Phosphorodiamidate morpholino oligomer conjugates

Abstract

Novel antisense oligonucleotide conjugates that cause exon skipping in the human dystrophin gene and their use in methods of treating Duchenne muscular dystrophy (DMD) in patients suffering from the disease are described.

Description

[Technical field]

[0001] (Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 320,773, filed March 17, 2022. The entire teachings of the above applications are incorporated herein by reference in their entireties.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy created on March 16, 2023 is named 4140_055PC01_SequenceListing_ST26 and is 572,344 bytes in size.

[0003] The present disclosure relates to certain phosphorodiamidate morpholino oligomer antisense oligonucleotide conjugates. The present disclosure relates to methods for treating muscular dystrophy in patients suffering from Duchenne muscular dystrophy (DMD) using antisense oligonucleotide conjugates that cause exon skipping in the human dystrophin gene. [Background technology]

[0004] Dystrophin is a key structural protein that protects muscles from repetitive exercise-induced muscle damage, affecting skeletal, diaphragm, and cardiac muscles. Duchenne muscular dystrophy is a rare, severe, life-threatening, X-linked recessive neuromuscular degenerative disease caused by mutations in the dystrophin gene. These mutations disrupt the reading frame of the dystrophin messenger ribonucleic acid (mRNA) and prevent the translation of functional dystrophin protein. Mutations in any exon that change the reading frame of an exon or introduce a stop codon, or are characterized by the removal of an entire out-of-frame exon or the duplication of one or more exons, can prevent the production of functional dystrophin and result in DMD. The absence of dystrophin protein is the direct cause of the disease, and patients undergo a predictable disease course that begins in early childhood with an increasingly progressive deterioration of skeletal muscle function, leading to premature death, usually before the age of 30.

[0005] Duchenne muscular dystrophy (DMD) is caused by defective expression of protein dystrophin.The gene that codes for the protein contains 79 exons, spread over more than 2 million nucleotides of DNA.The mutation of any exon, which changes the reading frame of the exon or introduces a stop codon, or is characterized by the removal of the entire out-of-frame exon or the duplication of one or more exons, can prevent the production of functional dystrophin and lead to DMD.

[0006] Mutations, typically deletions of one or more exons, have been shown to result in a less severe form of muscular dystrophy, Becker muscular dystrophy (BMD), if they result in the correct reading frame along the entire dystrophin transcript and thus premature termination of translation of the mRNA into protein. If the joining of the upstream and downstream exons in the processing of the mutated dystrophin pre-mRNA maintains the correct reading frame of the gene, the result is an mRNA that codes for a protein with a short internal deletion that retains some activity, resulting in the BMD phenotype.

[0007] For many years, it has been known that exon deletions that do not change the reading frame of dystrophin protein cause BMD phenotypes, while exon deletions that cause frameshifts cause DMD (Monaco, Bertelson et al. 1988). In general, dystrophin mutations, including point mutations and exon deletions that change the reading frame and thus interrupt proper protein translation, result in DMD. It should also be noted that some BMD and DMD patients have exon deletions that span multiple exons.

[0008] Antisense oligonucleotides, such as splice-switching oligonucleotides (SSOs), have been successfully used in the treatment of DMD to induce alternative splicing of pre-mRNA by steric blocking of spliceosome.SSOs are specifically designed to target specific regions of pre-mRNA, typically exons, to induce skipping of mutations in DMD gene, thereby restoring these out-of-frame mutations in frame, allowing the production of internally truncated but functional dystrophin protein.Such antisense oligomers are known to target completely within exons (so-called exon internal sequences) or at splice donor or splice acceptor junctions crossing from exons to parts of introns.

[0009] For example, eteplirsen is a phosphorodiamidate morpholino oligomer (PMO) designed to skip exon 51 of the human dystrophin gene in patients with DMD, where exon 51 skipping is suitable to restore the reading frame and generate a functional truncated form of the dystrophin protein. In 2016, the US Food and Drug Administration (FDA) approved Exondys 51® (eteplirsen) for the treatment of DMD in patients with confirmed mutations of the DMD gene, where exon 51 skipping is suitable. As another example, golodirsen (Vyondys 53®), an antisense oligonucleotide of the PMO subclass, has also been approved for the treatment of DMD in patients with confirmed mutations of the DMD gene, where exon 53 skipping is suitable. In addition, casimersen (Amondys 45®), also an antisense oligonucleotide of the PMO subclass, was recently approved in the United States for the treatment of DMD in patients with confirmed mutations in the DMD gene who are amenable to exon 45 skipping.

[0010] The discovery and development of antisense oligomers (e.g., PPMOs) conjugated to cell membrane-permeable peptides for DMD has also been an area of ​​research (see, e.g., U.S. Pat. No. 10,888,578, U.S. Patent Application Serial No. 16 / 469,104, U.S. Pat. No. 11,000,600). Cell membrane-permeable peptides (CPPs), e.g., arginine-rich peptide transport moieties, are effective in enhancing the penetration of antisense oligomers into cells and have been shown to cause exon skipping in different muscle groups in animal models.

[0011] Thus, despite the success achieved in preclinical models with antisense oligomers conjugated to cell membrane-permeable peptides, there remains a need for safe and effective administration paradigms for treating DMD and BMD with such conjugates in human patients. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Pat. No. 10,888,578 [Patent Document 2] U.S. Patent Application Serial No. 16 / 469,104 [Patent Document 3] U.S. Patent No. 11,000,600 Summary of the Invention

[0013] The antisense oligomer conjugates according to formula (I) have been found to be pharmacologically active. Certain antisense oligomer conjugates according to formula (I) have also been found to distribute to different tissues, such as muscle and kidney tissue.

[0014] In some aspects, the present disclosure provides an antisense oligomer conjugate of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, n is 1 to 40; each Nu is a nucleobase that, taken together, forms a target sequence complementary to an exon annealing site in the dystrophin pre-mRNA; T' is [ka] and [ka] is a moiety selected from R 100is selected from the group consisting of RRRRRG-, RRRRG-, RRRG-, RRG-, RG-, and G-, where R is arginine and G is glycine; R 200 is hydrogen, R 1 is C1-C6 alkyl. In some embodiments, the target sequence of the antisense oligomer conjugate of Formula (I), or a pharma- ceutically acceptable salt thereof, is complementary to the exon 51 annealing site in the dystrophin pre-mRNA, designated as H51A(+66+95).

[0015] In some embodiments, the target sequence of the antisense oligomer conjugate of Formula (I), or a pharma- ceutically acceptable salt thereof, is complementary to the exon 45 annealing site in the dystrophin pre-mRNA, designated as H45A(-03+19).

[0016] In some embodiments, the target sequence of the antisense oligomer conjugate of Formula (I), or a pharma- ceutically acceptable salt thereof, is complementary to the exon 53 annealing site in the dystrophin pre-mRNA, designated as H53A(+36+60).

[0017] In some embodiments, in the antisense oligomer conjugate of Formula (I), or a pharma- ceutically acceptable salt thereof, each Nu is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0018] In some embodiments, in the antisense oligomer conjugate of Formula (I), or a pharma- ceutically acceptable salt thereof, T' is a moiety: [ka] In the formula, R 200 is hydrogen.

[0019] In some aspects, the disclosure provides antisense oligomer conjugates of formula (I), and pharma- ceutically acceptable salts thereof, wherein R 100 is RRRRRG-.

[0020] In some aspects, the disclosure provides antisense oligomer conjugates of formula (I), and pharma- ceutically acceptable salts thereof, wherein R 100 is RRRRG-.

[0021] In some aspects, the disclosure provides antisense oligomer conjugates of formula (I), and pharma- ceutically acceptable salts thereof, wherein R 100 is RRRG-.

[0022] In some aspects, the disclosure provides antisense oligomer conjugates of formula (I), and pharma- ceutically acceptable salts thereof, wherein R 100 is RRG-.

[0023] In some aspects, the disclosure provides antisense oligomer conjugates of formula (I), and pharma- ceutically acceptable salts thereof, wherein R 100 is RG-.

[0024] In some aspects, the disclosure provides antisense oligomer conjugates of formula (I), and pharma- ceutically acceptable salts thereof, wherein R 100 is G-.

[0025] In another aspect, the present disclosure provides an antisense oligomer conjugate having formula (V): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase that, together, forms a target sequence that is complementary to an exon annealing site in the dystrophin pre-mRNA, m is 0, 1, 2, 3, 4, or 5.

[0026] In some embodiments, the antisense oligonucleotide conjugate of formula (V) is according to formula (VA): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu from 1 to 30 and 5' to 3' is as follows: [Table 7] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0027] In another aspect, the present disclosure provides an antisense oligomer conjugate having formula (VII): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase that together form a target sequence that is complementary to an exon annealing site in the dystrophin pre-mRNA, m is 0, 1, 2, 3, 4, or 5.

[0028] In some embodiments, the antisense oligonucleotide conjugate of formula (VII) is according to formula (VIIA): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu from 1 to 22 and 5' to 3' is as follows: [Table 8] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0029] In another aspect, the present disclosure provides an antisense oligomer conjugate having formula (IX): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase that, together, forms a target sequence that is complementary to an exon annealing site in the dystrophin pre-mRNA, m is 0, 1, 2, 3, 4, or 5.

