary to the target sequence. For example, in an analog having 20 bases, only 12 to 14 may be the targeting sequence. Typically, the targeting sequence is formed by consecutive bases in the analog, but alternatively, it may be formed by non-contiguous sequences that, when placed together from both ends of the analog, constitute a sequence spanning the target sequence.

[0061] As used herein, "target sequence" refers to a nucleotide sequence within the genome of the human Duchenne muscular dystrophy (DMD) gene to which an antisense compound binds under conditions favorable for such binding, e.g., physiological conditions. Examples of potential target sequences include sequences comprising all or at least a portion of the 5'-terminal region, transcriptional regulatory sequence (TRS), translation initiation region, or AUG region. The target sequence may typically encompass about 10 to about 30, about 20 to about 30, or about 20 to about 25 contiguous nucleotides of the viral genome sequence.

[0062] As used herein, a "slow intron" is an intron that is retained for a longer period in a dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA downstream of the slow intron.

[0063] As used herein, a "fast intron" is an intron that is retained for a shorter period of time in a dystrophin pre-mRNA compared to the average retention time of the intron in the dystrophin pre-mRNA upstream of the intron.

[0064] The identification of slow and fast introns can be determined by the relative frequency with which a particular intron is present in a sample (e.g., a pre-mRNA sample isolated from multiple cells expressing a gene of interest (e.g., DMD)). Determining intron frequency can be determined by any method known in the art, including, but not limited to, high-throughput sequencing. An intron can be classified as a slow intron if the normalized frequency of the intron is high. An intron can be classified as a fast intron if the normalized frequency of the intron is low.

[0065] The identification and characterization of slow and fast introns is described in further detail in Gazzoli et al. (RNA Biology. 13(3):290-305. 2016).

[0066] As used herein, a "cell-penetrating peptide" (CPP) or "carrier peptide" is a relatively short peptide that can facilitate cellular uptake of a PMO, thereby delivering the PMO to the interior (cytoplasm) of the cell. A CPP or carrier peptide is typically about 12 to about 40 amino acids in length. The length of the carrier peptide is not particularly limited and varies in different embodiments. In some embodiments, the carrier peptide comprises 4 to 40 amino acid subunits. In other embodiments, the carrier peptide comprises 6 to 30, 6 to 20, 8 to 25, or 10 to 20 amino acid subunits.

[0067] In certain embodiments, a carrier peptide, when conjugated to an antisense oligonucleotide having a substantially uncharged backbone, is effective to enhance the activity of the antisense oligonucleotide compared to the antisense oligonucleotide in its unconjugated form, as evidenced by the following: (i) a reduction in expression of the encoded protein compared to that provided by the unconjugated oligomer, when binding of the antisense oligonucleotide to its target sequence is effective to block the translation initiation codon of the encoded protein; or (ii) When binding of the antisense oligonucleotide to its target sequence is effective in blocking an aberrant splice site in a pre-mRNA that, when correctly spliced, encodes said protein, increased expression of the encoded protein compared to that provided by the unconjugated oligomer. Suitable assays for measuring these effects are further described below. In one embodiment, conjugation of the peptide provides this activity in a cell-free translation assay, as described herein. In some embodiments, activity is enhanced by at least 2-fold, at least 5-fold, or at least 10-fold.

[0068] Alternatively or additionally, the carrier peptide is effective to enhance transport of the nucleic acid analog into the cell compared to the unconjugated form of the analog, hi certain embodiments, transport is enhanced by at least 2-fold, at least 2-fold, at least 5-fold, or at least 10-fold.

[0069] As used herein, "peptide-linked phosphorodiamidate-linked morpholino oligomer," "conjugate," or "PPMO" refers to a PMO covalently attached to a peptide, such as a cell membrane-penetrating peptide (CPP) or carrier peptide. The cell membrane-penetrating peptide facilitates cellular uptake of the PMO, thereby delivering the PMO to the interior (cytoplasm) of the cell. Depending on its amino acid sequence, the CPP can be generally effective or can be specifically or selectively effective in delivering the PMO to a particular cell type. The PMO and CPP are typically linked at their termini; for example, the C-terminus of the CPP can be linked to the 5'-terminus of the PMO, or the 3'-terminus of the PMO can be linked to the N-terminus of the CPP. PPMOs can include uncharged PMOs, charged (e.g., cationic) PMOs, and mixtures thereof.

[0070] The carrier peptide can be linked to the nucleic acid analog directly or via an optional linker, e.g., one or more additional amino acids such as cysteine ​​(C), glycine (G), or proline (P), or additional amino acid analogs, e.g., 6-aminohexanoic acid (X), beta-alanine (B), or XB.

[0071] An "amino acid subunit" is generally an α-amino acid residue (-CO-CHR-NH-), but can be a β- or other amino acid residue (e.g., -CO-CHCHR-NH-), where R is an amino acid side chain.

[0072] The term "naturally occurring amino acid" refers to an amino acid that occurs in proteins found in nature, and examples include alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y). The term "unnatural amino acid" refers to an amino acid that does not occur in proteins found in nature, and examples include beta-alanine (β-Ala) and 6-aminohexanoic acid (Ahx).

[0073] A representative oligomer-peptide conjugate is shown below. [ka]

[0074] Each morpholino oligomer is conjugated to a carrier peptide at the 5' or 3' end. W represents O, and each X represents OH and -NR 3 R 4 wherein each R 3 and R 4 is independently -C in each occurrence 1-6 is alkyl, Y is O, and each Pi is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein the nucleobase is independently selected at each occurrence from pyridine, pyrimidine, triazinane, purine, and deazapurine. 3-6 It contains a heterocycle, and x is an integer of 6 to 38.

[0075] An agent is "actively taken up by a mammalian cell" if the agent can enter the cell by a mechanism other than passive diffusion across the cell membrane. The agent can be transported, for example, by "active transport," which refers to the transport of the agent across the mammalian cell membrane by an ATP-dependent transport mechanism, or by "facilitated transport," which refers to the transport of the antisense agent across the cell membrane by a transport mechanism that requires binding of the agent to a transport protein, which in turn facilitates passage of the bound agent across the membrane.

[0076] As used herein, "effective amount" refers to any amount of a substance that is sufficient to achieve a desired biological result. A "therapeutically effective amount" refers to any amount of a substance that is sufficient to achieve a desired therapeutic result.

[0077] As used herein, a "subject" is a mammal, which may include a mouse, rat, hamster, guinea pig, rabbit, goat, sheep, cat, dog, pig, cow, horse, monkey, non-human primate, or human. In certain embodiments, the subject is a human.

[0078] As used herein, the term "treatment" refers to inhibiting or ameliorating a disease, condition, or disorder in a subject experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder. For example, inhibiting a disease, condition, or disorder refers to stopping further development of the pathology and / or symptoms of said disease, condition, or disorder. Furthermore, ameliorating a disease, condition, or disorder refers to reversing the pathology and / or symptoms, such as, for example, reducing the severity of the disease.

[0079] As used herein, the terms "prevent, preventing, prevention" include the prevention of at least one symptom associated with or caused by the disease, condition, or disorder being prevented.

[0080] As used herein, "subject" refers to an animal, preferably a mammal, particularly a human or non-human animal, including livestock animals and pets, including, but not limited to, cows, horses, sheep, pigs, goats, rabbits, cats, dogs, and other mammals in need of treatment. In some embodiments, the subject is a human.

[0081] As used herein, the term "administration" and variations thereof (e.g., "administering") with respect to antisense oligonucleotides means providing a compound to a subject in need of treatment. As used herein, "orally" and variations thereof (e.g., "oral") refer to administration via the mouth, i.e., administration of an antisense oligonucleotide through the oral cavity.

[0082] Administration of an antisense oligonucleotide to a subject includes both self-administration and administration to another subject. A subject may need or desire treatment for an existing disease or medical condition, or may need or desire prophylactic treatment to prevent or reduce the risk of developing a disease or medical condition. As used herein, a subject "in need" of treatment or prophylactic treatment for an existing condition encompasses both a medical professional's determination of need and the patient's desire for such treatment.

[0083] II. Antisense Oligonucleotides As shown herein, targeting the exon whose upstream intron is maintained longer in transcript (slow intron) generally results in higher exon skipping efficiency than when the exon is preceded by a rapidly spliced ​​(fast) intron.Furthermore, targeting the exon close to the 5' end results in more efficient exon skipping than targeting the 3' of the same exon.The optimal exon targeting region is 5', which can guide the future design of AON-based exon skipping therapy.

[0084] Accordingly, provided herein are modified antisense oligonucleotides, the modified antisense oligonucleotides being 18 to 40 subunits in length and comprising a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene;

[0085] The modified antisense oligonucleotide comprises a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2'O-Me-phosphorothioate oligomer, or any combination of the foregoing;

[0086] each subunit together from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form a targeting sequence;

[0087] The target region is within an exon of a human dystrophin pre-mRNA, the exon being flanked at the exon's upstream 5' splice site by a slow intron, which is an intron that is retained for a longer period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA that are downstream of the slow intron.

[0088] In one embodiment, the downstream 3' splice site of the exon is adjacent to a slow intron or a fast intron, a fast intron being an intron that is retained for a shorter period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA upstream of the intron.

[0089] In certain embodiments, the modified antisense oligonucleotides may be 20-40 subunits, 18-35 subunits, 20-35 subunits, 18-30 subunits, 20-30 subunits, 18-26 subunits, 20-26 subunits, 22-26 subunits, 23-26 subunits, or 24-25 subunits, including all integers within these ranges. In certain embodiments, the modified antisense oligonucleotides are 24-25 subunits in length.

[0090] In one embodiment, the modified antisense oligonucleotides comprise a guanine / cytosine (G / C) content of 30-70%. In a further embodiment, the modified antisense oligonucleotides comprise a G / C content of 40-60%.

[0091] In one embodiment, the modified antisense oligonucleotide may have a melting temperature of about 60-80°C, about 62-80°C, about 64-80°C, about 60-78°C, about 62-78°C, about 64-78°C, about 60-76°C, about 62-76°C, about 64-76°C, about 65-75°C, about 64-74°C, or about 65-74°C, including all integers within these ranges. In a further embodiment, the modified antisense oligonucleotide has a melting temperature of about 64-75°C.

[0092] In one embodiment, the exon is selected from exon 10, exon 14, exon 17, exon 18, exon 21, exon 22, exon 42, exon 50, exon 53, and exon 70.

[0093] In certain embodiments, the exon is exon 10. In further embodiments, the targeting sequence is SEQ ID NO: 1 ACTTGTCTTCAGGAGCTTCCAAATG SEQ ID NO: 2 AAATGACTTGTCTTCAGGAGCTTCC SEQ ID NO: 3 CTGCCAAATGACTTGTCTTCAGGAG SEQ ID NO: 4 CTCCATCAATGAACTGCCAAATGAC SEQ ID NO: 5 CTCTCCTTGTGCTTGCAATGTGTCC SEQ ID NO: 6 GAAATCTCTCCTTGTGCTTGCAATG SEQ ID NO: 7 ACTGGTCTTTCACCACTTCCACATC SEQ ID NO: 8 ATGAAACTGGTCTTTCACCACTTCC The sequence may comprise or consist of a sequence selected from:

[0094] In another embodiment, the exon is exon 14. In a further embodiment, the targeting sequence is SEQ ID NO: 9 TGTTTGCCCATCGATCTCCCAATAC SEQ ID NO: 10 TACAGATGTTTGCCCATCGATCTCC SEQ ID NO: 11 CCATCTACAGATGTTTGCCCATCGA SEQ ID NO: 12 TCTGTCCATCTACAGATGTTTGCCC SEQ ID NO: 13 ACGTTGCCATTTGAGAAGGATGTCT SEQ ID NO: 14 GTAAGACGTTGCCATTTGAGAAGGA SEQ ID NO: 15 CTTCAGTAAGACGTTGCCATTTGAG SEQ ID NO: 16 CTGTTCTTCAGTAAGACGTTGCCAT The sequence may comprise or consist of a sequence selected from:

[0095] In another embodiment, the exon is exon 17. In a further embodiment, the targeting sequence is SEQ ID NO: 17 GAGTGGTGGTGACAGCCTGTGAAAT SEQ ID NO: 18 TCTGTGTTAGTGATGGCTGAGTGGT SEQ ID NO: 19 CCCTTGTGGTCACCGTAGTTACTGT SEQ ID NO: 20 GTTCCTCTTGAGCATGCTTTACCAG SEQ ID NO: 21 ACAGTAATCTGCCTCTTCTTTTGGG SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC The sequence may comprise or consist of a sequence selected from:

[0096] In another embodiment, the exon is exon 18. In a further embodiment, the targeting sequence is SEQ ID NO: 23 GCGAGTAATCCAGCTGTGAAGTTCA SEQ ID NO: 24 TCTGAGCGAGTAATCCAGCTGTGAA SEQ ID NO: 25 AGCTTCTGAGCGAGTAATCCAGCTG SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC SEQ ID NO: 27 GCAAATTCAGGACTCTGCAACACAG SEQ ID NO: 28 GCCTTCCTTCCGAAAGATTGCAAAT SEQ ID NO: 29 AGTTGCCTTCCTTCCGAAAGATTGC SEQ ID NO: 30 TGAGAAGTTGCCTTCCTTCCGAAAG SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC The sequence may comprise or consist of a sequence selected from:

[0097] In another embodiment, the exon is exon 21. In a further embodiment, the targeting sequence is SEQ ID NO: 32 GATCTGATAGCCGGTTGACTTCATC SEQ ID NO: 33 GTTGAAGATCTGATAGCCGGTTGAC SEQ ID NO: 34 GTCCTTGTCCTTTCTCTTTCAGGGC SEQ ID NO: 35 GAACATGGGTCCTTGTCCTTTCTC SEQ ID NO: 36 TGTAAAGGCCACAAAGTCTGCATCC SEQ ID NO: 37 TCTGGCCTGCACATCAGAAAAGACT SEQ ID NO: 38 TGTCTGTAGCTCTTTCTCTCTGGCC The sequence may comprise or consist of a sequence selected from:

[0098] In another embodiment, the exon is exon 22. In a further embodiment, the targeting sequence is SEQ ID NO: 39 CGCATTGGTGGCAAAGTGTCAAAA SEQ ID NO: 40 CTGATAGCGCATTGGTGGCAAAGT SEQ ID NO: 41 ATGGTCTCCTGATAGCGCATTGGTG SEQ ID NO: 42 CACTCATGGTCTCCTGATAGCGCAT SEQ ID NO: 43 CTGATGGCACTCATGGTCTCCTGAT SEQ ID NO: 44 GAGAGTTTGGTTTCTGACTGCTGGA SEQ ID NO: 45 GCTCCATGATTTCATAGTCGGTGAC SEQ ID NO: 46 TCTCTGCTCCATGATTTCATAGTCG SEQ ID NO: 47 CCGAGTCTCTGCTCCATGATTTCAT SEQ ID NO: 48 ATTCCCCGAGTCTCTGCTCCATGAT SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT The sequence may comprise or consist of a sequence selected from:

[0099] In another embodiment, the exon is exon 42. In a further embodiment, the targeting sequence is SEQ ID NO: 50 TCATCGTTTCTTCACGGACAGTGTG SEQ ID NO: 51 CACCATCATCGTTTCTTCACGGACA SEQ ID NO: 52 GTCATCACCATCATCGTTTCTTCAC SEQ ID NO: 53 CTTCAGTCATCACCATCATCGTTTC SEQ ID NO: 54 AGCACAGAGGTCAGGAGCATTGAGA SEQ ID NO: 55 TCCTTAGCACAGAGGTCAGGAGCAT SEQ ID NO: 56 CAAAGTCCTTAGCACAGAGGTCAGG SEQ ID NO: 57 ATCTTCAAAGTCCTTAGCACAGAGG The sequence may comprise or consist of a sequence selected from:

[0100] In another embodiment, the exon is exon 50. In a further embodiment, the targeting sequence is SEQ ID NO: 58 CCTTCCACTCAGAGCTCAGATCTTC SEQ ID NO: 59 AAGTAAACGGTTTACCGCCTTCCAC SEQ ID NO: 60 CTGCTTTGCCCTCAGCTCTTGAAGT SEQ ID NO: 61 TCCAATAGTGGTCAGTCCAGGAGCT The sequence may comprise or consist of a sequence selected from:

[0101] In another embodiment, the exon is exon 53. In a further embodiment, the targeting sequence is SEQ ID NO: 62 CTTGTACTTCATCCCACTGATTCTG SEQ ID NO: 63 GTGTTCTTGTACTTCATCCCACTGA SEQ ID NO: 64 TGAAGGTGTTCTTGTACTTCATCCC SEQ ID NO: 65 CGGTTCTGAAGGTGTTCTTGTACTT SEQ ID NO: 66 CTCCTTCCATGACTCAAGCTTGGCT SEQ ID NO: 67 TATAGGGACCCTCCTTCCATGACTC SEQ ID NO: 68 TACTGTATAGGGACCCTCCTTCCAT SEQ ID NO: 69 TGCATCTACTGTATAGGGACCCTCC The sequence may comprise or consist of a sequence selected from:

[0102] In another embodiment, the exon is exon 70. In a further embodiment, the targeting sequence is SEQ ID NO: 70 GCAAAGTCTCGAACATCTTCTCCTG SEQ ID NO: 71 GTACCTTGGCAAAGTCTCGAACATC SEQ ID NO: 72 GGGGATGCTTCGCAAAATACCTTT SEQ ID NO: 73 TTGTCCCCCTCTAAGACAGTCTGCA SEQ ID NO: 74 TTCCATGTTGTCCCCCTCTAAGACA The sequence may comprise or consist of a sequence selected from:

[0103] In one aspect, provided herein is a conjugate comprising a modified antisense oligonucleotide and a cell membrane-permeable peptide, wherein the modified antisense oligonucleotide is 18 to 40 subunits in length and comprises a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene;

[0104] The modified antisense oligonucleotide comprises a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2'O-Me-phosphorothioate oligomer, or any combination of the foregoing;

[0105] Each subunit, taken together from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide, forms a targeting sequence, the target region being within an exon of a human dystrophin pre-mRNA, the exon being flanked by a 5' splice site upstream of the exon by a slow intron, the slow intron being an intron that is retained for a longer period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA that are downstream of the slow intron.

[0106] In one embodiment of a conjugate comprising a modified antisense oligonucleotide and a cell membrane-permeable peptide, the downstream 3' splice site of the exon is adjacent to a slow intron or a fast intron, a fast intron being an intron that is retained for a shorter period in dystrophin pre-mRNA compared to the average retention time of introns in dystrophin pre-mRNA upstream of the intron.

[0107] In certain embodiments, a conjugate comprising a modified antisense oligonucleotide and a cell membrane-permeable peptide may be 20 to 40 subunits, 18 to 35 subunits, 20 to 35 subunits, 18 to 30 subunits, 20 to 30 subunits, 18 to 26 subunits, 20 to 26 subunits, 22 to 26 subunits, 23 to 26 subunits, or 24 to 25 subunits, including all integers within these ranges. In certain embodiments, the conjugate is 24 to 25 subunits.

[0108] In one embodiment, the conjugate comprising the modified antisense oligonucleotide and the cell membrane-penetrating peptide has a guanine / cytosine (G / C) content of 30-70%. In a further embodiment, the conjugate has a G / C content of 40-60%.

[0109] In one embodiment, a conjugate comprising a modified antisense oligonucleotide and a cell membrane-permeable peptide can have a melting temperature of about 60-80°C, about 62-80°C, about 64-80°C, about 60-78°C, about 62-78°C, about 64-78°C, about 60-76°C, about 62-76°C, about 64-76°C, about 65-75°C, about 64-74°C, or about 65-74°C, including all integers within these ranges. In a further embodiment, the conjugated oligonucleotide has a melting temperature of about 64-75°C.

[0110] In one embodiment of the conjugate comprising a modified antisense oligonucleotide and a cell membrane-permeable peptide, the exon is selected from exon 10, exon 14, exon 17, exon 18, exon 21, exon 22, exon 42, exon 50, exon 53, and exon 70.

[0111] In certain embodiments, the exon is exon 10. In further embodiments, the targeting sequence is SEQ ID NO: 1 ACTTGTCTTCAGGAGCTTCCAAATG SEQ ID NO: 2 AAATGACTTGTCTTCAGGAGCTTCC SEQ ID NO: 3 CTGCCAAATGACTTGTCTTCAGGAG SEQ ID NO: 4 CTCCATCAATGAACTGCCAAATGAC SEQ ID NO: 5 CTCTCCTTGTGCTTGCAATGTGTCC SEQ ID NO: 6 GAAATCTCTCCTTGTGCTTGCAATG SEQ ID NO: 7 ACTGGTCTTTCACCACTTCCACATC SEQ ID NO: 8 ATGAAACTGGTCTTTCACCACTTCC The sequence may comprise or consist of a sequence selected from:

[0112] In another embodiment, the exon is exon 14. In a further embodiment, the targeting sequence is SEQ ID NO: 9 TGTTTGCCCATCGATCTCCCAATAC SEQ ID NO: 10 TACAGATGTTTGCCCATCGATCTCC SEQ ID NO: 11 CCATCTACAGATGTTTGCCCATCGA SEQ ID NO: 12 TCTGTCCATCTACAGATGTTTGCCC SEQ ID NO: 13 ACGTTGCCATTTGAGAAGGATGTCT SEQ ID NO: 14 GTAAGACGTTGCCATTTGAGAAGGA SEQ ID NO: 15 CTTCAGTAAGACGTTGCCATTTGAG SEQ ID NO: 16 CTGTTCTTCAGTAAGACGTTGCCAT The sequence may comprise or consist of a sequence selected from:

[0113] In another embodiment, the exon is exon 17. In a further embodiment, the targeting sequence is SEQ ID NO: 17 GAGTGGTGGTGACAGCCTGTGAAAT SEQ ID NO: 18 TCTGTGTTAGTGATGGCTGAGTGGT SEQ ID NO: 19 CCCTTGTGGTCACCGTAGTTACTGT SEQ ID NO: 20 GTTCCTCTTGAGCATGCTTTACCAG SEQ ID NO: 21 ACAGTAATCTGCCTCTTCTTTTGGG SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC The sequence may comprise or consist of a sequence selected from:

[0114] In another embodiment, the exon is exon 18. In a further embodiment, the targeting sequence is SEQ ID NO: 23 GCGAGTAATCCAGCTGTGAAGTTCA SEQ ID NO: 24 TCTGAGCGAGTAATCCAGCTGTGAA SEQ ID NO: 25 AGCTTCTGAGCGAGTAATCCAGCTG SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC SEQ ID NO: 27 GCAAATTCAGGACTCTGCAACACAG SEQ ID NO: 28 GCCTTCCTTCCGAAAGATTGCAAAT SEQ ID NO: 29 AGTTGCCTTCCTTCCGAAAGATTGC SEQ ID NO: 30 TGAGAAGTTGCCTTCCTTCCGAAAG SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC The sequence may comprise or consist of a sequence selected from:

[0115] In another embodiment, the exon is exon 21. In a further embodiment, the targeting sequence is SEQ ID NO: 32 GATCTGATAGCCGGTTGACTTCATC SEQ ID NO: 33 GTTGAAGATCTGATAGCCGGTTGAC SEQ ID NO: 34 GTCCTTGTCCTTTCTCTTTCAGGGC SEQ ID NO: 35 GAACATGGGTCCTTGTCCTTTCTC SEQ ID NO: 36 TGTAAAGGCCACAAAGTCTGCATCC SEQ ID NO: 37 TCTGGCCTGCACATCAGAAAAGACT SEQ ID NO: 38 TGTCTGTAGCTCTTTCTCTCTGGCC The sequence may comprise or consist of a sequence selected from:

[0116] In another embodiment, the exon is exon 22. In a further embodiment, the targeting sequence is SEQ ID NO: 39 CGCATTGGTGGCAAAGTGTCAAAA SEQ ID NO: 40 CTGATAGCGCATTGGTGGCAAAGT SEQ ID NO: 41 ATGGTCTCCTGATAGCGCATTGGTG SEQ ID NO: 42 CACTCATGGTCTCCTGATAGCGCAT SEQ ID NO: 43 CTGATGGCACTCATGGTCTCCTGAT SEQ ID NO: 44 GAGAGTTTGGTTTCTGACTGCTGGA SEQ ID NO: 45 GCTCCATGATTTCATAGTCGGTGAC SEQ ID NO: 46 TCTCTGCTCCATGATTTCATAGTCG SEQ ID NO: 47 CCGAGTCTCTGCTCCATGATTTCAT SEQ ID NO: 48 ATTCCCCGAGTCTCTGCTCCATGAT SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT The sequence may comprise or consist of a sequence selected from:

[0117] In another embodiment, the exon is exon 42. In a further embodiment, the targeting sequence is SEQ ID NO: 50 TCATCGTTTCTTCACGGACAGTGTG SEQ ID NO: 51 CACCATCATCGTTTCTTCACGGACA SEQ ID NO: 52 GTCATCACCATCATCGTTTCTTCAC SEQ ID NO: 53 CTTCAGTCATCACCATCATCGTTTC SEQ ID NO: 54 AGCACAGAGGTCAGGAGCATTGAGA SEQ ID NO: 55 TCCTTAGCACAGAGGTCAGGAGCAT SEQ ID NO: 56 CAAAGTCCTTAGCACAGAGGTCAGG SEQ ID NO: 57 ATCTTCAAAGTCCTTAGCACAGAGG The sequence may comprise or consist of a sequence selected from:

[0118] In another embodiment, the exon is exon 50. In a further embodiment, the targeting sequence is SEQ ID NO: 58 CCTTCCACTCAGAGCTCAGATCTTC SEQ ID NO: 59 AAGTAAACGGTTTACCGCCTTCCAC SEQ ID NO: 60 CTGCTTTGCCCTCAGCTCTTGAAGT SEQ ID NO: 61 TCCAATAGTGGTCAGTCCAGGAGCT The sequence may comprise or consist of a sequence selected from:

[0119] In another embodiment, the exon is exon 53. In a further embodiment, the targeting sequence is SEQ ID NO: 62 CTTGTACTTCATCCCACTGATTCTG SEQ ID NO: 63 GTGTTCTTGTACTTCATCCCACTGA SEQ ID NO: 64 TGAAGGTGTTCTTGTACTTCATCCC SEQ ID NO: 65 CGGTTCTGAAGGTGTTCTTGTACTT SEQ ID NO: 66 CTCCTTCCATGACTCAAGCTTGGCT SEQ ID NO: 67 TATAGGGACCCTCCTTCCATGACTC SEQ ID NO: 68 TACTGTATAGGGACCCTCCTTCCAT SEQ ID NO: 69 TGCATCTACTGTATAGGGACCCTCC The sequence may comprise or consist of a sequence selected from:

[0120] In another embodiment, the exon is exon 70. In a further embodiment, the targeting sequence is SEQ ID NO: 70 GCAAAGTCTCGAACATCTTCTCCTG SEQ ID NO: 71 GTACCTTGGCAAAGTCTCGAACATC SEQ ID NO: 72 GGGGATGCTTCGCAAAATACCTTT SEQ ID NO: 73 TTGTCCCCCTCTAAGACAGTCTGCA SEQ ID NO: 74 TTCCATGTTGTCCCCCTCTAAGACA The sequence may comprise or consist of a sequence selected from:

[0121] In one embodiment, the cell membrane penetrating peptide is rTAT, TAT, R9F2, R5F2R4, R4, R5, R6, R7, R8, R9, (RXR)4, (RXR)5, (RXRRBR) 2、 (RAR)4F2, (RGR)4F2.