[0030] In some embodiments, the formula (IX) antisense oligonucleotide conjugate is according to formula (IXA): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5; In the formula, each Nu from 1 to 25 and from 5' to 3' is as follows: [Table 9] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0031] In certain aspects, the disclosure provides an antisense oligomer conjugate of any of formulas (V), (VA), (VII), (VIIA), (IX), or (IXA), or a pharma- ceutically acceptable salt thereof, wherein m is 0.

[0032] In certain aspects, the disclosure provides an antisense oligomer conjugate of any of formulas (V), (VA), (VII), (VIIA), (IX), or (IXA), or a pharma- ceutically acceptable salt thereof, wherein m is 1.

[0033] In certain aspects, the disclosure provides an antisense oligomer conjugate of any of formulas (V), (VA), (VII), (VIIA), (IX), or (IXA), or a pharma- ceutically acceptable salt thereof, wherein m is 2.

[0034] In certain aspects, the disclosure provides an antisense oligomer conjugate of any of formulas (V), (VA), (VII), (VIIA), (IX), or (IXA), or a pharma- ceutically acceptable salt thereof, wherein m is 3.

[0035] In certain aspects, the disclosure provides an antisense oligomer conjugate of any of formulas (V), (VA), (VII), (VIIA), (IX), or (IXA), or a pharma- ceutically acceptable salt thereof, wherein m is 4.

[0036] In certain aspects, the disclosure provides an antisense oligomer conjugate of any of formulas (V), (VA), (VII), (VIIA), (IX), or (IXA), or a pharma- ceutically acceptable salt thereof, wherein m is 5.

[0037] In certain embodiments, the antisense oligomer conjugates of the present disclosure are provided in free base form. In certain embodiments, the antisense oligomer conjugates of the present disclosure provided herein are pharma- ceutically acceptable salts, such as hydrochloride salts.

[0038] The present disclosure also provides pharmaceutical compositions comprising an antisense oligomer conjugate described herein, such as an antisense oligomer conjugate of formula (I), or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for parenteral use.

[0039] The present disclosure provides a method of treating a patient with Duchenne muscular dystrophy (DMD) having a mutation suitable for exon skipping in a patient in need thereof, the method comprising administering to the patient an antisense oligomer conjugate described herein, such as an antisense oligomer conjugate of formula (I), or a pharma- ceutically acceptable salt thereof. In some embodiments, the antisense oligomer conjugate causes exon skipping in the human dystrophin gene. In some embodiments, the exon is selected from exons 44, 45, 50, 51, 52, or 53. In some embodiments, the exon is selected from exons 45, 51, or 53.

[0040] The present disclosure provides a method of treating a patient having Duchenne muscular dystrophy (DMD) with an antisense oligomer conjugate, the method comprising administering to the patient an antisense oligomer conjugate described herein, such as an antisense oligomer conjugate of formula (I), or a pharma- ceutically acceptable salt thereof.

[0041] In certain aspects, the disclosure provides a method of treating a patient with DMD, having a mutation amenable to exon 51 skipping, in a patient in need thereof, the method comprising administering to a patient an antisense oligomer conjugate having formula (VI): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0042] In certain aspects, the disclosure provides a method of treating a patient with DMD, having a mutation amenable to exon 45 skipping, in a patient in need thereof, the method comprising administering to a patient an antisense oligomer conjugate having formula (VIII): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0043] In certain aspects, the disclosure provides a method of treating a patient with DMD, having a mutation amenable to exon 53 skipping, in a patient in need thereof, the method comprising administering to a patient an antisense oligomer conjugate having formula (X): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5. [Brief description of the drawings]

[0044] [Figure 1] Exon 51 skipping (%) - Concentration (μM) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present disclosure relates to antisense oligonucleotide conjugates and their use in methods for treating muscular dystrophies, such as DMD and BMD, in patients, comprising administering an antisense oligomer conjugate described herein, or a pharma- ceutically acceptable salt thereof, to induce exon skipping in the human dystrophin gene.

[0046] definition "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0047] As used herein, the term "alkyl" refers to a saturated straight or branched chain hydrocarbon, unless otherwise specified. In certain embodiments, an alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, an alkyl group is a group having 1 to 10 carbon atoms, i.e., C1 to C6. 10and alkyl. In certain embodiments, the alkyl group comprises 1 to 6 carbon atoms, i.e., C1 to C6 alkyl. In certain embodiments, the alkyl group is selected from the group consisting of methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term includes both substituted and unsubstituted alkyl groups, including halogenated alkyl groups. In certain embodiments, the alkyl group is a fluorinated alkyl group. Non-limiting examples of moieties with which an alkyl group may be substituted are selected from the group consisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected or optionally protected, as known to those of skill in the art, e.g., as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, incorporated herein by reference.

[0048] "Suitable for exon skipping", as used herein with respect to a subject or patient, is intended to include subjects and patients who have one or more mutations in the dystrophin gene that, without skipping of a particular exon in the dystrophin pre-mRNA, result in an out-of-frame reading frame, thereby preventing translation of the pre-mRNA and rendering the subject or patient unable to produce functional or semi-functional dystrophin. It is well within the understanding of one of ordinary skill in the art to determine whether a patient has a mutation in the dystrophin gene that is suitable for exon skipping (see, for example, Aartsma-Rus et al. (2009) Hum Mutat. 30:293-299, Gurvich et al., Hum Mutat. 2009; 30(4) 633-640, and Fletcher et al. (2010) Molecular Therapy 18(6) 1218-1223).

[0049] The terms "oligomer" and "oligonucleotide" are used interchangeably and refer to a sequence of subunits linked by intersubunit bonds. In certain instances, the term "oligomer" is used in connection with "antisense oligomers." For "antisense oligomers," each subunit consists of (i) a ribose sugar or derivative thereof, and (ii) a nucleic acid base bound thereto, whereby the order of base-pairing moieties forms a base sequence that is complementary to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence, provided that either the subunits, the intersubunit bonds, or both, are not naturally occurring. In certain embodiments, the antisense oligomer is a PMO.

[0050] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each containing a nucleobase sequence) related to each other by Watson-Crick base pairing rules. For example, the nucleobase sequence "TGA(5'→3')" is complementary to the nucleobase sequence "ACT(3'→5')". Complementarity may be "partial", where less than all of the nucleobases of a given nucleobase sequence are matched with another nucleobase sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleobase sequence and another nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Alternatively, continuing the example, there may be "complete" or "perfect" (100%) complementarity between a given nucleobase sequence and another nucleobase sequence. The degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between sequences.

[0051] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a therapeutic compound, such as an antisense oligomer conjugate, administered to a mammalian subject, either as a single dose or as part of a series of doses, that is effective to produce a desired therapeutic effect. For antisense oligomer conjugates, this effect is typically produced by inhibiting translation or natural splice processing of a selected target sequence, or by producing a clinically significant (statistically significant) amount of dystrophin.

[0052] "Enhance" or "enhancing", or "increase" or "increasing", or "stimulate" or "stimulating" generally refers to the ability of one or more antisense oligomer conjugates or pharmaceutical compositions to produce or cause a greater physiological response (i.e., a downstream effect) in a cell or subject compared to the response caused by either no antisense oligomer conjugate, or a control compound. A greater physiological response can include increased expression of a functional form of dystrophin protein, or increased dystrophin-related biological activity in muscle tissue, among other responses apparent from the understanding in the art and the description herein. Increases in muscle function may also be measured, including an increase or improvement in muscle function of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of muscle fibers expressing functional dystrophin can also be measured, including increasing dystrophin expression by about 1%, 2%, 5%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibers. For example, it has been shown that when 25-30% of fibers express dystrophin, an improvement in muscle function of about 40% can occur (see, e.g., DelloRusso et al., Proc Natl Acad Sci USA 99: 12979-12984, 2002). An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50-fold or more (e.g., 500, 1000-fold, including all integers and decimals therebetween and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the amount produced without the antisense oligomer conjugate (absence of agent) or by a control compound.

[0053] As used herein, the terms "function", "functionality", and the like refer to biological, enzymatic, or therapeutic functions.

[0054] A "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to reduce the progressive degradation of muscle tissue otherwise characteristic of muscular dystrophies, as compared to altered or "defective" forms of dystrophin protein typically present in certain subjects with DMD or BMD. In certain embodiments, a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween) of the in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art. Truncated forms of dystrophin are included, such as those forms produced after administration of certain exon-skipping antisense oligomer conjugates of the present disclosure.

[0055] The term "mismatch" refers to one or more nucleobases (whether contiguous or separated) in an oligomer nucleobase sequence that do not match the target pre-mRNA according to base pairing rules. While full complementarity is often desired, some embodiments may include one or more, but preferably 6, 5, 4, 3, 2, or 1 mismatches to the target pre-mRNA. Variations at any position within the oligomer are included. In certain embodiments, the antisense oligomer conjugates of the present disclosure include nucleobase sequence variations near the internal terminal variations, when present, typically within about 6, 5, 4, 3, 2, or 1 subunit at the 5' and / or 3' end.

[0056] The terms "morpholino," "morpholino oligomer," and "PMO" have the following general structure: [ka] and as described in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997). Morpholino as described herein includes 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.

[0057] In some embodiments, morpholino oligos (PMOs) are conjugated with a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include: [ka] Includes.

[0058] Among the above exemplary tail portions, "TEG" or "EG3" refers to the following tail portions: [ka]

[0059] Of the exemplary tail moieties above, "GT" refers to the following tail moieties: [ka]

[0060] As used herein, the term "RRRRRG-" refers to the following structure: [ka]

[0061] As used herein, the term "RRRRG-" refers to the following structure: [ka]

[0062] As used herein, the term "RRRG-" refers to the following structure: [ka]

[0063] As used herein, the term "RRG-" refers to the following structure: [ka]

[0064] As used herein, the term "RG-" refers to the following structure: [ka]

[0065] As used herein, the term "G-" refers to the following structure: [ka]

[0066] The synthesis of arginine peptides and methods of conjugating to oligomers are further described in U.S. Pat. Nos. 9,161,948, 10,888,578, and 11,000,600, U.S. Patent Application Publication No. 2012 / 0289457, and International Patent Application Publication Nos. 2004 / 097017, 2009 / 005793, and 2012 / 150960, the disclosures of which are incorporated by reference in their entireties.

[0067] The terms "nucleobase" (Nu), "base-pairing moiety," or "base" are used interchangeably to refer to a purine or pyrimidine base found in naturally occurring or "natural" DNA or RNA (e.g., uracil, thymine, adenine, cytosine, and guanine), as well as analogs of these naturally occurring purines and pyrimidines. These analogs may confer improved properties, such as binding affinity, to an oligomer. Exemplary analogs include hypoxanthine (the base component of inosine), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl-modified pyrimidines, 10-(9-(aminoethoxy)phenoxazinyl) (G-clamp), and the like.

[0068] Further examples of base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine (inosine) (with their respective amino groups protected by acyl protecting groups), pyrimidine analogs such as 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 are natural decomposition products). Also contemplated are modified nucleobases disclosed in the following: Chiu and Rana, RNA, 2003, 9, 1034-1048; Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196, and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, the contents of which are incorporated herein by reference.

[0069] Further examples of base pairing moieties include, but are not limited to, extended size nucleobases with one or more additional benzene rings. Nucleobase substitutions described in the Glen Research catalog (www.glenresearch.com), Krueger AT et al., Acc. Chem. Res., 2007, 40, 141-150, Kool, ET, Acc. Chem. Res., 2002, 35, 936-943, Benner SA, et al., Nat. Rev. Genet., 2005, 6, 553-543, Romesberg, FE, et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733, and Hirao, I., Curr. Opin. Chem. Chem.Biol.,2006,10,622-627, the contents of which are incorporated herein by reference, are contemplated as useful in the antisense oligomer conjugates described herein. Examples of extended size nucleobases include those shown below, as well as tautomers thereof. [ka]

[0070] The term "exposure" refers to dose (PPMO input into the body) and various measures of acute or integrated PPMO concentration in plasma and other biological fluids (e.g., Cmax, Cmin, Css, AUC). The term "response" refers to a direct measure of the pharmacological effect of the drug. Responses include a wide range of endpoints or biomarkers, ranging from potential or acceptable surrogates (e.g., effects on blood pressure, magnesium levels, or cardiac output) to short-term or long-term clinical effects related to efficacy and safety.

[0071] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0072] For clarity, the structures of the present disclosure are continuous from 5' to 3' and include various diagrammatic breaks, designated "break A," "break B," and "break C," for the convenience of depicting the entire structure in a compact form. As will be understood by those skilled in the art, for example, each designation of "break A" indicates the diagrammatic continuation of the structure at these points. Those skilled in the art will understand that the same is true for each instance of "break B" and "break C" in the above structures. However, none of the diagrammatic breaks are intended to indicate an actual discontinuity in the above structures, and those skilled in the art would not understand them to mean so.

[0073] As used herein, a set of brackets used within a structural formula indicates that the structural feature between the brackets is repeated. In some embodiments, the brackets used may be "[" and "]", and in certain embodiments, the brackets used to indicate a repeating structural feature may be "(" and ")". In some embodiments, the number of repeating repetitions of the structural feature between the brackets is the number shown outside the brackets, e.g., 2, 3, 4, 5, 6, 7, etc. In various embodiments, the number of repeating repetitions of the structural feature between the brackets is indicated by a variable shown outside the brackets, such as "n".

[0074] As used herein, a straight or squiggly bond drawn to a chiral carbon or phosphorus atom in a structural formula indicates that the stereochemistry of the chiral carbon or phosphorus is undefined and is intended to include all forms of the chiral center. Examples of such diagrams are shown below. [ka]

[0075] Antisense oligomer conjugates of the present disclosure In various aspects, the present disclosure provides an antisense oligomer conjugate according to formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: n is 1 to 40; each Nu is a nucleobase which together form the target sequence, is complementary to an exon annealing site in the dystrophin pre-mRNA; T' is a moiety selected from [ka] During the ceremony, R 100 is selected from the group consisting of RRRRRG-, RRRRG-, RRRG-, RRG-, RG-, and G-, where R is arginine and G is glycine; R 200 is hydrogen, R 1 is C1-C6 alkyl.

[0076] In some embodiments, T' is the moiety: [ka] In the formula, R 200 is hydrogen.

[0077] In some embodiments, the antisense oligomer conjugate is according to formula (I), or a pharma- ceutically acceptable salt thereof, wherein R 100 is RRRRRG-.

[0078] In some embodiments, the antisense oligomer conjugate is according to formula (I), or a pharma- ceutically acceptable salt thereof, wherein R100 is RRRRG-.

[0079] In some embodiments, the antisense oligomer conjugate is according to formula (I), or a pharma- ceutically acceptable salt thereof, wherein R 100 is RRRG-.

[0080] In some embodiments, the antisense oligomer conjugate is according to formula (I), or a pharma- ceutically acceptable salt thereof, wherein R 100 is RRG-.

[0081] In some embodiments, the antisense oligomer conjugate is according to formula (I), or a pharma- ceutically acceptable salt thereof, wherein R 100 is RG-.

[0082] In some embodiments, the antisense oligomer conjugate is according to formula (I), or a pharma- ceutically acceptable salt thereof, wherein R 100 is G-.

[0083] In some embodiments, the antisense oligomer of the antisense oligomer conjugate has n+2 base pairs, where n in Formula (I) is 1 to 40, optionally 13 to 38, optionally 13 to 28, optionally 13 to 23, or optionally 13 to 18. In other words, the oligomer is 15 to 40, 15 to 35, 15 to 30, 15 to 25, or 15 to 20 nucleotides in length.

[0084] In some embodiments, the antisense oligomer conjugate of Formula (I) induces exon skipping in the human dystrophin gene. In some embodiments, the exon is selected from exons 44, 45, 50, 51, 52, or 53. In certain embodiments, the exon is selected from exons 45, 51, or 53.

[0085] In various embodiments, the antisense oligomer conjugate is according to formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase which together form the target sequence, T' is a moiety selected from: [ka] R 1 is C1-C6 alkyl, m is 0, 1, 2, 3, 4, or 5; The target sequence is complementary to an annealing site in the dystrophin pre-RNA.

[0086] In various embodiments, the antisense oligonucleotide conjugate is according to formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase which together form the target sequence, T' is a moiety selected from: [ka] R 1 is C1-C6 alkyl, m is 0, 1, 2, 3, 4, or 5; The target sequence is complementary to an annealing site in the dystrophin pre-RNA.

[0087] In various embodiments, the antisense oligonucleotide conjugate is according to formula (IV): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase which together form the target sequence, T' is a moiety selected from: [ka] R 1 is C1-C6 alkyl, m is 0, 1, 2, 3, 4, or 5; The target sequence is complementary to an annealing site in the dystrophin pre-RNA.

[0088] In some embodiments, the antisense oligonucleotide conjugate in the composition comprises a sequence complementary to 15-35 nucleobases of a target region of dystrophin pre-mRNA in exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53. Oligonucleotide sequences designed to target and skip these dystrophin exons have been described in the art. For example, WO2018 / 129384, WO2019 / 060775, WO2020 / 219820, WO2018 / 007475, WO2018 / 091544, WO2020 / 089325, WO2004 / 048570, WO2020 / 028832, WO2017 / 062862, U.S. Patent No. 10,683,322, U.S. Patent No. 8,969,551, U.S. Patent No. 10,781,448, U.S. Patent No. 9,988,629, U.S. Patent No. 9,840,706, U.S. Patent No. Nos. 10,851,373, WO2020 / 004675, and WO2020 / 0158792, and issued U.S. patents, the sequence disclosures of which are incorporated herein.

[0089] A number of exemplary target sequences are described below. These sequences are provided as morpholino target sequences and can be incorporated into the antisense oligonucleotide conjugates of formula (I).