[0122] In another aspect, provided herein are compounds of formula I: [ka] (I) or a pharmaceutically acceptable salt thereof, During the ceremony, A' is -N(H)CH2C(O)NH2, -N(C 1-6 -alkyl)CH2C(O)NH2, [ka] , and [ka] is selected from: R 5 is -C(O)(O-alkyl) x -OH, wherein x is 3 to 10, and each alkyl group in each occurrence is independently selected from the group consisting of C 2-6 - alkyl or or R 5 is H, -C(O)C 1-6 -Alkyl, trityl, monomethoxytrityl, -(C 1-6 -alkyl)-R 6 , -(C 1-6 -heteroalkyl)-R 6 , aryl-R 6 , heteroaryl-R 6 , -C(O)O-(C 1-6 -alkyl)-R 6 , -C(O)O-aryl-R 6 , —C(O)O-heteroaryl-R 6, and selected from: [ka] ; R 6 is selected from OH, SH, and NH2, or R 6 is O, S, or NH, each of which is covalently attached to a solid support; Each R 1 is OH and -N(R 3 )(R 4 ) independently selected from 3 and R 4 is independently in each occurrence H or -C 1-6 -alkyl, Each R 2 is independently selected at each occurrence from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein said nucleobase is independently selected at each occurrence from pyridine, pyrimidine, purine, and deaza-purine. 3-6 heterocyclic ring, wherein each R 2 together form the targeting sequence, z is 8 to 40; E' is H, -C 1-6 -Alkyl, -C(O)C 1-6 -Alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, [ka] , and [ka] is selected from During the ceremony, Q is —C(O)(CH)C(O)— or —C(O)(CH)S(CH)C(O)—; R 7 is -(CH2)2OC(O)N(R 8 )2, where R 8 is -(CH2)6NHC(=NH)NH2, L is glycine, proline, W, WW, or R 9 and L is covalently linked by an amide bond to the N-terminus or C-terminus of J; W is -C(O)-(CH2) m -NH-, where m is 2 to 12; R 9 teeth, [ka] , [ka] , and [ka] is selected from the group consisting of n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, or 5; R 10 is selected from a bond, glycine, proline, W, or WW; R 11 is glycine, proline, W, WW, and [ka] is selected from the group consisting of R 16 is selected from a bond, glycine, proline, W, or WW; R 16 is covalently attached by an amide bond to the N-terminus or C-terminus of J, wherein J is a cell membrane penetrating peptide; G is H, -C(O)C 1-6 -alkyl, benzoyl, and stearoyl; G is covalently bonded to J; The targeting sequence is SEQ ID NO: 1 ACTTGTCTTCAGGAGCTTCCAAATG SEQ ID NO: 2 AAATGACTTGTCTTCAGGAGCTTCC SEQ ID NO: 3 CTGCCAAATGACTTGTCTTCAGGAG SEQ ID NO: 4 CTCCATCAATGAACTGCCAAATGAC SEQ ID NO: 5 CTCTCCTTGTGCTTGCAATGTGTCC SEQ ID NO: 6 GAAATCTCTCCTTGTGCTTGCAATG SEQ ID NO: 7 ACTGGTCTTTCACCACTTCCACATC SEQ ID NO: 8 ATGAAACTGGTCTTTCACCACTTCC SEQ ID NO: 9 TGTTTGCCCATCGATCTCCCAATAC SEQ ID NO: 10 TACAGATGTTTGCCCATCGATCTCC SEQ ID NO: 11 CCATCTACAGATGTTTGCCCATCGA SEQ ID NO: 12 TCTGTCCATCTACAGATGTTTGCCC SEQ ID NO: 13 ACGTTGCCATTTGAGAAGGATGTCT SEQ ID NO: 14 GTAAGACGTTGCCATTTGAGAAGGA SEQ ID NO: 15 CTTCAGTAAGACGTTGCCATTTGAG SEQ ID NO: 16 CTGTTCTTCAGTAAGACGTTGCCAT SEQ ID NO: 17 GAGTGGTGGTGACAGCCTGTGAAAT SEQ ID NO: 18 TCTGTGTTAGTGATGGCTGAGTGGT SEQ ID NO: 19 CCCTTGTGGTCACCGTAGTTACTGT SEQ ID NO: 20 GTTCCTCTTGAGCATGCTTTACCAG SEQ ID NO: 21 ACAGTAATCTGCCTCTTCTTTTGGG SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC SEQ ID NO: 23 GCGAGTAATCCAGCTGTGAAGTTCA SEQ ID NO: 24 TCTGAGCGAGTAATCCAGCTGTGAA SEQ ID NO: 25 AGCTTCTGAGCGAGTAATCCAGCTG SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC SEQ ID NO: 27 GCAAATTCAGGACTCTGCAACACAG SEQ ID NO: 28 GCCTTCCTTCCGAAAGATTGCAAAT SEQ ID NO: 29 AGTTGCCTTCCTTCCGAAAGATTGC SEQ ID NO: 30 TGAGAAGTTGCCTTCCTTCCGAAAG SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC SEQ ID NO: 32 GATCTGATAGCCGGTTGACTTCATC SEQ ID NO: 33 GTTGAAGATCTGATAGCCGGTTGAC SEQ ID NO: 34 GTCCTTGTCCTTTCTCTTTCAGGGC SEQ ID NO: 35 GAACATGGGTCCTTGTCCTTTCTC SEQ ID NO: 36 TGTAAAGGCCACAAAGTCTGCATCC SEQ ID NO: 37 TCTGGCCTGCACATCAGAAAAGACT SEQ ID NO: 38 TGTCTGTAGCTCTTTCTCTCTGGCC SEQ ID NO: 39 CGCATTGGTGGCAAAGTGTCAAAA SEQ ID NO: 40 CTGATAGCGCATTGGTGGCAAAGT SEQ ID NO: 41 ATGGTCTCCTGATAGCGCATTGGTG SEQ ID NO: 42 CACTCATGGTCTCCTGATAGCGCAT SEQ ID NO: 43 CTGATGGCACTCATGGTCTCCTGAT SEQ ID NO: 44 GAGAGTTTGGTTTCTGACTGCTGGA SEQ ID NO: 45 GCTCCATGATTTCATAGTCGGTGAC SEQ ID NO: 46 TCTCTGCTCCATGATTTCATAGTCG SEQ ID NO: 47 CCGAGTCTCTGCTCCATGATTTCAT SEQ ID NO: 48 ATTCCCCGAGTCTCTGCTCCATGAT SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT SEQ ID NO: 50 TCATCGTTTCTTCACGGACAGTGTG SEQ ID NO: 51 CACCATCATCGTTTCTTCACGGACA SEQ ID NO: 52 GTCATCACCATCATCGTTTCTTCAC SEQ ID NO: 53 CTTCAGTCATCACCATCATCGTTTC SEQ ID NO: 54 AGCACAGAGGTCAGGAGCATTGAGA SEQ ID NO: 55 TCCTTAGCACAGAGGTCAGGAGCAT SEQ ID NO: 56 CAAAGTCCTTAGCACAGAGGTCAGG SEQ ID NO: 57 ATCTTCAAAGTCCTTAGCACAGAGG SEQ ID NO: 58 CCTTCCACTCAGAGCTCAGATCTTC SEQ ID NO: 59 AAGTAAACGGTTTACCGCCTTCCAC SEQ ID NO: 60 CTGCTTTGCCCTCAGCTCTTGAAGT SEQ ID NO: 61 TCCAATAGTGGTCAGTCCAGGAGCT SEQ ID NO: 62 CTTGTACTTCATCCCACTGATTCTG SEQ ID NO: 63 GTGTTCTTGTACTTCATCCCACTGA SEQ ID NO: 64 TGAAGGTGTTCTTGTACTTCATCCC SEQ ID NO: 65 CGGTTCTGAAGGTGTTCTTGTACTT SEQ ID NO: 66 CTCCTTCCATGACTCAAGCTTGGCT SEQ ID NO: 67 TATAGGGACCCTCCTTCCATGACTC SEQ ID NO: 68 TACTGTATAGGGACCCTCCTTCCAT SEQ ID NO: 69 TGCATCTACTGTATAGGGACCCTCC SEQ ID NO: 70 GCAAAGTCTCGAACATCTTCTCCTG SEQ ID NO: 71 GTACCTTGGCAAAGTCTCGAACATC SEQ ID NO: 72 GGGGATGCTTCGCAAAATACCTTT SEQ ID NO: 73 TTGTCCCCCTCTAAGACAGTCTGCA SEQ ID NO: 74 TTCCATGTTGTCCCCCTCTAAGACA The sequence comprises or consists of:

[0123] In certain embodiments, one of the following definitions occurs in an oligomer of Formula I: 1) A' is [ka] ; or 2) E' is [ka] is.

[0124] In another embodiment, E' is H, -C 1-6 -Alkyl, -C(O)C 1-6 -alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and [ka] is selected from.

[0125] In yet another embodiment, A' is -N(C 1-6 -alkyl)CH2C(O)NH2, [ka] , [ka] , and [ka] is selected from.

[0126] In further embodiments, E' is H, -C(O)CH3, benzoyl, stearoyl, trityl, 4-methoxytrityl, and [ka] is selected from.

[0127] In another embodiment, A' is -N(C 1-6 -alkyl)CH2C(O)NH2, [ka] , and [ka] is selected from E' is [ka] is.

[0128] In one embodiment, A' is [ka] and E' is selected from H, -C(O)CH3, trityl, 4-methoxytrityl, benzoyl, and stearoyl.

[0129] In certain embodiments, the compound of formula I is [ka] (Ia); and [ka] (Ib),

[0130] In the formula, E' is H, C 1-6 -alkyl, -C(O)CH3, benzoyl, and stearoyl.

[0131] In one embodiment, the phosphorodiamidate morpholino oligomer is of Formula (Ia): In another embodiment, the phosphorodiamidate morpholino oligomer is of Formula (Ib):

[0132] In one embodiment of the phosphorodiamidate morpholino oligomer of Formula I, the cell membrane penetrating peptide is selected from rTAT, Tat, R9F2, R5F2R4, R4, R5, R6, R7, R8, R9, (RAhxR)4, (RAhxR)5, (RAhxRRBR)2, (RAR)4F2, and (RGR)4F2.

[0133] In one embodiment, each R 1 is -N(CH3)2.

[0134] In one embodiment, L is glycine. In another embodiment, L is proline. In yet another embodiment, L is -C(O)-(CH2)5-NH-. In yet another embodiment, L is -C(O)-(CH2)2-NH-. In one embodiment, L is -C(O)-(CH2)2-NH-C(O)-(CH2)5-NH-.

[0135] In one embodiment, L is [ka] and R 10 is a bond and R 11 is glycine and [ka] is selected from.

[0136] In another embodiment, L is [ka] and R 10 is a bond and R 11 is glycine and [ka] is selected from.

[0137] In yet another embodiment, L is [ka] and R 10 is a bond and R 11 is glycine and [ka] is selected from.

[0138] In one embodiment, J is selected from rTAT, TAT, R9F2, R5F2R4, R4, R5, R6, R7, R8, R9, (RXR)4, (RXR)5, (RXRRBR)2, (RAR)4F2, (RGR)4F2.

[0139] In further embodiments, G is selected from H, C(O)CH3, benzoyl, and stearoyl.

[0140] In one embodiment, G is H or —C(O)CH 3 .

[0141] In a further embodiment, G is H.

[0142] In still further embodiments, G is —C(O)CH 3 .

[0143] In one aspect, provided herein are antisense oligomeric compounds and a pharmaceutically acceptable carrier.

[0144] III. Characteristics of Oligomeric Chemicals The antisense oligonucleotide of the present disclosure can use various antisense oligonucleotide chemicals.Examples of oligomer chemicals include, but are not limited to, morpholino oligomers, phosphorothioate-modified oligomers, 2'-O-methyl-modified oligomers, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate oligomers, 2'-O-MOE-modified oligomers, 2'-fluoro-modified oligomers, 2'-O,4'C-ethylene-bridged nucleic acid (ENA), tricyclo-DNA, tricyclo-DNA phosphorothioate subunits, 2'-O-[2-(N-methylcarbamoyl)ethyl]-modified oligomers (including any combination of the above).Phosphorothioate and 2'-O-Me-modified chemicals can be combined to produce a 2'-O-Me-phosphorothioate backbone. See, e.g., PCT Publication Nos. 2013 / 112053 and 2009 / 008725, which are incorporated by reference in their entireties.

[0145] In some embodiments, the nucleobases of the modified antisense oligonucleotides are linked to morpholino ring structures that are joined by phosphorus-containing intersubunit linkages connecting the morpholino nitrogen of one ring structure to the 5' exocyclic carbon of an adjacent ring structure.

[0146] In some embodiments, the nucleobases of the antisense oligonucleotide are linked to peptide nucleic acids (PNAs), in which the phosphate-sugar polynucleotide backbone is replaced by a flexible pseudo-peptide polymer to which the nucleobases are linked. In some aspects, at least one of the nucleobases of the antisense oligonucleotide is linked to a locked nucleic acid (LNA), in which the locked nucleic acid structure is a chemically modified nucleotide analog in which the ribose moiety has an extra bridge connecting the 2' oxygen and the 4' carbon.

[0147] In some embodiments, at least one of the nucleobases of the antisense oligonucleotide is linked to a bridged nucleic acid (BNA), where the sugar conformation is restricted or locked by the introduction of an additional bridge structure to the furanose backbone. In some aspects, at least one of the nucleobases of the antisense oligonucleotide is linked to a 2'-O,4'-C-ethylene-bridged nucleic acid (ENA).

[0148] In some embodiments, modified antisense oligonucleotides may contain unlocked nucleic acid (UNA) subunits. UNAs and UNA oligomers are analogs of RNA in which the C2'-C3' bond of the subunit has been truncated.

[0149] In some embodiments, modified antisense oligonucleotides contain one or more phosphorothioates (or S-oligos) in which one of the non-bridging oxygens is replaced by sulfur. In some aspects, modified antisense oligonucleotides contain one or more 2'O-methyl, 2'O-MOE, MCE, and 2'-F in which the 2'-OH of the ribose is replaced with a methyl, methoxyethyl, 2-(N-methylcarbamoyl)ethyl, or fluoro group, respectively.

[0150] In some embodiments, the modified antisense oligonucleotide is tricyclo-DNA (tc-DNA), a constrained DNA analogue in which each nucleotide is modified by the introduction of a cyclopropane ring to limit the conformational flexibility of the backbone and optimize the backbone geometry for torsion angle g.

[0151] In some embodiments, at least one of the nucleobases of the antisense oligonucleotide is linked to a bridged nucleic acid (BNA), and the sugar conformation is restricted or locked by the introduction of an additional bridge structure to the furanose backbone. In some aspects, at least one of the nucleobases of the antisense oligonucleotide is linked to a 2'-O,4'-C-ethylene-bridged nucleic acid (ENA). In such aspects, each nucleobase linked to the BNA or ENA contains a 5-methyl group.

[0152] 1. Peptide Nucleic Acid (PNA) Peptide nucleic acids (PNAs) are analogs of DNA whose backbone is structurally isomorphous with a deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligomers according to Watson-Crick base-pairing rules, mimicking DNA in terms of base pair recognition. PNA backbones are formed by peptide bonds rather than phosphodiester bonds, making them well suited for antisense applications (see structure below). The backbone is uncharged, resulting in PNA / DNA or PNA / RNA duplexes that exhibit exceptional thermal stability. PNAs are not recognized by nucleases or proteases.

[0153] Non-limiting examples of PNAs are shown below. [ka]

[0154] Despite their radical structural changes relative to their native structure, PNAs are capable of sequence-specific binding to DNA or RNA in a helical configuration. PNA characteristics include high binding affinity to complementary DNA or RNA, destabilization caused by single-base mismatches, resistance to nucleases and proteases, salt-independent hybridization with DNA or RNA, and triplex formation with homopurine DNA. PANAGENE™ has developed its proprietary Bts PNA monomer (Bts, benzothiazole-2-sulfonyl group) and proprietary oligomerization process. PNA oligomerization using the Bts PNA monomer consists of repeated cycles of deprotection, coupling, and capping. PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Patent Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. Also, for the preparation of PNAs, see U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500, 1991. Each of the above is incorporated by reference in its entirety.

[0155] 2. Locked Nucleic Acid (LNA) Antisense oligonucleotides may also contain "locked nucleic acid" subunits (LNA). "LNA" is a member of a class of modifications called bridged nucleic acids (BNA). BNAs are characterized by a covalent bond that locks the conformation of the ribose ring to the C30-endo (northern) sugar pucker. In the case of LNA, the bridge consists of a methylene between the 2'-O and 4'-C positions. LNAs enhance backbone preorganization and base stacking, improving hybridization and thermal stability.

[0156] The structure of LNA can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998) 54: 3607; Jesper Wengel, Accounts of Chem. Research (1999) 32: 301; Obika, et al., Tetrahedron Letters (1997) 38: 8735; Obika, et al., Tetrahedron Letters (1998) 39: 5401; and Obika, et al., Bioorganic Medicinal Chemistry (2008) 16: 9230, which are incorporated herein by reference in their entirety. Non-limiting examples of LNA are shown below. [ka]

[0157] The antisense oligonucleotides of the present disclosure may incorporate one or more LNAs. In some cases, the antisense oligonucleotide may be composed entirely of LNAs. Methods for synthesizing individual LNA nucleoside subunits and incorporating them into oligomers are described, for example, in U.S. Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated by reference in its entirety. Typical intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers may be used. Further embodiments include LNA-containing antisense oligonucleotides, in which each LNA subunit is separated by a DNA subunit. Certain antisense oligonucleotides are composed of alternating LNA and DNA subunits, with the intersubunit linker being phosphorothioate.

[0158] 3. Ethylene-bridged nucleic acid (ENA) 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs) are another member of the BNA class. Non-limiting examples are shown below. [ka]

[0159] ENA oligomers and their preparation are described in Obika et al., Tetrahedron Lett (1997) 38(50):8735, which is incorporated herein by reference in its entirety. The antisense oligonucleotides of the present disclosure may incorporate one or more ENA subunits.

[0160] 4. Unlocked Nucleic Acid (UNA) Antisense oligonucleotides may also contain unlocked nucleic acid (UNA) subunits. UNA and UNA oligomers are analogs of RNA in which the C2'-C3' bond of the subunit is cleaved. LNAs are conformationally restricted (compared to DNA and RNA), while UNAs are highly flexible. UNAs are disclosed, for example, in WO2016 / 070166. Non-limiting examples of UNAs are shown below. [ka]

[0161] Typical intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers can be used.

[0162] 5. Phosphorothioates "Phosphorothioates" (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens has been replaced with a sulfur. Non-limiting examples of phosphorothioates are shown below. [ka]

[0163] Sulfurization of internucleotide bonds reduces the action of endonucleases and exonucleases, including 5' to 3' and 3' to 5' DNA POL 1 exonucleases, nucleases SI and PI, RNases, serum nucleases, and snake venom phosphodiesterases. Phosphorothioates are produced by two major routes: the action of a solution of elemental sulfur in carbon disulfide on hydrogen phosphonates, or sulfurization of phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990, incorporated herein by reference in its entirety). The latter method avoids the insolubility of elemental sulfur in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also result in higher purity phosphorothioates.

[0164] 6. Tricyclo-DNA and tricyclo-phosphorothioate subunits Tricyclo-DNA (tc-DNA) is a class of constrained DNA analogues in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry for torsion angle g. Homobasic adenine- and thymine-containing tc-DNA forms highly stable AT base pairs with complementary RNA. Tricyclo-DNA and its synthesis are described in International Patent Application Publication No. 2010 / 115993, which is incorporated herein by reference in its entirety. The antisense oligomers of the present disclosure may incorporate one or more tricyclo-DNA subunits. In some cases, the antisense oligomer may be composed entirely of tricyclo-DNA subunits.