[0090] In some embodiments, the target sequence is complementary to an exon 51 annealing site in a dystrophin pre-mRNA. In some embodiments, the site is designated as H51A(+66+95). In some embodiments, the target sequence is complementary to an exon 45 annealing site in a dystrophin pre-mRNA. In some embodiments, the site is designated as H45A(-03+19). In some embodiments, the target sequence is complementary to an exon 53 annealing site in a dystrophin pre-mRNA. In some embodiments, the site is designated as H53A(+36+60).

[0091] In various embodiments, T' is [ka] It is.

[0092] In various embodiments, R 1 is methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, or 2,3-dimethylbutyl.

[0093] In some embodiments, the antisense oligomer conjugate of Formula (I) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a .5 HCl salt. In certain embodiments, the HCl salt is a .4 HCl salt. In certain embodiments, the HCl salt is a .3 HCl salt. In certain embodiments, the HCl salt is a .2 HCl salt. In certain embodiments, the HCl salt is a .1 HCl salt.

[0094] In some embodiments, each Nu is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0095] In some embodiments, the target sequence is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO:1), where each thymine (T) is optionally a uracil (U).

[0096] In various embodiments, T' is [ka] and the target sequence is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO:1), where each thymine (T) is optionally a uracil (U).

[0097] In various embodiments, T' is [ka] and the target sequence is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO:1).

[0098] In some embodiments, the target sequence is 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 2), where each thymine (T) is optionally a uracil (U).

[0099] In various embodiments, T' is [ka] and the target sequence is 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 2), where each thymine (T) is optionally a uracil (U).

[0100] In various embodiments, T' is [ka] and the target sequence is 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 2).

[0101] In some embodiments, the target sequence is 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 3), where each thymine (T) is optionally a uracil (U).

[0102] In various embodiments, T' is [ka] and the target sequence is 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 3), where each thymine (T) is optionally a uracil (U).

[0103] In various embodiments, T' is [ka] and the target sequence is 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 3).

[0104] For example, in some embodiments, including some embodiments of Formula (I), and Formula (II), the antisense oligomer conjugates of the present disclosure are according to Formula (V): [ka] or a pharma- ceutically acceptable salt thereof, wherein:

[0105] each Nu is a nucleobase that together form a target sequence that is complementary to an exon 51 annealing site in the dystrophin pre-mRNA, designated as H51A(+66+95), m is 0, 1, 2, 3, 4, or 5.

[0106] For example, in some embodiments, including some embodiments of formula (V), the antisense oligomer conjugates of the present disclosure are according to formula (VA): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase that together form a target sequence that is complementary to an exon 51 annealing site in the dystrophin pre-mRNA, designated as H51A(+66+95), m is 0, 1, 2, 3, 4, or 5.

[0107] In some embodiments, each Nu in formula (V) or formula (VA) is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0108] In various embodiments, each Nu from 1 to 30 and 5' to 3' is as follows: [Table 10] Here, A is [ka] and C is [ka] and G is [ka] and X is [ka] or [ka] In certain embodiments, each X is independently [ka] It is.

[0109] In some embodiments, the antisense oligomer conjugate of Formula (V) or Formula (VA) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0110] For example, in some embodiments, including some embodiments of formula (VA), the antisense oligomer conjugates of the present disclosure are according to formula (VB) or formula (VC): [ka] or [ka] or a pharma- ceutically acceptable salt thereof (such as the HCl salt), where each Nu is a nucleobase that, taken together, forms a target sequence that is complementary to an exon 51 annealing site in dystrophin pre-mRNA, designated as H51A(+66+95).

[0111] For example, in some embodiments, including some embodiments of formula (VA), the antisense oligomer conjugates of the present disclosure are according to formula (VD) or formula (VE): [ka] or [ka] where each Nu is a nucleobase that together form a target sequence that is complementary to an exon 51 annealing site in the dystrophin pre-mRNA, designated as H51A(+66+95).

[0112] In some embodiments, each Nu in formulas (VB), (VC), (VD), and (VE) is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0113] In various embodiments, each Nu from 1 to 30 and 5' to 3' is as follows: [Table 11] Here, A is [ka] and C is [ka] and G is [ka] and X is [ka] or [ka] In certain embodiments, each X is [ka] It is.

[0114] In some embodiments, including, for example, embodiments of antisense oligomeric conjugates of Formula (V), Formula (VA), Formula (VB), Formula (VC), Formula (VD), and Formula (VE), the target sequence is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 1), where each thymine (T) is optionally a uracil (U). In some embodiments, including, for example, embodiments of antisense oligomeric conjugates of Formula (V), Formula (VA), Formula (VB), Formula (VC), Formula (VD), and Formula (VE), the target sequence is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 1).

[0115] In some embodiments, including, for example, embodiments of the antisense oligomer conjugates of Formula (I) or Formula (II), the antisense oligomer conjugates of the present disclosure are according to Formula (V): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu of 1 to 30 and 5' to 3' is as follows: [Table 12]

[0116] Here, A is [ka] and C is [ka] and G is [ka] and T is represented by formula (VI) [ka] is: [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0117] In some embodiments, the antisense oligomer conjugate of formula (VI) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0118] In some embodiments, including for example, embodiments of antisense oligomer conjugates of formula (VI), the antisense oligomer conjugates of the present disclosure are according to formula (VIA), or formula (VIB): [ka] or [ka] or a pharma- ceutically acceptable salt thereof, such as the HCl salt.

[0119] In some embodiments, including for example, embodiments of the antisense oligomer conjugates of Formula (I) and Formula (II), the antisense oligomer conjugates of the present disclosure are according to Formula (VA): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu of 1 to 30 and 5' to 3' is as follows: [Table 13] Here, A is [ka] and C is [ka] and G is [ka] and T is represented by formula (VIC) [ka] is: [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0120] In some embodiments, the antisense oligomer conjugate of formula (VIC) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0121] In some embodiments, including, for example, embodiments of the antisense oligomer conjugate of formula (VIC), the antisense oligomer conjugates of the present disclosure are according to formula (VIC), or formula (VIE): [ka] or [ka] or a pharma- ceutically acceptable salt thereof, such as the HCl salt.

[0122] In some embodiments, including for example, embodiments of the antisense oligomer conjugate of formula (VIC), the antisense oligomer conjugates of the present disclosure are according to formula (VIF), or formula (VIG): [ka] or [ka]

[0123] For example, in some embodiments, including some embodiments of Formula (I), or Formula (III), the antisense oligomer conjugates of the present disclosure are according to Formula (VII): [ka] or a pharma- ceutically acceptable salt thereof, wherein: Each Nu is a nucleobase that together form a target sequence that is complementary to an exon 45 annealing site in the dystrophin pre-mRNA, designated as H45A(-03+19), and m is 0, 1, 2, 3, 4, or 5.

[0124] For example, in some embodiments, including some embodiments of formula (VII), the antisense oligomer conjugates of the present disclosure are according to formula (VIIA): [ka] or a pharma- ceutically acceptable salt thereof, wherein: Each Nu is a nucleobase that together form a target sequence that is complementary to an exon 45 annealing site in the dystrophin pre-mRNA, designated as H45A(-03+19), and m is 0, 1, 2, 3, 4, or 5.

[0125] In some embodiments, each Nu of formula (VII) or formula (VIIA) is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0126] In various embodiments, each Nu from 1 to 22 and 5' to 3' is as follows: [Table 14] Here, A is [ka] and C is [ka] and G is [ka] and X is [ka] or [ka] In certain embodiments, each X is independently [ka] It is.

[0127] In some embodiments, the antisense oligomer conjugate of Formula (VII) or Formula (VIIA) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0128] For example, in some embodiments, including some embodiments of formula (VIIA), the antisense oligomer conjugates of the present disclosure are according to formula (VIIB), or formula (VIIC): [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu is a nucleobase that, taken together, forms a target sequence that is complementary to an exon 45 annealing site in dystrophin pre-mRNA, designated as H45A(-03+19).

[0129] In some embodiments, each Nu in formula (VIIB) or formula (VIIC) is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0130] In various embodiments, each Nu from 1 to 22 and 5' to 3' is as follows: [Table 15] Here, A is [ka] and C is [ka] and G is [ka] and X is [ka] or [ka] In certain embodiments, each X is [ka] It is.

[0131] In some embodiments, including, for example, embodiments of the antisense oligomeric conjugates of formula (VII), formula (VIIA), formula (VIIB), and formula (VIIC), the target sequence is 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 2), where each thymine (T) is optionally a uracil (U). In various embodiments, including, for example, embodiments of the antisense oligomeric conjugates of formula (VII), formula (VIIA), formula (VIIB), and formula (VIIC), the target sequence is 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 2).

[0132] In some embodiments, including, for example, embodiments of the antisense oligomer conjugates of Formula (I), or Formula (III), the antisense oligomer conjugates of the present disclosure are according to Formula (VII): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu from 1 to 22 and 5' to 3' is as follows: [Table 16] Here, A is [ka] and C is [ka] and G is [ka] and T is represented by formula (VIII) [ka] is: [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0133] In some embodiments, the antisense oligomer conjugate of formula (VIII) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0134] In some embodiments, including for example, embodiments of antisense oligomer conjugates of formula (VIII), the antisense oligomer conjugates of the present disclosure are according to formula (VIIIA), or formula (VIIIB): [ka] or [ka] or a pharma- ceutically acceptable salt thereof, such as the HCl salt.