[0165] A tricyclo-phosphorothioate subunit is a tricyclo-DNA subunit with a phosphorothioate intersubunit linkage. Tricyclo-phosphorothioate subunits and their synthesis are described in International Patent Application Publication No. 2013 / 053928, which is incorporated herein by reference in its entirety. The antisense oligomer of the present disclosure may incorporate one or more tricyclo-DNA subunits. In some cases, the antisense oligomer may be entirely composed of tricyclo-DNA subunits. Non-limiting examples of tricyclo-DNA / tricyclo-phosphorothioate subunits are shown below. [ka]

[0166] 7. 2'-O-Methyl, 2'-O-MOE, and 2'-F Oligomers A "2'-O-Me oligomer" molecule has a methyl group at the 2'-OH residue of the ribose molecule. 2'-O-Me-RNA behaves the same as (or similar to) DNA, but is protected from nuclease degradation. 2'-O-Me-RNA can also be combined with a phosphorothioate oligomer (PTO) for further stabilization. 2'O-Me oligomers (phosphodiester or phosphorothioate) can be synthesized according to conventional techniques in the art (see, for example, Yoo et al., Nucleic Acids Res. 32:2008-16, 2004, which is incorporated herein by reference in its entirety). Non-limiting examples of 2'O-Me oligomers are shown below. [ka]

[0167] 2'-O-Methoxyethyl oligomers (2'-O-MOE) have a methoxyethyl group at the 2'-OH residue of the ribose molecule and are discussed in Martin et al., Helv. Chim. Acta, 78, 486-504, 1995, which is incorporated herein by reference in its entirety. Non-limiting examples of 2'-O-MOE subunits are shown below. [ka]

[0168] 2'-Fluoro (2'-F) oligomers have a fluorescent radical at the 2' position instead of 2'-OH. Non-limiting examples of 2'-F oligomers are shown below. [ka]

[0169] 2'-Fluoro oligomers are further described in WO2004 / 043977, which is incorporated herein by reference in its entirety.

[0170] The 2'-O-methyl, 2'-O-MOE, and 2'-F oligomers may also contain one or more phosphorothioate (PS) linkages, as shown below. [ka]

[0171] Additionally, 2'-O-methyl, 2'-O-MOE, and 2'-F oligomers may contain PS intersubunit linkages throughout the oligomer, such as, for example, the 2'-O-methyl PS oligomer drisapersen shown below. [ka]

[0172] Alternatively, the 2'-O-methyl, 2'-O-MOE, and / or 2'-F oligomers may contain a PS linkage at the terminus of the oligomer, as shown below. [ka]

[0173] During the ceremony, R is CH2CH2OCH3 (methoxyethyl or MOE), X, Y, and Z represent the number of nucleotides contained within the designated 5'-wing, central gap, and 3'-wing regions, respectively.

[0174] The antisense oligomers of the present disclosure may incorporate one or more 2'-O-methyl, 2'-O-MOE, and 2'-F subunits and may utilize any of the intersubunit linkages described herein. In some cases, the antisense oligomers of the present disclosure may be composed entirely of 2'-O-methyl, 2'-O-MOE, or 2'-F subunits. One embodiment of the antisense oligomer of the present disclosure consists entirely of 2'-O-methyl subunits.

[0175] 8. 2'-O-[2-(N-methylcarbamoyl)ethyl] oligomer (MCE) MCE is another example of a 2'-O modified ribonucleoside useful in the antisense oligomers of the present disclosure, where the 2'-OH is derivatized to a 2-(N-methylcarbamoyl)ethyl moiety to enhance nuclease resistance. Non-limiting examples of MCE oligomers are shown below. [ka]

[0176] MCE and its synthesis are described in Yamada et al., J. Org. Chem. (2011) 76(9):3042-53, which is incorporated herein by reference in its entirety. Antisense oligonucleotides of the present disclosure may incorporate one or more MCE subunits.

[0177] 9. Stereospecific Oligomers Stereospecific oligomers are those in which the stereochemistry of each phosphorus-containing linkage is fixed by synthetic methods such that a substantially stereopure oligomer is produced. Non-limiting examples of stereospecific oligomers are shown below. [ka]

[0178] In the above examples, each phosphorus of the oligomer has the same stereochemistry. Additional examples include the oligomers described herein. For example, LNA, ENA, tricyclo-DNA, MCE, 2'-O-methyl, 2'-O-MOE, 2'-F, and morpholino-based oligomers can be prepared using stereospecific phosphorus-containing internucleoside linkages, such as phosphorothioate, phosphodiester, phosphoramidate, phosphorodiamidate, or other phosphorus-containing internucleoside linkages. Stereospecific oligomers, methods of preparation, chiral controlled synthesis, chiral designs, and chiral auxiliaries for use in preparing such oligomers are described, for example, in WO2017 / 192664, WO2017 / 192679, WO2017 / 062862, WO2017 / 015575, WO2017 / 015555, WO2015 / 107425, WO2015 / 108048, WO 2015 / 108046, WO2015 / 108047, WO2012 / 039448, WO2010 / 064146, WO2011 / 034072, WO2014 / 010250, WO2014 / 012081, WO2013 / 0127858, and WO2011 / 005761, each of which is incorporated herein by reference in its entirety.

[0179] The stereospecific oligomers are R P Placement or S PThe oligomer may have phosphorus-containing internucleoside linkages in any configuration. Chiral phosphorus-containing linkages in which the configuration of the linkage is controlled are referred to as "stereo-pure," while chiral phosphorus-containing linkages in which the configuration of the linkage is not controlled are referred to as "stereoirregular." In certain embodiments, oligomers of the present disclosure include multiple stereo-pure and stereoirregular linkages, such that the resulting oligomer has stereo-pure subunits at pre-designated positions in the oligomer. Examples of the locations of stereo-pure subunits are provided in Figures 7A and 7B of International Patent Application Publication No. 2017 / 062862(A2). In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereo-irregular. In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereo-pure.

[0180] In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), all n chiral phosphorus-containing linkages in the oligomer are stereoirregular. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), all n chiral phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), at least 10% (rounded to the nearest integer) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), at least 20% (rounded to the nearest integer) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), at least 30% (rounded to the nearest integer) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 40% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 50% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 60% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer greater than or equal to 1) chiral phosphorus-containing linkages, at least 70% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), at least 80% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer greater than or equal to 1), at least 90% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure.

[0181] In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), the oligomers have the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least two consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least three consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least four consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least five consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least six consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 7 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 8 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R PIn one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 9 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 10 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 11 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 12 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 13 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 14 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 15 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 16 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., SP or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 17 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 18 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least 19 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P contains at least 20 consecutive stereopure phosphorus-containing linkages of

[0182] In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), the oligomers have the same stereo orientation (i.e., S P or R P and at least two consecutive stereopure phosphorus-containing linkages of the other stereoorientation. For example, the oligomer may be P at least two consecutive stereopure phosphorus-containing linkages in the orientation P and at least two consecutive stereopure phosphorus-containing linkages in the oriented direction.

[0183] In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), the oligomer contains at least two consecutive stereopure phosphorus-containing linkages of the same stereoorientation in an alternating pattern. For example, the oligomer may contain, in order: two or more R P , two or more S P , and two or more R P etc.

[0184] 10. Morpholino Oligomers Exemplary embodiments of the present disclosure include those having the following general structure: [ka] and as shown in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997). The morpholino described herein is intended to include all stereoisomers and tautomers of the above general structure. The synthesis, structure, and binding properties of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476, and 8,299,206, all of which are incorporated herein by reference.

[0185] In certain embodiments, the morpholino is conjugated to a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include: [ka] , [ka] , and [ka] Includes:

[0186] In various aspects, the present disclosure provides an antisense oligomer according to Formula I, or a pharmaceutically acceptable salt thereof.

[0187] IV. Target Sequences and Target Regions In some embodiments for antisense applications, oligonucleotides can be 100% complementary to the nucleic acid target sequence, or can contain mismatches, for example, to accommodate variants, as long as the heteroduplex formed between the oligonucleotide and the nucleic acid target sequence is sufficiently stable to withstand the action of cellular nucleases and other degradation modes that may occur in vivo. If present, mismatches are less destabilizing toward the end regions of the hybrid duplex than toward the center. The number of mismatches tolerated depends on the length of the oligonucleotide, the proportion of G:C base pairs in the duplex, and the position of the mismatch in the duplex, in accordance with well-understood principles of duplex stability. Such antisense oligonucleotides are not necessarily 100% complementary to the nucleic acid target sequence, but are effective in stably and specifically binding to the target sequence so as to modulate the biological activity of the nucleic acid target, for example, the expression of the encoded protein.

[0188] The stability of the duplex formed between the oligonucleotide and the target sequence is determined by the binding T m The T of an antisense compound relative to a complementary sequence RNA is a function of the T and indicates the susceptibility of the duplex to cellular enzymatic cleavage. m can be measured by conventional methods such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or Miyada CG. and Wallace RB (1987) Oligonucleotide hybridization techniques, Methods Enzymol. Vol. 154 pp. 94-107.

[0189] In some embodiments, each antisense oligonucleotide has a binding T with respect to the complementary sequence RNA that is greater than body temperature, or in other embodiments, greater than 50°C. m In another embodiment, T m According to well-known principles, the T of an oligonucleotide compound with respect to its complementary base RNA hybrid is in the range of 60-80°C or higher. Mcan be increased by increasing the ratio of C:G base pairs in the duplex and / or by increasing the length (in base pairs) of the heteroduplex. At the same time, it may be advantageous to limit the size of the oligonucleotide in order to optimize cellular uptake. For this reason, high T m (50°C or higher) m These are generally preferred over compounds that require more than 20 bases for their value. In some applications, longer oligonucleotides, e.g., oligonucleotides longer than 20 bases, may have certain advantages.

[0190] The targeted sequence bases can be normal DNA bases or their analogs, such as uracil and inosine, which are capable of Watson-Crick base pairing to the targeted sequence RNA bases.

[0191] Antisense oligonucleotides can be designed to block, inhibit, or modulate translation of mRNA, inhibit or modulate splice processing of pre-mRNA, or induce degradation of targeted mRNA, and can be said to be "directed against" or "targeted to" the target sequence to which they hybridize. In certain embodiments, the target sequence includes a region containing a 3' or 5' splice site, branch point, or other sequence involved in regulating splicing of pre-processed mRNA. The target sequence can be within an exon, within an intron, or can span an intron / exon junction.

[0192] An antisense oligonucleotide having sufficient sequence complementarity to a target RNA sequence to modulate splicing of the target RNA means that the antisense agent has a sequence sufficient to induce masking of a binding site for a natural protein that would otherwise modulate splicing and / or alter the three-dimensional structure of the targeted RNA. Similarly, an oligonucleotide reagent having a sequence sufficiently complementary to a target RNA sequence to modulate splicing of the target RNA means that the oligonucleotide reagent has a sequence sufficient to induce masking of a binding site for a natural protein that would otherwise modulate splicing and / or alter the three-dimensional structure of the targeted RNA.

[0193] In certain embodiments, the degree of complementarity between the target sequence and the antisense targeting sequence is sufficient to form a stable duplex. The complementary region of the antisense oligonucleotide, excluding the abasic unit, containing the target RNA sequence can be as short as 8-11 bases, but can be 12-15 or more bases, for example, 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers between these ranges. An antisense oligonucleotide of about 14-15 bases is generally long enough to have a unique complementary sequence. In certain embodiments, as discussed herein, a minimum length of complementary bases may be required to achieve the required binding Tm.

[0194] In certain embodiments, oligonucleotides as long as 40 bases may be suitable, with at least a minimum number of bases, e.g., 10-12 bases, being complementary to the target sequence. In some embodiments, facilitated or active uptake in cells is optimized with oligonucleotide lengths of less than about 30 bases. For the PMO oligonucleotides described further herein, the optimal balance of binding stability and uptake generally occurs with lengths of 18-25 bases. The present disclosure includes antisense oligonucleotides consisting of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases. nucleotides (e.g., PMO, PMO-X base, LNA, 2'-OMe) of which at least about 6, 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, or 40 contiguous or non-contiguous bases are complementary to the desired target sequence.

[0195] In certain embodiments, antisense oligonucleotides can be 100% complementary to target sequences, or can contain mismatches, for example, to accommodate variants, as long as the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other degradation methods that may occur in vivo.Therefore, some oligonucleotides can have substantial complementarity, i.e., about or at least about 70% sequence complementarity, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity between the oligonucleotide and the target sequence. The oligonucleotide backbone discussed herein is less susceptible to nuclease cleavage.When mismatches exist, they are typically less destabilizing towards the end regions of hybrid double strands than in the middle.The number of mismatches that can be tolerated depends on the length of oligonucleotide, the proportion of G:C base pairs in the double strand, and the position of mismatches in the double strand, according to the well-understood principle of double strand stability.Such antisense oligonucleotides are not necessarily 100% complementary to target sequences, but they are effective in stably and specifically binding to target sequences so as to regulate the splicing of target RNA precursors.

[0196] The stability of the duplex formed between oligonucleotide and target sequence is a function of binding Tm, which indicates the susceptibility of duplex to cellular enzymatic cleavage.The Tm of oligonucleotide with respect to complementary sequence RNA can be measured by conventional methods such as those described in Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp.107-108, or those described in Miyada CG and Wallace RB, 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol.154 pp.94-107.

[0197] In certain embodiments, antisense oligonucleotides may have a binding Tm with respect to complementary sequence RNA that is higher than body temperature, preferably above about 45°C or 50°C. Tms in the range of 60-80°C are also included. According to well-known principles, the Tm of an oligonucleotide with respect to a complementary base RNA hybrid can be increased by increasing the ratio of C:G base pairs in the duplex and / or by increasing the length (in base pairs) of the heteroduplex. At the same time, it may be advantageous to limit the size of the oligonucleotide in order to optimize cellular uptake. For this reason, compounds that exhibit high Tm (45-50°C or higher) at lengths of 25 bases or less are generally preferred over compounds that require more than 25 bases for a high Tm value.

[0198] In one aspect, the present disclosure provides an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, capable of binding to a selected target and inducing exon skipping in a human dystrophin gene, wherein the antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, comprises a sequence of bases that are complementary to an exon target region of a dystrophin pre-mRNA designated as an annealing site, and each nucleobase R from 1 to t and 5' to 3' is set forth in Formula I and described throughout the specification. 2 teeth, [ka] , [ka] , [ka] , [ka] , [ka] , Methylated guanine, methylated adenine, and [ka] may be selected from:

[0199] Antisense oligomers can be designed to block, inhibit, or modulate translation of mRNA, inhibit or modulate pre-mRNA splice processing, or induce degradation of the targeted mRNA, and can be said to be "directed against" or "targeted to" the target sequence to which they hybridize. In certain embodiments, the target sequence includes a region containing a 3' or 5' splice site, branch point, or other sequence involved in regulating splicing of a preprocessed mRNA. The target sequence can be within an exon or an intron, or can span an intron / exon junction. An antisense oligomer having sufficient sequence complementarity to a target RNA sequence to modulate splicing of the target RNA means that the antisense agent has sufficient sequence to induce masking of a binding site for a natural protein that would otherwise modulate splicing and / or alter the three-dimensional structure of the targeted RNA. Similarly, an oligomeric reagent having a sequence sufficiently complementary to a target RNA sequence to modulate splicing of the target RNA means that the oligomeric reagent has sufficient sequence to induce masking of a binding site for a natural protein that would otherwise modulate splicing and / or alter the three-dimensional structure of the targeted RNA.