[0135] In some embodiments, including, for example, embodiments of the antisense oligomer conjugates of Formula (I), or Formula (III), the antisense oligomer conjugates of the present disclosure are according to Formula (VIIA): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu from 1 to 22 and 5' to 3' is as follows: [Table 17] Here, A is [ka] and C is [ka] and G is [ka] and T is represented by formula (VIIIC) [ka] is: [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0136] In some embodiments, the antisense oligomer conjugate of Formula (VIIIC) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0137] In some embodiments, including, for example, embodiments of the antisense oligomer conjugate of formula (VIIIC), the antisense oligomer conjugates of the present disclosure are according to formula (VIIID), or formula (VIIIE): [ka] or [ka] or a pharma- ceutically acceptable salt thereof, such as the HCl salt.

[0138] In some embodiments, including for example, embodiments of the antisense oligomer conjugate of formula (VIIIC), the antisense oligomer conjugates of the present disclosure are according to formula (VIIIF), or formula (VIIIG): [ka] or [ka]

[0139] For example, in some embodiments, including some embodiments of Formula (I), or Formula (IV), the antisense oligomer conjugates of the present disclosure are according to Formula (IX): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase that together form a target sequence that is complementary to an exon 53 annealing site in the dystrophin pre-mRNA, designated as H53A(+36+60); m is 0, 1, 2, 3, 4, or 5.

[0140] For example, in some embodiments, including some embodiments of formula (IX), the antisense oligomer conjugate of the present disclosure has the formula (IXA): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase that together form a target sequence that is complementary to an exon 53 annealing site in the dystrophin pre-mRNA, designated as H53A(+36+60); m is 0, 1, 2, 3, 4, or 5.

[0141] In some embodiments, each Nu in formula (IX) or formula (IXA) is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0142] In various embodiments, each Nu from 1 to 25 and 5' to 3' is as follows: [Table 18] Here, A is [ka] and C is [ka] and G is [ka] and X is [ka] or [ka] In certain embodiments, each X is independently [ka] It is.

[0143] In some embodiments, the antisense oligomer conjugate of Formula (IX) or Formula (IXA) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0144] For example, in some embodiments, including some embodiments of formula (IXA), the antisense oligomer conjugates of the present disclosure are according to formula (IXB), or formula (IXC): [ka] [ka] or a pharma- ceutically acceptable salt thereof, e.g., an HCl salt, wherein each Nu is a nucleobase that, taken together, forms a target sequence that is complementary to an exon 53 annealing site in dystrophin pre-mRNA, designated as H53A(+36+60).

[0145] In some embodiments, each Nu in formula (IXB) or formula (IXC) is independently selected from cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0146] In various embodiments, each Nu from 1 to 25 and 5' to 3' is as follows: [Table 19] Here, A is [ka] and C is [ka] and G is [ka] and X is [ka] or [ka] In certain embodiments, each X is [ka] It is.

[0147] In some embodiments, including, for example, embodiments of antisense oligomeric conjugates of formula (IX), formula (IXA), formula (IXB), and formula (IXC), the target sequence is 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 3), where each thymine (T) is optionally a uracil (U). In various embodiments, including, for example, embodiments of antisense oligomeric conjugates of formula (IX), formula (IXA), formula (IXB), and formula (IXC), the target sequence is 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 3).

[0148] In some embodiments, including, for example, embodiments of the antisense oligomer conjugates of Formula (I), or Formula (IV), the antisense oligomer conjugates of the present disclosure are according to Formula (IX): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5; Each Nu from 1 to 25 and 5' to 3' is as follows: [Table 20] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] and represented by formula (X): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0149] In some embodiments, the antisense oligomer conjugate of formula (X) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0150] In some embodiments, including for example, embodiments of antisense oligomer conjugates of formula (X), the antisense oligomer conjugates of the disclosure are according to formula (XA), or formula (XB): [ka] [ka] or a pharma- ceutically acceptable salt thereof, such as the HCl salt.

[0151] In some embodiments, including for example, embodiments of the antisense oligomer conjugates of formula (I), or (IV), the antisense oligomer conjugate is according to formula (IXA): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5; In the formula, each Nu from 1 to 25 and from 5' to 3' is as follows: [Table 21] Here, A is [ka] and C is [ka] and G is [ka] and T is represented by the formula (XC) [ka] is: [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

[0152] In some embodiments, the antisense oligomer conjugate of formula (XC) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0153] In some embodiments, including for example, embodiments of antisense oligomer conjugates of formula (XC), the antisense oligomer conjugates of the present disclosure are according to formula (XD), or formula (XE): [ka] [ka] or a pharma- ceutically acceptable salt thereof, such as the HCl salt.

[0154] In some embodiments, including for example, embodiments of the antisense oligomer conjugate of formula (XC), the antisense oligomer conjugates of the present disclosure are according to formula (XF), or formula (XG): [ka] [ka]

[0155] In one aspect, the present disclosure provides an antisense oligomer conjugate, or a pharma- ceutically acceptable salt thereof, capable of binding to a selected target and inducing exon skipping in a human dystrophin gene, wherein the antisense oligomer conjugate, or a pharma- ceutically acceptable salt thereof, comprises a sequence of bases, designated as an annealing site, that is complementary to an exon 51 target region of a dystrophin pre-mRNA, wherein the base sequence and the annealing site are selected from one of the following: [Table 22-1] [Table 22-2] [Table 22-3] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0156] In one embodiment, the base sequence and the annealing site are selected from one of the following: [Table 23-1] [Table 23-2]

[0157] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0158] In another aspect, the present disclosure provides an antisense oligomeric conjugate of formula (XI): [ka] or a pharma- ceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu from 1 to (n+1) and 5' to 3' is the following sequence: [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0159] In one embodiment, the base sequence and the annealing site are selected from one of the following: [Table 25-1] [Table 25-2]

[0160] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0161] In some embodiments, the antisense oligomer conjugate of formula (XI) is its HCl (hydrochloric acid) salt. In certain embodiments, m is 5 and the HCl salt is a .5 HCl salt. In certain embodiments, m is 4 and the HCl salt is a .4 HCl salt. In certain embodiments, m is 3 and the HCl salt is a .3 HCl salt. In certain embodiments, m is 2 and the HCl salt is a .2 HCl salt. In certain embodiments, m is 1 and the HCl salt is a .HCl salt.

[0162] In another aspect, the disclosure provides an antisense oligomer of formula (XIA) or formula (XIB): [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu from 1 to (n+1) and 5' to 3' corresponds to a nucleobase in the following sequence: [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0163] In one embodiment, the base sequence and the annealing site are selected from one of the following: [Table 27-1] [Table 27-2]

[0164] Here, A is [ka] and C is [ka] and G is [ka] and T is [ka] and U is [ka] where Gm is a methylated guanine, Am is a methylated adenine, and mC is [ka] It is.

[0165] Nucleobase Modifications and Substitutions In certain embodiments, the antisense oligomer conjugates of the present disclosure are composed of RNA nucleobases and DNA nucleobases (often simply referred to in the art as "bases"). RNA bases are commonly known as adenine (A), uracil (U), cytosine (C), and guanine (G). DNA bases are commonly known as adenine (A), thymine (T), cytosine (C), and guanine (G). In various embodiments, the antisense oligomer conjugates of the present disclosure are composed of cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0166] In certain embodiments, one or more RNA or DNA bases in an oligomer may be modified or replaced with a base other than an RNA or DNA base. Oligomers containing modified or substituted bases include oligomers in which one or more of the purine or pyrimidine bases most commonly found in nucleic acids are replaced with a less common or unnatural base.

[0167] Purine bases contain a pyrimidine ring fused to an imidazole ring, as described by the general formula below: [ka]

[0168] Adenine and guanine are the two most commonly found purine nucleobases in nucleic acids. Other naturally occurring purines include N 6 -Methyladenine, N 2 Examples of methylguanine include, but are not limited to, -methylguanine, hypoxanthine, and 7-methylguanine.

[0169] The pyrimidine base contains a six-membered pyrimidine ring, as described by the general formula: [ka]

[0170] Cytosine, uracil, and thymine are the most commonly found pyrimidine bases in nucleic acids. Other naturally occurring pyrimidines include, but are not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligomers described herein contain thymine bases instead of uracil.