[0200] In one embodiment, the target region is within an exon of a human dystrophin pre-mRNA, the exon being flanked at the exon's upstream 5' splice site by a slow intron, the slow intron being an intron that is retained for a longer period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA that are downstream of the slow intron.

[0201] Table 2 below shows examples of targeting sequences that are complementary (in the 5' to 3' orientation) to the pre-mRNA sequence of the Duchenne muscular dystrophy gene. [Table 2-1] [Table 2-2] [Table 2-3]

[0202] V. Cell-penetrating peptides (CPPs) In some embodiments, the subject oligomers are conjugated to a peptide transporter moiety, e.g., a cell membrane-permeable peptide transport moiety (also referred to as a cell membrane-permeable peptide) effective to enhance transport of the oligomer into a cell. For example, in some embodiments, the cell membrane-permeable peptide moiety is an arginine-rich peptide. In further embodiments, the peptide moiety is attached to either the 5' or 3' end of the oligomer. When such a peptide is conjugated to either end, the opposite end is available for further conjugation to a modified end group described herein.

[0203] In some of the aforementioned embodiments, the cell membrane-permeable peptide portion comprises 6 to 16 subunits selected from X' subunits, Y' subunits, and Z' subunits; During the ceremony, (a) each X′ subunit independently represents a lysine, arginine, or arginine analog, said analog having the structure R 33 N=C(NH2)R 34 wherein R 33 is H or R, and R 34 is R 35 , NH2, NHR, or NR 34 where R 35 is lower alkyl or lower alkenyl and may further contain oxygen or nitrogen; R33 and R 34 can be taken together to form a ring, and the side chains are R 33 or R 34 is linked to the amino acid via (b) Each Y' subunit consists of the neutral amino acid -C(O)-(CHR) n independently represents —NH—, where n is 2 to 7, and each R is independently H or methyl; (c) each Z' subunit independently represents an α-amino acid having a neutral aralkyl side chain; The peptide is (X'Y'X') p , (X'Y') m , and (X'Z'Z') p wherein p is 2 to 5 and m is 2 to 8.

[0204] In selected embodiments, for each X', the side chain moiety is guanidyl, such as the amino acid subunit arginine (Arg). In further embodiments, each Y' is -CO-(CH) n -CHR-NH-, where n is 2 to 7 and R is H. For example, when n is 5 and R is H, Y' is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx (or simply X), and when n is 2 and R is H, Y' is a β-alanine subunit (referred to herein as B).

[0205] In certain embodiments, peptides of this type include peptides comprising arginine dimers alternating with a single Y' subunit, where Y' is Ahx. Examples include peptides of the formula (RY'R): p or formula (RRY') p wherein Y' is Ahx. In one embodiment, Y' is a 6-aminohexanoic acid subunit, R is arginine, and p is 4.

[0206] In a further embodiment, each Z' is phenylalanine and m is 3 or 4.

[0207] In some embodiments, the conjugated peptide is linked to the terminus of the oligomer via the linker Ahx-B, where Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit.

[0208] In selected embodiments, for each X', the side chain moiety is independently selected from the group consisting of guanidyl (HN=C(NH2)NH-), amidinyl (HN=C(NH2)C-), 2-aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl, and is preferably selected from guanidyl and amidinyl. In one embodiment, the side chain moiety is guanidyl, as in the amino acid subunit arginine (Arg(R)).

[0209] In some embodiments, the Y' subunits are contiguous in that no X' subunits are interposed between the Y' subunits or are singly interspersed between the X' subunits. However, in some embodiments, linking subunits may be present between the Y' subunits. In one embodiment, the Y' subunits are at the termini of the cell membrane-penetrating peptide moiety, while in other embodiments, they are adjacent to the X' subunits. In further embodiments, each Y' is -CO-(CH2) n‐CHR-NH-, where n is 2-7 and R is H. For example, when n is 5 and R is H, Y' is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx. In selected embodiments of this group, each X' contains a guanidyl side chain moiety, such as an arginine subunit. Exemplary peptides of this type include peptides containing arginine dimers alternating with single Y' subunits, where Y' is preferably Ahx. Examples include peptides having the formula (RY'R)4 or (RRY')4, where Y' is preferably Ahx. In some embodiments, the nucleic acid analog is linked to the terminal Y' subunit, preferably at the C-terminus. In other embodiments, the linker is of the structure AhxB, where Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit.

[0210] Cell membrane-penetrating peptide moieties such as those described above have been shown to significantly enhance cellular entry of the attached oligomer relative to uptake of the oligomer in the absence of an attached transport moiety, and relative to uptake by an attached transport moiety lacking the hydrophobic subunit Y'. Such enhanced uptake may be evidenced by at least a two-fold increase, or in other embodiments, a four-fold increase, in mammalian cell uptake of the compound relative to uptake of the drug by an attached transport moiety lacking the hydrophobic subunit Y'. In some embodiments, uptake is enhanced by at least 20-fold and at least 40-fold relative to the unconjugated compound.

[0211] An additional benefit of the cell membrane-permeable peptide moiety is its predicted ability to stabilize the duplex between the antisense oligomer and its target nucleic acid sequence. Without wishing to be bound by theory, this ability to stabilize the duplex may result from electrostatic interactions between the positively charged transport moiety and the negatively charged nucleic acid. In some embodiments, the number of charged subunits in the transporter is less than 14, or in other embodiments, 8 to 11, since too many subunits can result in reduced sequence specificity.

[0212] As mentioned above, the cell membrane penetrating peptides (CPPs) discussed herein within the scope of substituent J have been shown to be effective in enhancing cellular penetration of antisense oligomers and in inducing exon skipping in different muscle groups in animal models.

[0213] Exemplary peptides are shown in Table 3 below. [Table 3]

[0214] VI. Pharmaceutical Compositions The present disclosure also provides for the formulation and delivery of the disclosed oligonucleotides. Accordingly, one aspect of the present disclosure is a pharmaceutical composition comprising an oligonucleotide disclosed herein and a pharmaceutically acceptable carrier.

[0215] Effective delivery of oligonucleotide to target nucleic acid is an important aspect of treatment.Oligonucleotide delivery route includes, but is not limited to, oral and parenteral route, for example, intravenous, subcutaneous, intraperitoneal and intramuscular, and various systemic routes, including inhalation, transdermal and topical delivery.Appropriate route can be determined by those skilled in the art according to the condition of the subject being treated.

[0216] Antisense oligonucleotides can be administered in any convenient vehicle that is physiologically and / or pharmaceutically acceptable.Such compositions can contain any of a variety of standard pharmaceutically acceptable carriers used by those skilled in the art.Examples include, but are not limited to, saline, phosphate buffered saline (PBS), water (e.g., sterile water for injection), aqueous ethanol, emulsions such as oil / water emulsions or triglyceride emulsions, tablets, and capsules.The selection of suitable physiologically acceptable carriers depends on the selected administration method.

[0217] The present compounds (e.g., oligonucleotides) can generally be used as free acids or free bases. Alternatively, the present compounds can be used in the form of acid addition salts or base addition salts. Acid addition salts of free amino compounds can be prepared by methods well known in the art and can be formed from organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, and benzenesulfonic acid. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid. Base addition salts include salts formed with carboxylate anions, and include salts formed with organic and inorganic cations such as those selected from alkali metals and alkaline earth metals (e.g., lithium, sodium, potassium, magnesium, barium, and calcium), and ammonium ions and their substituted derivatives (e.g., dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, etc.). Thus, the term "pharmaceutically acceptable salts" of Formula I is intended to encompass any and all acceptable salt forms.

[0218] In addition, prodrugs are also included within the context of the present invention. A prodrug is any covalently bonded carrier that releases a compound of Formula I in vivo when such a prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in such a way that the modification is cleaved, either by routine manipulation or in vivo, to yield the parent compound. Prodrugs include, for example, compounds of the present invention in which a hydroxyl, amine, or sulfhydryl group is bonded to any group that cleaves to form a hydroxyl, amine, or sulfhydryl group when administered to a patient. Thus, representative examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in compounds of Formula I. Furthermore, in the case of carboxylic acids (—COOH), esters such as methyl esters and ethyl esters may be used.

[0219] VI. Preparation Method Preparation of oligomers with basic nitrogenous internucleotide linkers Morpholino subunits, modified intersubunit linkages, and oligomers containing them can be prepared as described, for example, in U.S. Patent Nos. 5,185,444 and 7,943,762, which are incorporated by reference in their entireties. Morpholino subunits can be prepared according to the following General Reaction Scheme 1.

[0220] Reaction Scheme 1. Preparation of Morpholino Subunits [ka]

[0221] Referring to Reaction Scheme 1, where B represents the base-pairing moiety and PG represents the protecting group, the morpholino subunit can be prepared from the corresponding ribonucleoside (1) as shown. The morpholino subunit (2) can be optionally protected by reaction with a suitable protecting group precursor, such as trityl chloride. The 3' protecting group is generally removed during solid-phase oligomer synthesis, as described in more detail below. The base-pairing moiety can be suitably protected for solid-phase oligomer synthesis. Suitable protecting groups include benzoyl for adenine and cytosine, phenylacetyl for guanine, and pivaloyloxymethyl for hypoxanthine (I). The pivaloyloxymethyl group can be introduced at the N1 position of the hypoxanthine heterocyclic base. It was used without isolation in the preparation. While unprotected hypoxanthine subunits can be used, the yield in the activation reaction is much better when the base is protected. Other suitable protecting groups include those disclosed in US Pat. No. 8,076,476, which is incorporated herein by reference in its entirety.

[0222] Reaction of 3 with activated phosphorus compound 4 yields a morpholino subunit bearing the desired linking moiety 5. Compounds of structure 4 can be prepared using any number of methods known to those skilled in the art. For example, such compounds can be prepared by reaction of the corresponding amine and phosphorus oxychloride. In this regard, the amine starting material can be prepared using any method known in the art, such as the methods described in the Examples and U.S. Pat. No. 7,943,762.

[0223] Compounds of structure 5 can be used in solid-phase automated oligomer synthesis to prepare oligomers containing intersubunit linkages. Such methods are well known in the art. Briefly, compounds of structure 5 can be modified at the 5' end to contain a linker to a solid support. For example, compound 5 can be linked to a solid support by a linker. Once supported, the protecting group (e.g., trityl) is removed, and the free amine is reacted with the activated phosphorus moiety of a second compound of structure 5. This sequence is repeated until an oligo of the desired length is obtained. The protecting group at the 3' end of the terminal can either be removed or left in place if a 3' modification is desired.

[0224] The preparation of modified morpholino subunits and morpholino oligomers is described in more detail in the Examples. Morpholino oligomers containing any number of modified linkages can be prepared using methods described herein, known in the art, and / or methods described herein by reference. Also described in the Examples are overall modifications of morpholino oligomers prepared as previously described (see, e.g., PCT Publication No. 2008 / 036127).

[0225] The synthesis of PMOs, PMO+, PPMOs, and PMO-X containing the additional linkage modifications described herein can be carried out using methods known in the art and are described in pending U.S. Patent Nos. 8,299,206 and 8,076,476, and PCT Publication Nos. 2009 / 064471, 2011 / 150408, and 2012 / 150960, which are incorporated herein by reference in their entireties.

[0226] PMOs with 3' trityl modifications are synthesized essentially as described in PCT Publication No. 2009 / 064471, except that the detritylation step is omitted.

[0227] VII. Treatment method Provided herein are methods for treating neuromuscular diseases. The methods comprise administering to a patient in need thereof a therapeutically effective amount of an oligonucleotide conjugate disclosed herein or a pharmaceutical composition thereof. In one embodiment, the neuromuscular disease is Duchenne muscular dystrophy.

[0228] In certain embodiments, the method is an in vitro method. In certain other embodiments, the method is an in vivo method.

[0229] In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is a non-human primate cell. In certain embodiments, the host cell is a human cell.

[0230] In certain embodiments, the host cell is a naturally occurring cell. In certain other embodiments, the host cell is an engineered cell.

[0231] In certain embodiments, the conjugate is administered in a suitable pharmaceutical carrier to a mammalian subject, such as a human or a laboratory or domestic animal.

[0232] In certain embodiments, the conjugate is administered to a mammalian subject, e.g., a human or an experimental or domestic animal, together with an additional agent. The conjugate and the additional agent may be administered simultaneously or sequentially via the same or different routes and / or sites of administration. In certain embodiments, the conjugate and the additional agent may be co-formulated and administered together. In certain embodiments, the conjugate and the additional agent may be provided together in a kit.

[0233] In one embodiment, the oligonucleotide is a phosphorodiamidate morpholino oligomer contained in a pharmaceutically acceptable carrier and delivered intramuscularly. In another embodiment, the oligonucleotide is a peptide-conjugated phosphorodiamidate morpholino oligomer contained in a pharmaceutically acceptable carrier and delivered intramuscularly.

[0234] In another embodiment, the oligonucleotide is a phosphorodiamidate morpholino oligomer contained in a pharmaceutically acceptable carrier and delivered intravenously (iv). In another embodiment, the oligonucleotide is a peptide-conjugated phosphorodiamidate morpholino oligomer contained in a pharmaceutically acceptable carrier and delivered intravenously.

[0235] Additional routes of administration, such as oral, subcutaneous, intraperitoneal, and intrapulmonary, are also contemplated by the present disclosure.

[0236] In another application of the method, the subject is a livestock animal, such as a pig, cow, or goat, and the treatment is either prophylactic or therapeutic. Also contemplated is an improvement in a method of feeding livestock food, wherein the food is supplemented with an effective amount of the conjugate composition described above.

[0237] In one embodiment, the conjugate is administered in an amount and manner effective to provide a peak blood concentration of the conjugate of at least 200 nM. In one embodiment, the conjugate is administered in an amount and manner effective to provide a peak plasma concentration of the conjugate of at least 200 nM. In one embodiment, the conjugate is administered in an amount and manner effective to provide a peak serum concentration of the conjugate of at least 200 nM.

[0238] In one embodiment, the conjugate is administered in an amount and manner effective to provide a peak blood concentration of the conjugate of at least 400 nM. In one embodiment, the conjugate is administered in an amount and manner effective to provide a peak plasma concentration of the conjugate of at least 400 nM. In one embodiment, the conjugate is administered in an amount and manner effective to provide a peak serum concentration of the conjugate of at least 400 nM.

[0239] Typically, one or more doses of the conjugate are administered, generally at regular intervals, for a period of about 1 to 2 weeks. A preferred dose for oral administration is about 0.01 to 15 mg of conjugate per kg of body weight. In some cases, more than 15 mg / kg of conjugate / kg may be required. For intravenous (iv) administration, a preferred dose is about 0.005 to 15 mg of conjugate per kg of body weight. The conjugate may be administered at regular intervals for a short period of time, for example, daily for up to two weeks. However, in some cases, the conjugate is administered intermittently for a longer period of time. Administration may be followed or accompanied by the administration of antibiotics or other therapeutic treatments. The treatment regimen may be adjusted (dosage, frequency, route, etc.) as indicated based on the results of immunoassays, other biochemical tests, and physiological tests of the subject being treated.

[0240] Effective in vivo therapeutic regimens using conjugates can vary depending on the duration, dose, frequency, and route of administration, as well as the condition of the subject being treated (i.e., prophylactic administration versus administration in response to a local or systemic infection). Thus, such in vivo therapies often require laboratory monitoring during treatment and corresponding adjustments of the dose or therapeutic regimen to achieve optimal therapeutic outcomes.

[0241] In some embodiments, the conjugate is actively taken up by mammalian cells. In further embodiments, the conjugate can be conjugated to a transport moiety (e.g., a transport peptide) described herein to facilitate such uptake.

[0242] Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antisense oligonucleotide, or a pharmaceutical composition of an antisense oligonucleotide. In one embodiment, the disease is a neuromuscular disease. In a further embodiment, The neuromuscular disease is Duchenne muscular dystrophy.