[0171] Other suitable bases include 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymine), G-clamp and derivatives thereof, 5-substituted pyrimidine (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-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine or derivatives thereof, N 2 -Cyclopentylguanine (cPent-G), N 2 -cyclopentyl-2-aminopurine (cPent-AP), and N 2Examples of Super A, Super G, and Super T derivatives include, but are not limited to, -propyl-2-aminopurine (Pr-AP), pseudouracil, or derivatives thereof, and degenerate or universal bases such as 2,6-difluorotoluene, or the absence of a base at such a base site (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen is replaced with nitrogen (azaribose)). Examples of Super A, Super G, and Super T derivatives can be found in U.S. Pat. No. 6,683,173 (Epoch Biosciences), which is incorporated herein by reference in its entirety. cPent-G, cPent-AP, and Pr-AP have been shown to reduce immunostimulatory effects when incorporated into siRNA (Peacock H. et al. J. Am. Chem. Soc. 2011, 133, 9200). Pseudouracil is a naturally occurring isomerized version of uracil, with the C-glycoside rather than the usual N-glycoside in uridine. Pseudouracil-containing synthetic mRNAs may have an improved safety profile compared to uridine-containing mPvNAs (WO2009127230, incorporated herein by reference in its entirety).

[0172] Certain nucleobases are particularly useful for increasing the binding affinity of the antisense oligomer conjugates 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-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C, and are currently the more preferred base substitutions, particularly when combined with 2'-O-methoxyethyl sugar modifications. Additional exemplary modified nucleobases include those in which at least one hydrogen atom of the nucleobase is replaced with fluorine.

[0173] Pharmaceutically acceptable salts of the antisense oligomer conjugates of the present disclosure Certain embodiments of the antisense oligomer conjugates described herein may contain basic functional groups, such as amino or alkylamino, and thus may form pharma- ceutically acceptable salts with pharma-ceutically acceptable acids. The term "pharma-ceutically acceptable salts" in this respect refers to relatively non-toxic, inorganic and organic acid addition salts of the antisense oligomer conjugates of the present disclosure. These salts may be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting the purified antisense oligomer conjugates of the present disclosure in their free base form with a suitable organic or inorganic acid, and then isolating the salts thus formed during purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, and the like (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0174] Pharmaceutically acceptable salts of the subject antisense oligomer conjugates include conventional non-toxic salts or quaternary ammonium salts of the antisense oligomer conjugates, e.g., from non-toxic organic or inorganic acids, such as those derived from inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as salts prepared from organic acids, e.g., acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothioic acid, and the like.

[0175] In certain embodiments, the antisense oligomer conjugates of the present disclosure may contain one or more acidic functional groups, and thus can form pharma- ceutically acceptable salts with pharma-ceutically acceptable bases.In these cases, the term "pharma-ceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic base addition salts of the antisense oligomer conjugates of the present disclosure.These salts can also be prepared in situ during the administration vehicle or dosage form manufacturing process, or can be prepared by separately reacting the purified antisense oligomer conjugate in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharma-ceutically acceptable metal cation, with ammonia, or with a pharma-ceutically acceptable organic primary, secondary, or tertiary amine.Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (See, for example, Berge et al., supra).

[0176] Formulations and Modes of Administration

[0177] In certain embodiments, the present disclosure provides a formulation or pharmaceutical composition suitable for therapeutic delivery of the antisense oligomer conjugates described herein. Pharmaceutical formulations comprising antisense oligomers (e.g., PPMOs) conjugated to cell membrane-permeable peptides for DMD are described, for example, in U.S. Pat. No. 10,888,578, the disclosure of which is incorporated herein by reference. In certain embodiments, the present disclosure provides a pharma- ceutically acceptable composition comprising a therapeutically effective amount of one or more of the antisense oligomer conjugates described herein, formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. Although it is possible to administer the antisense oligomer conjugates of the present disclosure alone, it is preferred to administer the antisense oligomer conjugates as a pharmaceutical formulation (composition). In one embodiment, the antisense oligomer conjugate of the formulation is according to formula (I).

[0178] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antisense oligomer of the present disclosure, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, in some embodiments, the pharma- ceutically acceptable carrier is saline, including phosphate buffer.

[0179] The phrase "pharmacologically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients of a formulation and / or the subject being treated therewith.

[0180] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any kind. Some examples of materials that can serve as pharmaceutically acceptable carriers include, according to the discretion of the formulator, sugars such as lactose, glucose, sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, oleic acid ... Leaves oil, corn oil, and soybean oil, glycols such as propylene glycol, esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffers, non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants.

[0181] Methods for delivery of nucleic acid molecules that may be applicable to the antisense oligomer conjugates of the present disclosure are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, CRC Press; and Sullivan et al., PCT WO94 / 02595. These and other protocols can be utilized for delivery of virtually any nucleic acid molecule, including the antisense oligomer conjugates of the present disclosure.

[0182] The pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., as a drench (aqueous or non-aqueous, solution, or suspension), tablet (targeted for buccal, sublingual, or systemic absorption), bolus, powder, granule, paste for application to the tongue; (2) parenteral administration, e.g., as a sterile solution or suspension, or sustained release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; (3) topical application, e.g., as a cream, ointment, or controlled release patch or spray for application to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; (5) sublingual administration; (6) ocular administration; (7) transdermal administration; or (8) nasal administration.

[0183] Some examples of materials that can serve as pharma- ceutically acceptable carriers include, but are not limited to, (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) cellulose acetate. glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0184] Additional non-limiting examples of drugs suitable for formulation with the antisense oligomer conjugates of the present disclosure include PEG-conjugated nucleic acids, phospholipid-conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (e.g., Pluronic® P85) which can enhance drug entry into various tissues, biodegradable polymers such as poly(D,L-lactide-coglycolide) microspheres for sustained delivery after implantation (Emerich, DF et al., 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass., and loaded nanoparticles such as those composed of polybutyl cyanoacrylate which can deliver drugs across the blood-brain barrier and alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

[0185] The present disclosure also features the use of compositions comprising surface-modified liposomes containing poly(ethylene glycol) ("PEG") lipids (PEG-modified, branched or unbranched, or combinations thereof, or long-circulating or stealth liposomes). The oligomeric conjugates of the present disclosure can also contain covalently attached PEG molecules of various molecular weights. These formulations provide a method for increasing drug accumulation in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear cell phagocytic system (MPS, or RES), thereby allowing for extended blood circulation time and enhanced tissue exposure of encapsulated drugs (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to selectively accumulate in tumors, presumably by extravasation and entrapment in angiogenic target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes, which are known to accumulate in MPS tissues (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International Patent Publication No. WO96 / 10391; Ansell et al., International Patent Publication No. WO96 / 10390; Holland et al., International Patent Publication No. WO96 / 10392). Long-circulating liposomes also likely protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

[0186] In further embodiments, the present disclosure includes antisense oligomer pharmaceutical compositions prepared for delivery, as described in U.S. Patent Nos. 6,692,911, 7,163,695, and 7,070,807. In this regard, in one embodiment, the present disclosure provides an antisense oligomer conjugate of the present disclosure in a composition comprising any of the above-mentioned copolymers of lysine and histidine (HK) (described in U.S. Patent Nos. 7,163,695, 7,070,807, and 6,692,911), alone or in combination with PEG (e.g., branched or unbranched PEG, or a mixture of both), in combination with PEG and a targeting moiety, or in combination with a crosslinker. In certain embodiments, the present disclosure provides an antisense oligomer conjugate in a pharmaceutical composition comprising gluconate-modified polyhistidine, or gluconylated polyhistidine / transferrin-polylysine. One of skill in the art will also recognize that amino acids with properties similar to His and Lys can be substituted within the compositions.

[0187] Wetting agents, emulsifying agents, and lubricants (e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0188] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0189] Methods of preparing these formulations or pharmaceutical compositions include the step of bringing into association an antisense oligomer conjugate of the present disclosure with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an antisense oligomer conjugate of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0190] Formulations of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous solution, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (an inert base, such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of an antisense oligomer conjugate of the present disclosure as an active ingredient. The antisense oligomer conjugates of the present disclosure may also be administered as a bolus, electuary, or paste.

[0191] Pharmaceutical compositions suitable for parenteral administration may contain one or more oligomer conjugates of the present disclosure in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending agents, or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In one embodiment, the antisense oligomer conjugate of the pharmaceutical composition is according to formula (I):

[0192] These pharmaceutical compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms on the subject oligomeric conjugates may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable dosage form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0193] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of the drug.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility, among other methods known in the art.The rate of absorption of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0194] Injectable depot forms can be made by forming microencapsule matrices of the subject oligomer conjugates in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of oligomer to polymer and the nature of the particular polymer used, the rate of oligomer release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0195] When the antisense oligomer conjugates of the present disclosure are administered to humans and animals as pharmaceuticals, they may be administered per se or as a pharmaceutical composition containing, for example, 0.1-99% (more preferably 10-30%) of the antisense oligomer conjugate in combination with a pharma- ceutically acceptable carrier.

[0196] The formulations or preparations of the present disclosure can be administered orally, parenterally, topically, or rectally. They are typically administered in a form suitable for each administration route. For example, they are administered in the form of tablets or capsules, by injection, inhalation, eye lotion, ointment, suppository, or infusion, topically by lotion or ointment, or rectally by suppository.

[0197] Regardless of the route of administration selected, the antisense oligomer conjugates of the present disclosure, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present disclosure, may be formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those skilled in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being unacceptably toxic to the patient.

[0198] The selected dosage level will depend on a variety of factors, including the activity of the particular antisense oligomer conjugate of the present disclosure, or ester, salt, or amide thereof, being employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular oligomer being employed, the rate and extent of absorption, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular oligomer being employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical art.