[0243] VIII. Methods for identifying targeting sequences Provided herein is a method for identifying a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene, wherein the target region is within an exon of a human dystrophin pre-mRNA, the exon being flanked by a slow intron at a 5' splice site upstream of the exon, the slow intron being an intron that is retained for a longer period in the dystrophin pre-mRNA compared to the average retention time of the intron in the dystrophin pre-mRNA downstream of the slow intron. The targeting sequence provided herein allows for higher exon skipping efficiency than when the 3' splice site downstream of the exon is flanked by a slow intron or a fast intron, and the fast intron is an intron that is retained for a shorter period in the dystrophin pre-mRNA compared to the average retention time of the intron in the dystrophin pre-mRNA upstream of the intron.

[0244] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Example]

[0245] Examples are described below for illustrative purposes to describe certain specific embodiments of the present disclosure. However, the scope of the claims is in no way limited by the examples described herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications, including but not limited to, those relating to the chemical structures, substituents, derivatives, formulations or methods of the present disclosure, can be made without departing from the spirit of the present disclosure and the scope of the appended claims. The definitions of variables in the structures in the schemes herein are equivalent to those of the corresponding positions in the formulas presented herein.

[0246] Example 1 - Methods and Materials Electroporation For delivery of PMOs to myoblasts, we used the Lonza Amaxa 4D nucleofector with X units, as previously described. 14 Briefly, cells were trypsinized and plated at 1 x 10 cells per 20 μl of buffer. 6 Cells were resuspended in Nucleofection P1 buffer at a concentration of 100 μM. 20 μl of the P1 cell suspension was then transferred to each well of a 16-well Nucleofection cuvette, after which 1 μl of 1 mM PMO was added to the well for a final concentration of 50 μM. Cells were electroporated at 100 μM using program CM-137 and allowed to recover for 10 minutes at room temperature (RT). After recovery, cells were carefully resuspended in 200 μl of growth medium and transferred to a 6-well plate containing equilibrated growth medium. Cells were grown for at least 48 hours, after which confluent cultures were differentiated into myotubes for 72 hours as described.

[0247] RNA isolation and cDNA synthesis For RNA isolation, myotubes from each well were lysed in 500 μl of Tri-Sure lysis reagent, followed by the addition of 200 μl of chloroform and phase separation by centrifugation at 16,200 relative centrifugal force (RCF) for 15 minutes at 4°C. The aqueous phase was transferred to 500 μl of 2-propanol and the RNA was precipitated by centrifugation at 16,200 RCF for 15 minutes at 4°C. The pellet was washed twice with 70% ethanol, air-dried, and resuspended in 25 μl of RNAse-free Milli-Q (MQ). RNA concentration and purity were determined using an ND-1000 Nanodrop (Thermo Scientific) and analyzed by A. 260 / A 230 and A 260 / A 280 The ratios were provided as 1:1. For cDNA synthesis, 1 μg of total RNA was used in a 20 μl reaction using BioScript Tetro (Biotin BIO-65050) according to the manufacturer's instructions. Samples were diluted with MQ to a final volume of 100 μl.

[0248] RT-PCR exon skipping analysis RT-PCR analysis was used to determine the efficiency of DMD exon skipping. Ten percent of the cDNA generated per reaction was used with a specific set of intron-spanning primers (see Table 4A) and DreamTaq polymerase (Thermo Scientific EP0713). A total of 25 μl of reaction consisted of 2.5 μl of 10× reaction buffer (green), 0.2 μl of DreamTaq polymerase (1 unit), 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, 1 μl of DNTP mix (10 μM per nucleotide), and 9.3 μl of MQ. Amplification was performed in a T-100 thermal cycler (Bio-Rad) using the following parameters: 1: 95°C, 2 min; 2: 95°C, 30 sec; 3: 60°C, 30 sec; 4: 72°C, 45 sec; 5: Proceed to step 2, 34 additional cycles; 6: 72°C, 5 min. Fractions from each sample were analyzed using standard agarose-TRIS-borate-EDTA (TBE) gel electrophoresis. DNA content in PCR samples was assessed using Qubit dsDNA broad-range reagents measured on a Spectramax ID3 instrument. Samples were diluted to 0.2 ng / μl and analyzed using an Agilent Femto Pulse with NGS separation gels. Peaks were called using the accompanying Prosize Data Analysis software, version 4.0.2.7. Skipping efficiency was calculated by determining the ratio of the skipped product concentration (nmoles / L) to the total concentration (nmoles / L) of the sum of full-length and skipped products. Statistical analysis was performed using GraphPad Prism 8. [Table 4A]

[0249] RT-qPCR analysis For gene expression analysis, 2% of the cDNA samples generated above were used per reaction. RT-qPCR reactions consisted of 4 μl of SensiMix 2x SYBR Master Mix (Biotin QT605-05), 2 μl of cDNA, 1 μl of forward primer (10 μM), and 1 μl of reverse primer (10 μM) (see Table 4B). Each sample / primer combination was measured in technical triplicate. Samples were manually pipetted into 384-well plates (Framestar 480 / 384, 4ti-0381 4titude) and run in a CFX-384 Real-time PCR system (Bio-Rad). Cycle conditions were as follows: 1: 95°C, 5:00; 2: 95°C, 0:10; 3: 60°C, 0:30 (plate read); 4: Proceed to step 2, 39 additional cycles; 5: Melt curve from 60°C to 95°C, 0.05°C increments per cycle. Data were analyzed using CFX-Maestro software version 2.0, with baseline and Cq value calculations set to "automatic calculation mode." CFX-Maestro was also used to determine run quality by studying the melt curves generated for each product, as well as the internal QC function. Expression values ​​were normalized to the housekeeping genes GAPDH and GUSB using the ΔΔCt method, followed by statistical analysis and visualization using Graphpad Prism version 8. [Table 4B]

[0250] Example 2 - Oligonucleotide Design PMOs were designed to specifically target selected DMD exons while exhibiting similar physical properties, such as G / C content, nucleotide number, and melting temperature. RNAstructure 6.2 was used to determine the free energies of potential secondary structures (intramolecular and homodimer formation). Potential 24 / 25-mer antisense oligonucleotides targeting each of the 47 DMD exons outlined below were designed and analyzed (a total of approximately 11,213 antisense oligonucleotides). The average guanine / cytosine (G / C) content and melting temperature were calculated for the entire set, and antisense oligonucleotides that deviated by more than 1-fold standard deviation were excluded. Exons with fewer than 25 potential antisense oligonucleotides remaining were excluded from further consideration.

[0251] To prevent efficiency bias from the physical properties of antisense oligonucleotides (AONs), we designed a panel of PMOs with similar physical properties, such as nucleotide number, GC content, melting temperature, and potential free energy for forming secondary structures.

[0252] All possible 24 / 25-mer AONs were considered for eligible exons, resulting in 11,213 potential AONs. After calculating the average GC content and melting temperature, only PMOs that were within 1 SD of the average GC content and melting temperature were considered. After removing AONs with unfavorable predicted free energies and secondary structures, sets of PMOs were selected for exons 17, 21, and 70 (5'Slow + 3'Slow), 18, 22, and 50 (5'Slow + 3'Fast), 57, 65, and 67 (5'Fast + 3'Slow), and 51, 55, and 59 (5'Fast + 3'Fast) (Figure 1A, Table 5A), with larger exons covered by a larger number of PMOs (approximately 1 PMO per 30 nucleotides). Overlap between PMO target sequences was avoided as much as possible. [Table 5A-1] [Table 5A-2] [Table 5A-3] [Table 5A-4]

[0253] Example 3 - Optimization of PMO delivery to myotube cultures To facilitate reliable and reproducible delivery of PMOs into control myoblast cell lines, electroporation in an Amaxa 4D Nucleofector X unit was used with 16-well nucleofector cuvette strips (Aung-Htut, MT, McIntosh, CS, West, KA, Fletcher, S. & Wilton, SD. In Vitro Validation of Phosphorodiamidate Morpholino Oligomers. Molecules (Basel, Switzerland) 24, doi:10.3390 / molecules24162922 (2019)). Delivery of PMOs was validated using this system in cell lines with a single exon 51 targeting PMO using various buffer systems and pulse programs. The condition with the highest detectable skipping of exon 51 was selected using RT-qPCR (Figure 2A), and this condition (buffer set P1, pulse program CM-137) was used for the remainder of the project. Electroporation of myoblasts still allowed differentiation into DMD-expressing myotubes, as determined by RT-qPCR of the myogenic markers MYOG and MYH3 (Figure 2A). The top method used for semi-quantification of exon-skipping PCR products was the Agilent Bioanalyzer 2100 using a DNA-1000 chip. To facilitate the large volume of generated samples, Bioanalyzer 2100 data was compared with data generated by the Agilent Femto Pulse system, which allows for higher automated sample throughput. Running the same RT-PCR samples on both systems showed no discernible differences in estimated exon-skipping efficiency (Figure 2B).

[0254] Example 4 - Exons with slow 5'-introns are more likely to be skippable A set of PMOs targeting the 12 selected exons outlined above was electroporated into HC myoblasts, and the cells were differentiated into myotubes. After RT-PCR and analysis of exon skipping efficiency (Figure 3), clear and statistically significant differences in skipping efficiency were observed between different exon classes (Figure 1B). The 5'Slow-'3Slow and 5'Slow-3'Fast exon classes showed higher mean skipping efficiencies than the 5'Fast-3'Slow and 5'Fast-3'Fast exon classes. No significant differences were observed between 5'Slow-3'Slow and 5'Slow-3'Fast, or between 5'Fast-3'Slow and 5'Fast-3'Fast. Confirming this, when we reanalyzed the data and grouped exons based solely on the splicing rates of their 5'- or 3'-flanking introns, we observed significant differences in skipping efficiency only when grouping by splicing rate of the 5'-intron (Fig. 1C), but not for the 3'-intron (Fig. 1D). Together, the data indicate that longer retention of upstream introns has a positive effect on exon skipping efficiency.

[0255] Example 5 – AONs targeting 5' regions within exons are more efficient To test whether optimal targets within exons were determined by the presence of slow introns (5' or 3'), another set of PMOs was designed using only the 5'Slow-3'Fast (exons 10, 14, 18, 22, 42, and 53) and 5'Fast-3'Slow (exons 9, 27, 52, 57, 65, and 67) exon classes (Figure 4A, Table 5B). Eight AONs per exon were desired: four AONs in the proximal 30% and four AONs in the distal 30% of the exon. Using these requirements, it was not possible to select only out-of-frame exons while still adhering to the PMO similarity design criteria. Therefore, six DMD exons were selected for each class from both out-of-frame exons (exons 18, 22, 52, 53, 57, 65, and 67) and in-frame exons (exons 9, 10, 14, 27, and 42).

[0256] After delivery of this set of PMOs to HC myoblasts and differentiation into DMD-expressing myotubes, the skipping efficiency of each PMO was analyzed as previously described using the appropriate RT-PCR primer set for each target exon. The relative binding position of each AON was calculated as the most proximal or distal exon position possible for the 25-mer AON on an arbitrary 1-100 scale, and the skipping efficiency was plotted on this coordinate for the corresponding PMO. When analyzing the skipping efficiency of PMOs in the 5'Slow-3'Fast category (Figure 4B) or the 5'Fast-3'Slow category (Figure 4C), a trend was observed for almost all exons tested, with higher skipping efficiency the closer to the 5' end of the exon. This was confirmed by regression analysis of the skipping efficiency of each exon, which showed a negative slope for all skippable exons (Figures 4B, 4C, and 5). Exons that do not meet this observation are exons 65 and 10, which did not show skipping for any of the individual PMOs tested (Figures 5 and 6).

[0257] This correlation between AON targeting site and skipping efficiency was also observed when combining data from all PMOs used in this study (Figures 1A-1D and 4A-4D) and when the regression analysis was plotted (Figure 4D). A significant negative trend was observed toward the 3' end of the exon, indicating that AONs targeting the 5' end of the exon are generally more likely to be effective, regardless of the splicing dynamics of the adjacent intron.

[0258] Out-of-frame transcripts can be unstable and are degraded by nonsense-mediated mRNA decay (NMD), a cellular mechanism used to degrade mRNA molecules containing premature stop codons. NMD degradation can introduce bias when targeting both in-frame and out-of-frame exons. Indeed, AONs targeting in-frame exons appear to have a tendency to have higher skipping efficiency (Figure 5D). [Table 5B-1] [Table 5B-2] [Table 5B-3] [Table 5B-4] [Table 5B-5]

[0259] Example 6 – Exon skipping can lead to transcript loss in control cells We investigated whether the low levels of exon skipping observed in control myotubes for exons 51 and 53 could be partially explained by the fact that these experiments generated out-of-frame DMD transcripts that could be targets of NMD. PMO-targeted sets of exon 51 or exon 53 were electroporated into two DMD patient-derived cell lines. These cells harbor a deletion of DMD exons 48–50 (line 8036) (Mamchaoui, K. et al. Skeleton Muscle 1, 34, doi:10.1186 / 2044-5040-1-34 (2011)) or exons 45–52 (line 6311) (Echigoya, Y. et al. Molecular Therapy 27, 2005–2017, doi:https: / / doi.org / 10.1016 / j.ymthe.2019.07.012 (2019)), and they are suitable for restoring the reading frame by skipping exon 51 or exon 53, respectively. When we compared the exon skipping levels observed in controls for exon 51 (Figure 7A) or exon 53 (Figure 7B) with the respective DMD cell lines (Figures 7C and 7D), it was clear that the majority of AON targeting resulted in higher exon skipping levels in DMD patient-derived cell lines. This indicated that the detectable level of exon skipping in control cells may be underestimated due to degradation of the skipped transcript. However, the trend in AON efficiency remained the same between control and patient cells, indicating that relative skipping efficiency can still be estimated by skipping out-of-frame exons in control cells. This is also suggested by comparing the levels of DMD transcripts measured by RT-qPCR using primers upstream and downstream of the targeted exons (Figure 8). As an example, in control cells, more efficient AONs skipping exon 51 (Figure 7A), such as PMO-027, -147, and -281, were observed to show reduced transcript levels when measuring exons 55-56 (Figure 8A) in these samples, suggesting a correlation between AON efficacy and transcript loss.This effect was not observed for exon 53 (Fig. 8B).

[0260] Example 7 – Nonsense-mediated mRNA decay can affect detectable skipping efficiency Skipping of exons 51 and 53 was more efficient in DMD patient cells, with exon skipping restoring the reading frame more efficiently than in HC cells, whereas exon skipping inhibits the reading frame. To test whether NMD plays a role in this observation, we nucleofected both HC and DMD muscle cells with PMOs and then treated the cells with cycloheximide (CHX), an inhibitor of NMD. As proof of principle, we included samples nucleofected with an equimolar mixture of all 12 available exon 65 targeting PMOs at the same terminal concentration (Figure 9A). While mixing the 12 PMOs resulted in minor levels of skipped exon 65, none of the individual PMOs resulted in detectable levels of exon skipping. Treatment of nucleofected HC cells with CHX caused an increase in the detected skipped DMD transcripts, indicating that NMD may degrade the skipped products, resulting in an underestimation of the efficiency of out-of-frame exon skipping. However, this effect was less pronounced in DMD exon Δ45-52 cells and completely absent in DMD exon Δ48-50 strains. RT-qPCR of the DMD gene in these samples showed a similar stabilizing effect on DMD transcripts, with a slight increase in the total expression level detected (Figure 9B). Similarly, exon skipping of exons 51 and 53 in HC (Figures 9C and 9D) and two DMD strains (Figures 9E and 9F) and treatment with CHX yielded inconclusive results. HC cells showed a slight increase in detectable skipped DMD after CHX, whereas DMD cells showed a detectable decrease in skipping efficiency for some PMOs.