[0199] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician or veterinarian can begin administration of the antisense oligomer conjugate of the present disclosure used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of the antisense oligomer conjugate of the present disclosure is the amount of the antisense oligomer conjugate that is the lowest dose effective to produce a therapeutic effect. Such effective doses generally depend on the factors described herein. In general, oral, intravenous, intracerebroventricular, and subcutaneous doses of the antisense oligomer conjugate of the present disclosure for a patient range from about 0.0001 to about 100 mg per kilogram of body weight per day when used for the indicated effects.

[0200] In some embodiments, the antisense oligomer conjugates of the present disclosure are generally administered at a dose of about 10-160 mg / kg, or 20-160 mg / kg. In some cases, doses greater than 160 mg / kg may be necessary. In some embodiments, the intravenous dose is about 0.5 mg-160 mg / kg. In some embodiments, the antisense oligomer conjugates are administered at a dose of about 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. In some embodiments, the antisense oligomer conjugate is administered at about 10 mg / kg, 11 mg / kg, 12 mg / kg, 15 mg / kg, 18 mg / kg, 20 mg / kg, 21 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, g, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg / kg, 54 mg / kg, 55 mg / kg, 56 mg / kg, 57 mg / kg, 58 mg / kg, 59 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 105 mg / kg, 110 mg / kg, 115 mg / kg, 120 mg / kg, 125 mg / kg, 130 mg / kg, 135 mg / kg, 140 mg / kg, 145 mg / kg, 150 mg / kg, 155 mg / kg, 160 mg / kg (including all integers therebetween). In some embodiments, the oligomer is administered at 10 mg / kg. In some embodiments, the oligomer is administered at 20 mg / kg. In some embodiments, the oligomer is administered at 30 mg / kg. In some embodiments, the oligomer is administered at 40 mg / kg.In some embodiments, the oligomer is administered at 60 mg / kg. In some embodiments, the oligomer is administered at 80 mg / kg. In some embodiments, the oligomer is administered at 160 mg / kg. In some embodiments, the oligomer is administered at 50 mg / kg.

[0201] In some embodiments, the antisense oligomer of formula (VI), formula (VIII), or formula (X), or a pharma- ceutically acceptable salt thereof, is generally administered at a dose of about 10 to about 160 mg / kg, or about 20 to about 160 mg / kg. In some embodiments, the dose of the antisense oligomer of formula (VI), formula (VIII), or formula (X), or a pharma- ceutically acceptable salt thereof, for intravenous administration, is about 0.5 mg to 160 mg / kg. In some embodiments, the antisense oligomer of formula (VI), formula (VIII), or formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 10 mg / kg, 11 mg / kg, 12 mg / kg, 15 mg / kg, 18 mg / kg, 20 mg / kg, 21 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg , 48mg / kg, 49mg / kg, 50mg / kg, 51mg / kg, 52mg / kg, 53mg / kg, 54mg / kg, 55mg / kg, 56mg / kg, 57mg / kg, 58mg / kg, 59mg / kg, 60mg / kg, 65mg / kg, 70mg / kg, 75mg / kg, 80mg / kg, 85mg / kg, 90mg / kg, 95mg / kg, 100mg / kg, 105mg / kg, 110mg / kg, 115mg / kg, 120mg / kg, 125mg / kg, 130mg / kg, 135mg / kg, 140mg / kg, 145mg / kg, 150mg / kg, 155mg / kg, 160mg / kg (including all integers therebetween).In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 10 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 20 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 30 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 40 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 60 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 80 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 160 mg / kg. In some embodiments, the antisense oligomer of Formula (VI), Formula (VIII), or Formula (X), or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 50 mg / kg.

[0202] If necessary, the effective daily dose of the active compound may be administered as 2, 3, 4, 5, 6 or more partial doses, which are administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In certain circumstances, administration is a single administration per day. In certain embodiments, administration is one or more administrations every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or one or more administrations every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or one or more administrations every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as necessary to maintain the desired expression of functional dystrophin protein. In certain embodiments, administration is one or more administrations every two weeks. In some embodiments, administration is one administration every two weeks. In various embodiments, administration is one or more administrations every month. In certain embodiments, administration is one administration every month.

[0203] In various embodiments, the antisense oligomer conjugate is administered at 10 mg / kg weekly. In various embodiments, the antisense oligomer conjugate is administered at 20 mg / kg weekly. In various embodiments, the antisense oligomer conjugate is administered at 30 mg / kg weekly. In various embodiments, the antisense oligomer conjugate is administered at 40 mg / kg weekly. In some embodiments, the antisense oligomer conjugate is administered at 60 mg / kg weekly. In some embodiments, the antisense oligomer conjugate is administered at 80 mg / kg weekly. In some embodiments, the antisense oligomer conjugate is administered at 100 mg / kg weekly. In some embodiments, the antisense oligomer conjugate is administered at 160 mg / kg weekly. As used herein, weekly is understood to have the art-accepted meaning of once a week.

[0204] In various embodiments, the antisense oligomer conjugate is administered at 10 mg / kg every other week. In various embodiments, the antisense oligomer conjugate is administered at 20 mg / kg every other week. In various embodiments, the antisense oligomer conjugate is administered at 30 mg / kg every other week. In various embodiments, the antisense oligomer conjugate is administered at 40 mg / kg every other week. In some embodiments, the antisense oligomer conjugate is administered at 60 mg / kg every other week. In some embodiments, the antisense oligomer conjugate is administered at 80 mg / kg every other week. In some embodiments, the antisense oligomer conjugate is administered at 100 mg / kg every other week. In some embodiments, the antisense oligomer conjugate is administered at 160 mg / kg every other week. As used herein, every other week is understood to have the art-accepted meaning of every two weeks.

[0205] In various embodiments, the antisense oligomer conjugate is administered at 10 mg / kg every three weeks. In various embodiments, the antisense oligomer conjugate is administered at 20 mg / kg every three weeks. In various embodiments, the antisense oligomer conjugate is administered at 30 mg / kg every three weeks. In various embodiments, the antisense oligomer conjugate is administered at 40 mg / kg every three weeks. In some embodiments, the antisense oligomer conjugate is administered at 60 mg / kg every three weeks. In some embodiments, the antisense oligomer conjugate is administered at 80 mg / kg every three weeks. In some embodiments, the antisense oligomer conjugate is administered at 100 mg / kg every three weeks. In some embodiments, the antisense oligomer conjugate is administered at 160 mg / kg every three weeks. As used herein, every three weeks is understood to have the art-accepted meaning of once every three weeks.

[0206] In various embodiments, the antisense oligomer conjugate is administered at 10 mg / kg monthly. In various embodiments, the antisense oligomer conjugate is administered at 20 mg / kg monthly. In various embodiments, the antisense oligomer conjugate is administered at 30 mg / kg monthly. In various embodiments, the antisense oligomer conjugate is administered at 40 mg / kg monthly. In some embodiments, the antisense oligomer conjugate is administered at 60 mg / kg monthly. In some embodiments, the antisense oligomer conjugate is administered at 80 mg / kg monthly. In some embodiments, the antisense oligomer conjugate is administered at 100 mg / kg monthly. In some embodiments, the antisense oligomer conjugate is administered at 160 mg / kg monthly. As used herein, monthly is understood to have the art-accepted meaning of monthly.

[0207] As is understood in the art, weekly, biweekly, triweekly, or monthly administration may be single or multiple administrations, or sub-administrations, as discussed herein.

[0208] The nucleic acid molecules and antisense oligomers described herein can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, encapsulation in liposomes, iontophoresis, or incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art. In certain embodiments, microemulsion technology can be used to improve the bioavailability of lipophilic (water-insoluble) pharmaceuticals. Examples include Trimetrine (Dordunoo, SK, et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991), and REV 5901 (Sheen, PC, et al., J Pharm Sci 80(7), 712-714, 1991). Among other advantages, microemulsification provides enhanced bioavailability by preferentially directing absorption into the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing destruction of the compound in the hepatobiliary circulation.

[0209] In one aspect of the disclosure, the formulation contains micelles formed from the oligomers provided herein and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter of less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter of less than about 30 nm, or even less than about 20 nm.

[0210] While all suitable amphiphilic carriers are contemplated, currently preferred carriers are generally those that have a generally regarded as safe (GRAS) status and are capable of both solubilizing the antisense oligomer conjugates of the present disclosure and microemulsifying them at a later stage when the solution comes into contact with a complex aqueous phase (such as that found in the human gastrointestinal tract). Typically, amphiphilic components that meet these requirements have an HLB (hydrophilic-lipophilic balance) value of 2-20, and their structures contain linear aliphatic groups ranging from C-6 to C-20. Examples include polyethylene glycolated fatty acid glycerides, and polyethylene glycol.