Claims

1. 1. A modified antisense oligonucleotide, comprising: the modified antisense oligonucleotide is 18 to 40 subunits in length and comprises a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene; the modified antisense oligonucleotide comprises a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2'O-Me-phosphorothioate oligomer, or any combination of the foregoing; each subunit taken together from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide forms the targeting sequence; 1. A modified antisense oligonucleotide, wherein the target region is within an exon of a human dystrophin pre-mRNA, the exon being flanked at a 5' splice site upstream of the exon by a slow intron, the slow intron being an intron that is retained for a longer period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA that are downstream of the slow intron.

2. 2. The modified antisense oligonucleotide of claim 1, wherein the downstream 3' splice site of the exon is adjacent to a slow intron or a fast intron, the fast intron being an intron that is retained for a shorter period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA upstream of the intron.

3. The modified antisense oligonucleotide of claim 1 or 2, wherein the modified antisense oligonucleotide is 24 to 25 subunits.

4. The modified antisense oligonucleotide of any one of claims 1 to 3, wherein the modified antisense oligonucleotide comprises a guanine / cytosine content of 40-60%.

5. The modified antisense oligonucleotide of any one of claims 1 to 4, wherein the modified antisense oligonucleotide has a melting temperature of about 64 to 75°C.

6. 6. The modified antisense oligonucleotide of claim 1, wherein the exon is selected from exon 10, exon 14, exon 17, exon 18, exon 21, exon 22, exon 42, exon 50, exon 53, and exon 70.

7. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 10.

8. the targeting sequence is SEQ ID NO: 1 ACTTGTCTTCAGGAGCTTCCAAATG SEQ ID NO: 2 AAATGACTTGTCTTCAGGAGCTTCC SEQ ID NO: 3 CTGCCAAATGACTTGTCTTCAGGAG SEQ ID NO: 4 CTCCATCAATGAACTGCCAAATGAC SEQ ID NO: 5 CTCTCCTTGTGCTTGCAATGTGTCC SEQ ID NO: 6 GAAATCTCTCCTTGTGCTTGCAATG SEQ ID NO: 7 ACTGGTCTTTCACCACTTCCACATC SEQ ID NO: 8 ATGAAACTGGTCTTTCACCACTTCC The modified antisense oligonucleotide of any one of claims 1 to 7, comprising or consisting of a sequence selected from:

9. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 14.

10. the targeting sequence is SEQ ID NO: 9 TGTTTGCCCATCGATCTCCCAATAC SEQ ID NO: 10 TACAGATGTTTGCCCATCGATCTCC SEQ ID NO: 11 CCATCTACAGATGTTTGCCCATCGA SEQ ID NO: 12 TCTGTCCATCTACAGATGTTTGCCC SEQ ID NO: 13 ACGTTGCCATTTGAGAAGGATGTCT SEQ ID NO: 14 GTAAGACGTTGCCATTTGAGAAGGA SEQ ID NO: 15 CTTCAGTAAGACGTTGCCATTTGAG SEQ ID NO: 16 CTGTTCTTCAGTAAGACGTTGCCAT 10. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 9, comprising or consisting of a sequence selected from:

11. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 17.

12. the targeting sequence is SEQ ID NO: 17 GAGTGGTGGTGACAGCCTGTGAAAT SEQ ID NO: 18 TCTGTGTTAGTGATGGCTGAGTGGT SEQ ID NO: 19 CCCTTGTGGTCACCGTAGTTACTGT SEQ ID NO: 20 GTTCCTCTTGAGCATGCTTTACCAG SEQ ID NO: 21 ACAGTAATCTGCCTCTTCTTTTGGG SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC 12. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 11, comprising or consisting of a sequence selected from:

13. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 18.

14. the targeting sequence is SEQ ID NO: 23 GCGAGTAATCCAGCTGTGAAGTTCA SEQ ID NO: 24 TCTGAGCGAGTAATCCAGCTGTGAA SEQ ID NO: 25 AGCTTCTGAGCGAGTAATCCAGCTG SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC SEQ ID NO: 27 GCAAATTCAGGACTCTGCAACACAG SEQ ID NO: 28 GCCTTCCTTCCGAAAGATTGCAAAT SEQ ID NO: 29 AGTTGCCTTCCTTCCGAAAGATTGC SEQ ID NO: 30 TGAGAAGTTGCCTTCCTTCCGAAAG SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC 14. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 13, comprising or consisting of a sequence selected from:

15. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 21.

16. the targeting sequence is SEQ ID NO: 32 GATCTGATAGCCGGTTGACTTCATC SEQ ID NO: 33 GTTGAAGATCTGATAGCCGGTTGAC SEQ ID NO: 34 GTCCTTGTCCTTTCTCTTTCAGGGC SEQ ID NO: 35 GAACATGGGTCCTTGTCCTTTCTC SEQ ID NO: 36 TGTAAAGGCCACAAAGTCTGCATCC SEQ ID NO: 37 TCTGGCCTGCACATCAGAAAGACT SEQ ID NO: 38 TGTCTGTAGCTCTTTCTCTCTGGCC.

16. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 15, comprising or consisting of a sequence selected from:

17. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 22.

18. the targeting sequence is SEQ ID NO: 39 CGCATTGGTGGCAAAGTGTCAAAA SEQ ID NO: 40 CTGATAGCGCATTGGTGGCAAAGT SEQ ID NO: 41 ATGGTCTCCTGATAGCGCATTGGTG SEQ ID NO: 42 CACTCATGGTCTCCTGATAGCGCAT SEQ ID NO: 43 CTGATGGCACTCATGGTCTCCTGAT SEQ ID NO: 44 GAGAGTTTGGTTTCTGACTGCTGGA SEQ ID NO: 45 GCTCCATGATTTCATAGTCGGTGAC SEQ ID NO: 46 TCTCTGCTCCATGATTTCATAGTCG SEQ ID NO: 47 CCGAGTCTCTGCTCCATGATTTCAT SEQ ID NO: 48 ATTCCCCGAGTCTCTGCTCCATGAT SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT 18. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 17, comprising or consisting of a sequence selected from:

19. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 42.

20. the targeting sequence is SEQ ID NO: 50 TCATCGTTTCTTCACGGACAGTGTG SEQ ID NO: 51 CACCATCATCGTTTCTTCACGGACA SEQ ID NO: 52 GTCATCACCATCATCGTTTCTTCAC SEQ ID NO: 53 CTTCAGTCATCACCATCATCGTTTC SEQ ID NO: 54 AGCACAGAGGTCAGGAGCATTGAGA SEQ ID NO: 55 TCCTTAGCACAGAGGTCAGGAGCAT SEQ ID NO: 56 CAAAGTCCTTAGCACAGAGGTCAGG SEQ ID NO: 57 ATCTTCAAAGTCCTTAGCACAGAGG 20. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 19, comprising or consisting of a sequence selected from:

21. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 50.

22. the targeting sequence is SEQ ID NO: 58 CCTTCCACTCAGAGCTCAGATCTTC SEQ ID NO: 59 AAGTAAACGGTTTACCGCCTTCCAC SEQ ID NO: 60 CTGCTTTGCCCTCAGCTCTTGAAGT SEQ ID NO: 61 TCCAATAGTGGTCAGTCCAGGAGCT 22. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 21, comprising or consisting of a sequence selected from:

23. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 53.

24. the targeting sequence is SEQ ID NO: 62 CTTGTACTTCATCCCACTGATTCTG SEQ ID NO: 63 GTGTTCTTGTACTTCATCCCACTGA SEQ ID NO: 64 TGAAGGTGTTCTTGTACTTCATCCC SEQ ID NO: 65 CGGTTCTGAAGGTGTTCTTGTACTT SEQ ID NO: 66 CTCCTTCCATGACTCAAGCTTGGCT SEQ ID NO: 67 TATAGGGACCCTCCTTCCATGACTC SEQ ID NO: 68 TACTGTATAGGGACCCTCCTTCCAT SEQ ID NO: 69 TGCATCTACTGTATAGGGACCCTCC 24. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 23, comprising or consisting of a sequence selected from:

25. The modified antisense oligonucleotide of any one of claims 1 to 6, wherein the exon is exon 70.

26. the targeting sequence is SEQ ID NO: 70 GCAAAGTCTCGAACATCTTCTCCTG SEQ ID NO: 71 GTACCTTGGCAAAGTCTCGAACATC SEQ ID NO: 72 GGGGATGCTTCGCAAAAATACCTTT SEQ ID NO: 73 TTGTCCCCCCTCTAAGACAGTCTGCA SEQ ID NO: 74 TTCCATGTTGTCCCCCCTAAGACA 26. The modified antisense oligonucleotide of any one of claims 1 to 6 or claim 25, comprising or consisting of a sequence selected from:

27. The modified antisense oligonucleotide of any one of claims 1 to 24, wherein the target region is the 5' region of an exon.

28. A conjugate comprising a modified antisense oligonucleotide and a cell membrane-permeable peptide, the modified antisense oligonucleotide is 18 to 40 subunits in length and comprises a targeting sequence complementary to a target region of the Duchenne muscular dystrophy (DMD) gene; the modified antisense oligonucleotide comprises a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligomer, a tricyclo-DNA oligomer, a tricyclo-phosphorothioate oligomer, a 2'O-Me-phosphorothioate oligomer, or any combination of the foregoing; each subunit together from the 5' end of the antisense oligonucleotide to the 3' end of the antisense oligonucleotide to form the targeting sequence; 1. A conjugate wherein the target region is within an exon of a human dystrophin pre-mRNA, the exon being flanked at a 5' splice site upstream of the exon by a slow intron, the slow intron being an intron that is retained for a longer period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA downstream of the slow intron.

29. 29. The conjugate of claim 28, wherein the downstream 3' splice site of the exon is adjacent to a slow intron or a fast intron, wherein the fast intron is an intron that is retained for a shorter period in the dystrophin pre-mRNA compared to the average retention time of introns in the dystrophin pre-mRNA upstream of the intron.

30. 30. The conjugate of claim 28 or 29, wherein the modified antisense oligonucleotide comprises 24 to 25 subunits.

31. The conjugate of any one of claims 28 to 30, wherein the modified antisense oligonucleotide comprises 40 to 60% GC content.

32. The conjugate of any one of claims 28 to 31, wherein the modified antisense oligonucleotide has a melting temperature of about 64 to 75°C.

33. 33. The conjugate of any one of claims 28 to 32, wherein the exons are selected from exon 10, exon 14, exon 17, exon 18, exon 21, exon 22, exon 42, exon 50, exon 53, and exon 70.

34. The conjugate of any one of claims 28 to 33, wherein the exon is exon 10.

35. the targeting sequence is SEQ ID NO: 1 ACTTGTCTTCAGGAGCTTCCAAATG SEQ ID NO: 2 AAATGACTTGTCTTCAGGAGCTTCC SEQ ID NO: 3 CTGCCAAATGACTTGTCTTCAGGAG SEQ ID NO: 4 CTCCATCAATGAACTGCCAAATGAC SEQ ID NO: 5 CTCTCCTTGTGCTTGCAATGTGTCC SEQ ID NO: 6 GAAATCTCTCCTTGTGCTTGCAATG SEQ ID NO: 7 ACTGGTCTTTCACCACTTCCACATC SEQ ID NO: 8 ATGAAACTGGTCTTTCACCACTTCC 35. The conjugate of any one of claims 28 to 34, comprising or consisting of a sequence selected from:

36. The conjugate of any one of claims 28 to 33, wherein the exon is exon 14.

37. the targeting sequence is SEQ ID NO: 9 TGTTTGCCCATCGATCTCCCAATAC SEQ ID NO: 10 TACAGATGTTTGCCCATCGATCTCC SEQ ID NO: 11 CCATCTACAGATGTTTGCCCATCGA SEQ ID NO: 12 TCTGTCCATCTACAGATGTTTGCCC SEQ ID NO: 13 ACGTTGCCATTTGAGAAGGATGTCT SEQ ID NO: 14 GTAAGACGTTGCCATTTGAGAAGGA SEQ ID NO: 15 CTTCAGTAAGACGTTGCCATTTGAG SEQ ID NO: 16 CTGTTCTTCAGTAAGACGTTGCCAT 37. The conjugate of any one of claims 28 to 33 or claim 36, comprising or consisting of a sequence selected from:

38. The conjugate of any one of claims 28 to 33, wherein the exon is exon 17.

39. the targeting sequence is SEQ ID NO: 17 GAGTGGTGGTGACAGCCTGTGAAAT SEQ ID NO: 18 TCTGTGTTAGTGATGGCTGAGTGGT SEQ ID NO: 19 CCCTTGTGGTCACCGTAGTTACTGT SEQ ID NO: 20 GTTCCTCTTGAGCATGCTTTACCAG SEQ ID NO: 21 ACAGTAATCTGCCTCTTCTTTTGGG SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC 39. The conjugate of any one of claims 28 to 33 or claim 38, comprising or consisting of a sequence selected from:

40. The conjugate of any one of claims 28 to 33, wherein the exon is exon 18.

41. the targeting sequence is SEQ ID NO: 23 GCGAGTAATCCAGCTGTGAAGTTCA SEQ ID NO: 24 TCTGAGCGAGTAATCCAGCTGTGAA SEQ ID NO: 25 AGCTTCTGAGCGAGTAATCCAGCTG SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC SEQ ID NO: 27 GCAAATTCAGGACTCTGCAACACAG SEQ ID NO: 28 GCCTTCCTTCCGAAAGATTGCAAAT SEQ ID NO: 29 AGTTGCCTTCCTTCCGAAAGATTGC SEQ ID NO: 30 TGAGAAGTTGCCTTCCTTCCGAAAG SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC 41. The conjugate of any one of claims 28 to 33 or claim 40, comprising or consisting of a sequence selected from:

42. The conjugate of any one of claims 28 to 33, wherein the exon is exon 21.

43. the targeting sequence is SEQ ID NO: 32 GATCTGATAGCCGGTTGACTTCATC SEQ ID NO: 33 GTTGAAGATCTGATAGCCGGTTGAC SEQ ID NO: 34 GTCCTTGTCCTTTCTCTTTCAGGGC SEQ ID NO: 35 GAACATGGGTCCTTGTCCTTTCTC SEQ ID NO: 36 TGTAAAGGCCACAAAGTCTGCATCC SEQ ID NO: 37 TCTGGCCTGCACATCAGAAAGACT SEQ ID NO: 38 TGTCTGTAGCTCTTTCTCTCTGGCC 43. The conjugate of any one of claims 28 to 33 or claim 42, comprising or consisting of a sequence selected from:

44. The conjugate of any one of claims 28 to 33, wherein the exon is exon 22.

45. the targeting sequence is SEQ ID NO: 39 CGCATTGGTGGCAAAGTGTCAAAA SEQ ID NO: 40 CTGATAGCGCATTGGTGGCAAAGT SEQ ID NO: 41 ATGGTCTCCTGATAGCGCATTGGTG SEQ ID NO: 42 CACTCATGGTCTCCTGATAGCGCAT SEQ ID NO: 43 CTGATGGCACTCATGGTCTCCTGAT SEQ ID NO: 44 GAGAGTTTGGTTTCTGACTGCTGGA SEQ ID NO: 45 GCTCCATGATTTCATAGTCGGTGAC SEQ ID NO: 46 TCTCTGCTCCATGATTTCATAGTCG SEQ ID NO: 47 CCGAGTCTCTGCTCCATGATTTCAT SEQ ID NO: 48 ATTCCCCGAGTCTCTGCTCCATGAT SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT 45. The conjugate of any one of claims 28 to 33 or claim 44, comprising or consisting of a sequence selected from:

46. The conjugate of any one of claims 28 to 33, wherein the exon is exon 42.

47. the targeting sequence is SEQ ID NO: 50 TCATCGTTTCTTCACGGACAGTGTG SEQ ID NO: 51 CACCATCATCGTTTCTTCACGGACA SEQ ID NO: 52 GTCATCACCATCATCGTTTCTTCAC SEQ ID NO: 53 CTTCAGTCATCACCATCATCGTTTC SEQ ID NO: 54 AGCACAGAGGTCAGGAGCATTGAGA SEQ ID NO: 55 TCCTTAGCACAGAGGTCAGGAGCAT SEQ ID NO: 56 CAAAGTCCTTAGCACAGAGGTCAGG SEQ ID NO: 57 ATCTTCAAAGTCCTTAGCACAGAGG 47. The conjugate of any one of claims 28 to 33 or claim 46, comprising or consisting of a sequence selected from:

48. The conjugate of any one of claims 28 to 33, wherein the exon is exon 50.

49. the targeting sequence is SEQ ID NO: 58 CCTTCCACTCAGAGCTCAGATCTTC SEQ ID NO: 59 AAGTAAACGGTTTACCGCCTTCCAC SEQ ID NO: 60 CTGCTTTGCCCTCAGCTCTTGAAGT SEQ ID NO: 61 TCCAATAGTGGTCAGTCCAGGAGCT 49. The conjugate of any one of claims 28 to 33 or claim 48, comprising or consisting of a sequence selected from:

50. The conjugate of any one of claims 28 to 33, wherein the exon is exon 53.

51. the targeting sequence is SEQ ID NO: 62 CTTGTACTTCATCCCACTGATTCTG SEQ ID NO: 63 GTGTTCTTGTACTTCATCCCACTGA SEQ ID NO: 64 TGAAGGTGTTCTTGTACTTCATCCC SEQ ID NO: 65 CGGTTCTGAAGGTGTTCTTGTACTT SEQ ID NO: 66 CTCCTTCCATGACTCAAGCTTGGCT SEQ ID NO: 67 TATAGGGACCCTCCTTCCATGACTC SEQ ID NO: 68 TACTGTATAGGGACCCTCCTTCCAT SEQ ID NO: 69 TGCATCTACTGTATAGGGACCCTCC 51. The conjugate of any one of claims 28 to 33 or claim 50, comprising or consisting of a sequence selected from:

52. The conjugate of any one of claims 28 to 33, wherein the exon is exon 70.

53. the targeting sequence is SEQ ID NO: 70 GCAAAGTCTCGAACATCTTCTCCTG SEQ ID NO: 71 GTACCTTGGCAAAGTCTCGAACATC SEQ ID NO: 72 GGGGATGCTTCGCAAAAATACCTTT SEQ ID NO: 73 TTGTCCCCCCTCTAAGACAGTCTGCA SEQ ID NO: 74 TTCCATGTTGTCCCCCCTAAGACA 53. The conjugate of any one of claims 28 to 33 or claim 52, comprising or consisting of a sequence selected from:

54. The conjugate of any one of claims 28 to 53, wherein the target region is the 5' region of an exon.

55. The cell membrane penetrating peptide is rTAT, TAT, R ９ F ２ , R ５ F ２ R ４ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , (RXR) ４ , (RXR) ５ , (RXRRBR) ２ , (RAR) ４ F ２ , (RGR) ４ F ２ The conjugate of any one of claims 28 to 54, selected from:

56. Compounds of Formula I: 【Chemistry 65】 (I) or a pharmaceutically acceptable salt thereof; During the ceremony, A' is -N(H)CH ２ C(O)NH ２ , -N(C １－６ -alkyl)CH ２ C(O)NH ２、 【Hua 66】 、 and 【Hua 67】 is selected from: R ５ is —C(O)(O-alkyl) ｘ -OH, wherein x is 3 to 10, and each alkyl group, independently in each occurrence, is selected from the group consisting of: ２－６ - alkyl or or R ５ H, -C(O)C １－６ -Alkyl, trityl, monomethoxytrityl, -(C １－６ -alkyl)-R ６ , -(C １－６ -heteroalkyl)-R ６ , aryl-R ６ , heteroaryl-R ６ , -C(O)O-(C １－６ -alkyl)-R ６ , —C(O)O-aryl-R ６ , —C(O)O-heteroaryl-R ６ , and selected from: 【Chemistry 68】 ； R ６ OH, SH, and NH ２ or R ６ is O, S, or NH, each of which is covalently attached to a solid support; Each R １ is OH and -N(R ３ ) (R ４ ) independently selected from ３ and R ４ is independently in each occurrence H or —C １－６ - alkyl, Each R ２ is independently selected at each occurrence from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein said nucleobase is independently selected at each occurrence from pyridine, pyrimidine, purine, and deaza-purine. ３－６ heterocyclic ring, wherein each R ２ together form the targeting sequence, z is 8 to 40; E' is H, -C １－６ -alkyl, -C(O)C １－６ - alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 【Chemical Formula 69】 、 and 【Chemistry 70】 is selected from During the ceremony, Q is —C(O)(CH ２ ) ６ C(O)- or -C(O)(CH ２ ) ２ S ２ (CH ２ ) ２ C(O)—, R ７ But -(CH ２ ) ２ OC(O)N(R ８ ) ２ wherein R ８ But -(CH ２ ) ６ NHC (=NH)NH ２ and L is glycine, proline, W, WW, or R ９ wherein L is covalently attached by an amide bond to the N-terminus or C-terminus of J; W is —C(O)—(CH ２ ) ｍ -NH-, wherein m is 2 to 12; R ９ but, 【Chemical Formula 71】 、 【Chemical Formula 72】 , and 【Chemical 73】 is selected from the group consisting of n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, or 5; R １０ is selected from a bond, glycine, proline, W, or W-W; R １１ is glycine, proline, W, W-W, and 【Chemical 74】 is selected from the group consisting of R １６ is selected from a bond, glycine, proline, W, or W-W; R １６ is covalently linked by an amide bond to the N-terminus or C-terminus of J, wherein J is a cell membrane penetrating peptide; G is H, -C(O)C １－６ - selected from alkyl, benzoyl, and stearoyl, wherein G is covalently bonded to J; The targeting sequence is SEQ ID NO: 1 ACTTGTCTTCAGGAGCTTCCAAATG SEQ ID NO: 2 AAATGACTTGTCTTCAGGAGCTTCC SEQ ID NO: 3 CTGCCAAATGACTTGTCTTCAGGAG SEQ ID NO: 4 CTCCATCAATGAACTGCCAAATGAC SEQ ID NO: 5 CTCTCCTTGTGCTTGCAATGTGTCC SEQ ID NO: 6 GAAATCTCTCCTTGTGCTTGCAATG SEQ ID NO: 7 ACTGGTCTTTCACCACTTCCACATC SEQ ID NO: 8 ATGAAACTGGTCTTTCACCACTTCC SEQ ID NO: 9 TGTTTGCCCATCGATCTCCCAATAC SEQ ID NO: 10 TACAGATGTTTGCCCATCGATCTCC SEQ ID NO: 11 CCATCTACAGATGTTTGCCCATCGA SEQ ID NO: 12 TCTGTCCATCTACAGATGTTTGCCC SEQ ID NO: 13 ACGTTGCCATTTGAGAAGGATGTCT SEQ ID NO: 14 GTAAGACGTTGCCATTTGAGAAGGA SEQ ID NO: 15 CTTCAGTAAGACGTTGCCATTTGAG SEQ ID NO: 16 CTGTTCTTCAGTAAGACGTTGCCAT SEQ ID NO: 17 GAGTGGTGGTGACAGCCTGTGAAAT SEQ ID NO: 18 TCTGTGTTAGTGATGGCTGAGTGGT SEQ ID NO: 19 CCCTTGTGGTCACCGTAGTTACTGT SEQ ID NO: 20 GTTCCTCTTGAGCATGCTTTACCAG SEQ ID NO: 21 ACAGTAATCTGCCTCTTCTTTTGGG SEQ ID NO: 22 TTCAGAATCCACAGTAATCTGCCTC SEQ ID NO: 23 GCGAGTAATCCAGCTGTGAAGTTCA SEQ ID NO: 24 TCTGAGCGAGTAATCCAGCTGTGAA SEQ ID NO: 25 AGCTTCTGAGCGAGTAATCCAGCTG SEQ ID NO: 26 AACACAGCTTCTGAGCGAGTAATCC SEQ ID NO: 27 GCAAATTCAGGACTCTGCAACACAG SEQ ID NO: 28 GCCTTCCTTCCGAAAGATTGCAAAT SEQ ID NO: 29 AGTTGCCTTCCTTCCGAAAGATTGC SEQ ID NO: 30 TGAGAAGTTGCCTTCCTTCCGAAAG SEQ ID NO: 31 AAGTCTGAGAAGTTGCCTTCCTTCC SEQ ID NO: 32 GATCTGATAGCCGGTTGACTTCATC SEQ ID NO: 33 GTTGAAGATCTGATAGCCGGTTGAC SEQ ID NO: 34 GTCCTTGTCCTTTCTCTTTCAGGGC SEQ ID NO: 35 GAACATGGGTCCTTGTCCTTTCTC SEQ ID NO: 36 TGTAAAGGCCACAAAGTCTGCATCC SEQ ID NO: 37 TCTGGCCTGCACATCAGAAAGACT SEQ ID NO: 38 TGTCTGTAGCTCTTTCTCTCTGGCC SEQ ID NO: 39 CGCATTGGTGGCAAAGTGTCAAAA SEQ ID NO: 40 CTGATAGCGCATTGGTGGCAAAGT SEQ ID NO: 41 ATGGTCTCCTGATAGCGCATTGGTG SEQ ID NO: 42 CACTCATGGTCTCCTGATAGCGCAT SEQ ID NO: 43 CTGATGGCACTCATGGTCTCCTGAT SEQ ID NO: 44 GAGAGTTTGGTTTCTGACTGCTGGA SEQ ID NO: 45 GCTCCATGATTTCATAGTCGGTGAC SEQ ID NO: 46 TCTCTGCTCCATGATTTCATAGTCG SEQ ID NO: 47 CCGAGTCTCTGCTCCATGATTTCAT SEQ ID NO: 48 ATTCCCCGAGTCTCTGCTCCATGAT SEQ ID NO: 49 CAATTCCCCGAGTCTCTGCTCCAT SEQ ID NO: 50 TCATCGTTTCTTCACGGACAGTGTG SEQ ID NO: 51 CACCATCATCGTTTCTTCACGGACA SEQ ID NO: 52 GTCATCACCATCATCGTTTCTTCAC SEQ ID NO: 53 CTTCAGTCATCACCATCATCGTTTC SEQ ID NO: 54 AGCACAGAGGTCAGGAGCATTGAGA SEQ ID NO: 55 TCCTTAGCACAGAGGTCAGGAGCAT SEQ ID NO: 56 CAAAGTCCTTAGCACAGAGGTCAGG SEQ ID NO: 57 ATCTTCAAAGTCCTTAGCACAGAGG SEQ ID NO: 58 CCTTCCACTCAGAGCTCAGATCTTC SEQ ID NO: 59 AAGTAAACGGTTTACCGCCTTCCAC SEQ ID NO: 60 CTGCTTTGCCCTCAGCTCTTGAAGT SEQ ID NO: 61 TCCAATAGTGGTCAGTCCAGGAGCT SEQ ID NO: 62 CTTGTACTTCATCCCACTGATTCTG SEQ ID NO: 63 GTGTTCTTGTACTTCATCCCACTGA SEQ ID NO: 64 TGAAGGTGTTCTTGTACTTCATCCC SEQ ID NO: 65 CGGTTCTGAAGGTGTTCTTGTACTT SEQ ID NO: 66 CTCCTTCCATGACTCAAGCTTGGCT SEQ ID NO: 67 TATAGGGACCCTCCTTCCATGACTC SEQ ID NO: 68 TACTGTATAGGGACCCTCCTTCCAT SEQ ID NO: 69 TGCATCTACTGTATAGGGACCCTCC SEQ ID NO: 70 GCAAAGTCTCGAACATCTTCTCCTG SEQ ID NO: 71 GTACCTTGGCAAAGTCTCGAACATC SEQ ID NO: 72 GGGGATGCTTCGCAAAAATACCTTT SEQ ID NO: 73 TTGTCCCCCCTCTAAGACAGTCTGCA SEQ ID NO: 74 TTCCATGTTGTCCCCCCTAAGACA A phosphorodiamidate morpholino oligomer comprising or consisting of a sequence selected from:

57. E' is H, -C １－６ -alkyl, -C(O)C １－６ - alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and 【Chemistry 75】 57. The phosphorodiamidate morpholino oligomer of claim 56, selected from:

58. A' is -N(C １－６ -alkyl)CH ２ C(O)NH ２ , 【Chemical 76】 、 [[IDID=0]]【Chemical 77】 、 and 【Chemical 78】 58. The phosphorodiamidate morpholino oligomer of claim 56 or 57, selected from:

59. E' is H, -C(O)CH ３ , benzoyl, stearoyl, trityl, 4-methoxytrityl, and 【Chemical 79】 59. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 58, selected from:

60. A' is -N(C １－６ -alkyl)CH ２ C(O)NH ２ , 【Chemistry 80】 、 and 【Chemistry 81】 is selected from E' is 【Chemistry 82】 The phosphorodiamidate morpholino oligomer according to any one of claims 56 to 59,

61. A' is 【Chemistry 83】 and E' is H, -C(O)CH ３ 60. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 59, wherein the aryl group is selected from the group consisting of aryl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.

62. The compound of formula I is 【Chemistry 84】 (Ia); and 【Chemistry 85】 (Ib) In the formula, E' is H, C １－６ -Alkyl, -C(O)CH ３ 57. The phosphorodiamidate morpholino oligomer of claim 56, wherein the aryl group is selected from the group consisting of benzoyl, benzoyl, and stearoyl.

63. 63. The phosphorodiamidate morpholino oligomer of claim 62, wherein the compound is a compound of formula (Ia):

64. 63. The phosphorodiamidate morpholino oligomer of claim 62, wherein the compound is a compound of formula (Ib):

65. Each R １ But -N(CH ３ ) ２ The phosphorodiamidate morpholino oligomer of any one of claims 56 to 64, wherein

66. 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, wherein L is glycine.

67. 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, wherein L is proline.

68. L is —C(O)—(CH ２ ) ５ 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, which is -NH-.

69. L is —C(O)—(CH ２ ) ２ 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, which is -NH-.

70. L is —C(O)—(CH ２ ) ２ -NH-C(O)-(CH ２ ) ５ 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, which is -NH-.

71. L, 【Chemistry 86】 and R １０ is a bond, and R １１ However, glycine and 【Hua 87】 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, selected from:

72. L, 【Hua 88】 and R １０ is a bond, and R １１ However, glycine and 【Chemistry 89】 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, selected from:

73. L, 【Chemistry 90】 and R １０ is a bond, and R １１ However, glycine and 【Chemistry 91】 66. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 65, selected from:

74. J is rTAT, TAT, R ９ F ２ , R ５ F ２ R ４ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , (RXR) ４ , (RXR) ５ , (RXRRBR) ２ , (RAR) ４ F ２ , (RGR) ４ F ２ 74. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 73, selected from:

75. G is H, C(O)CH ３ , benzoyl, and stearoyl.

76. G is H or —C(O)CH ３ The phosphorodiamidate morpholino oligomer according to any one of claims 56 to 75,

77. 77. The phosphorodiamidate morpholino oligomer of any one of claims 56 to 76, wherein G is H.

78. G is —C(O)CH ３ The phosphorodiamidate morpholino oligomer according to any one of claims 56 to 76,

79. A pharmaceutical composition comprising the antisense oligonucleotide of any one of claims 1 to 55, the phosphorodiamidate morpholino oligomer of claims 56 to 78, a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

80. 81. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antisense oligonucleotide of any one of claims 1 to 55, or the phosphorodiamidate morpholino oligomer of claims 56 to 78, or the pharmaceutical composition of claim 80.

81. 81. The method of claim 80, wherein the disease is a neuromuscular disease.

82. 82. The method of claim 81, wherein the neuromuscular disease is Duchenne muscular dystrophy.

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