[0211] Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various vegetable oils, fully or partially hydrogenated. Such oils may advantageously consist of tri-, di-, and mono-fatty acid glycerides and di- and mono-poly(ethylene glycol) esters of the corresponding fatty acids, with particularly preferred fatty acid compositions including 4-10% capric acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid, and 5-15% stearic acid. Another useful class of amphiphilic carriers includes partially esterified sorbitan and / or sorbitol with saturated or monounsaturated fatty acids (SPAN® series) or the corresponding ethoxylated analogues (TWEEN® series).

[0212] Commercially available amphiphilic carriers may be particularly useful, including the Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurate, and di-laurate, lecithin, polysorbate 80, and the like (manufactured and distributed by a number of companies in the United States and around the world).

[0213] In certain embodiments, delivery may occur by using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., to introduce the pharmaceutical compositions of the present disclosure into suitable host cells. In particular, the pharmaceutical compositions of the present disclosure may be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc. The formulation and use of such delivery vehicles may be carried out using known and conventional techniques.

[0214] Hydrophilic polymers suitable for use in the present disclosure are those that are readily water soluble, can be covalently attached to vesicle-forming lipids, and are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include poly(ethylene glycol) (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. In certain embodiments, the polymer has a weight average molecular weight of about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is a poly(ethylene glycol) having a weight average molecular weight of about 100 to about 5,000 daltons, or about 300 to about 5,000 daltons. In certain embodiments, the polymer is a poly(ethylene glycol) having a weight average molecular weight of about 750 daltons, e.g., PEG(750). A polymer may also be defined by the number of monomers therein; preferred embodiments of the present disclosure utilize polymers of at least about three monomers; such a PEG polymer consisting of three monomers has a molecular weight of about 132 daltons.

[0215] Other hydrophilic polymers that may be suitable for use in the present disclosure include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose, or hydroxyethylcellulose.

[0216] In certain embodiments, the formulations of the present disclosure comprise a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0217] Cyclodextrins are cyclic oligosaccharides consisting of 6, 7, or 8 glucose units, designated by the Greek letters α, β, or γ, respectively. The glucose units are linked by α-1,4-glucosidic bonds. As a result of the chair conformation of the sugar units, all the secondary hydroxyl groups (at C-2, C-3) are located on one side of the ring, while all the primary hydroxyl groups at C-6 are located on the other side. As a result, the exterior surface is hydrophilic, making cyclodextrins water-soluble. In contrast, the cavities of cyclodextrins are hydrophobic, since they are lined by hydrogens at atoms C-3 and C-5, as well as by ether-like oxygens. These matrices allow complexation with a variety of relatively hydrophobic compounds, including, for example, steroid compounds such as 17α-estradiol (see, for example, van Uden et al. Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). Complexation occurs through van der Waals interactions and hydrogen bond formation. For a general review of the chemistry of cyclodextrins, see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

[0218] The physicochemical properties of cyclodextrin derivatives depend largely on the type and degree of substitution. For example, their water solubility ranges from insoluble (e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w / v) (G-2-beta-cyclodextrin). Furthermore, they are soluble in many organic solvents. The properties of cyclodextrins allow the control of the solubility of various formulation components by increasing or decreasing their solubility.

[0219] Numerous cyclodextrins and methods for their preparation have been described. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259) and Gramera, et al. (U.S. Pat. No. 3,459,731) described electrically neutral cyclodextrins. Other derivatives include cyclodextrins with cationic character [Parmeter (II), U.S. Pat. No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), and cyclodextrins with anionic character [Parmeter (III), U.S. Pat. No. 3,426,011]. Among the cyclodextrin derivatives with anionic character, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids, and sulfonic acids are added to the parent cyclodextrin [Parmeter (III), see above]. Additionally, sulfoalkyl ether cyclodextrin derivatives have been described by Stella et al. (US Pat. No. 5,134,127).

[0220] Liposomes consist of at least one lipid bilayer membrane surrounding an aqueous interior compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically range between 0.02-0.05 μm in diameter, while large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large oligolamellar vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with several non-concentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are called multivesicular vesicles.

[0221] One aspect of the present disclosure relates to a formulation comprising a liposome containing an antisense oligomer of the present disclosure, wherein the liposome membrane is formulated to provide the liposome with increased loading capacity. Alternatively, or in addition, the antisense oligomer of the present disclosure can be contained within or adsorbed onto the liposome bilayer of the liposome. The antisense oligomer of the present disclosure can be aggregated with a lipid surfactant and loaded into the interior space of the liposome. In these cases, the liposome membrane is formulated to resist the disruptive effect of the active agent-surfactant aggregate.

[0222] According to one embodiment of the present disclosure, the lipid bilayer of the liposome contains lipids derivatized with poly(ethylene glycol) (PEG), such that PEG chains extend from the inner surface of the lipid bilayer into the interior space encapsulated by the liposome and from the exterior of the lipid bilayer into the surrounding environment.

[0223] The active agent contained within the liposomes of the present disclosure is in solubilized form. Surfactants and aggregates of active agent (such as emulsions or micelles containing the active agent of interest) may be encapsulated within the interior space of liposomes according to the present disclosure. Surfactants act to disperse and solubilize the active agent and may be selected from any suitable aliphatic, alicyclic, or aromatic surfactants, including but not limited to biocompatible lysophosphatidylcholine (LPG) of various chain lengths (e.g., from about C14 to about C20). Polymer-derivatized lipids such as PEG-lipids may also be utilized for micelle formation, as they act to inhibit micelle / membrane fusion and the addition of polymers to the surfactant molecule reduces the CMC of the surfactant and aids in micelle formation. Surfactants with CMOs in the micromolar range are preferred, and higher CMC surfactants may be utilized to prepare micelles encapsulated within the liposomes of the present disclosure.

[0224] Liposomes according to the present disclosure can be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent No. 4,235,871, published PCT application WO96 / 14057, New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; and Lasic DD, Liposomes from physics to applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, liposomes according to the present disclosure can be prepared by diffusing a lipid derivatized with a hydrophilic polymer into a preformed liposome, e.g., by exposing the preformed liposome to micelles composed of lipid-grafted polymers, at a lipid concentration corresponding to the final molar percentage of the desired derivatized lipid in the liposome. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques, as known in the art.

[0225] In another exemplary formulation procedure, the active agent is first dispersed by sonication in lysophosphatidylcholine or other low CMC surfactants (including polymer-grafted lipids) that easily solubilize hydrophobic molecules.The resulting micelle suspension of active agent is then used to rehydrate a dried lipid sample containing a suitable mole percentage of polymer-grafted lipid or cholesterol.The lipid and active agent suspension is then formed into liposomes using extrusion techniques known in the art, and the resulting liposomes are separated from the non-encapsulated solution by standard column separation.

[0226] In one aspect of the present disclosure, liposomes are prepared to have a substantially uniform size in a selected size range. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes with a selected uniform pore size, the pore size of the membrane roughly corresponding to the largest size of liposomes that will be produced by extrusion through the membrane. See, for example, U.S. Patent No. 4,737,323 (April 12, 1988). In certain embodiments, reagents such as DharmaFECT® and Lipofectamine® can be used to introduce polynucleotides or proteins into cells.

[0227] The release characteristics of the formulations of the present disclosure depend on the encapsulation material, the concentration of the encapsulated drug, and the presence of release modifiers. For example, release can be engineered to be pH-dependent using a pH-sensitive coating that releases only at low pH, such as in the stomach, or at high pH, ​​such as in the intestine. An enteric coating can be used so that release does not occur until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an early release in the stomach followed by a later release in the intestine. Release can also be engineered by including salts or pore formers, which can increase water uptake or release of the drug by diffusion from the capsule. Excipients that modify the solubility of the drug can also be used to control the release rate. Agents that enhance the degradation or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as particulates), or co-dissolved in the polymer phase depending on the compound. In most cases, the amount should be between 0.1 and 30 percent (w / w polymer). Types of degradation promoters include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, and surfactants such as Tween® and Pluronic®. Pore formers (i.e., water-soluble compounds such as inorganic salts and sugars) that impart microstructure to the matrix are added as microparticles. The range is typically between 1 and 30 percent (w / w polymer).

[0228] Uptake can also be manipulated by changing the residence time of the particles in the intestine. This can be accomplished, for example, by coating the particles with or selecting as the encapsulating material, a mucoadhesive polymer. Examples include most polymers with free carboxyl groups, such as chitosan, cellulose, and especially polyacrylates (as used herein, polyacrylate refers to polymers that contain acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).

[0229] The antisense oligomer conjugates may be formulated to be contained within or adapted to be released by a surgical or medical device or implant. In certain aspects, implants may be coated or otherwise treated with the antisense oligomer conjugates. For example, the pharmaceutical compositions of the present disclosure may be used to coat implants (i.e., the compositions may be adapted for use with medical devices by using hydrogels or other polymers, such as biocompatible and / or biodegradable polymers). Polymers and copolymers for coating medical devices with drugs are well known in the art. Examples of implants include, but are not limited to, stents, drug-eluting stents, sutures, prostheses, vascular catheters, dialysis catheters, vascular grafts, artificial heart valves, cardiac pacemakers, implantable cardioverter defibrillators, IV needles, bone and osteogenic devices, such as pins, screws, plates, and other devices, and artificial tissue matrices for wound healing.

[0230] In addition to the methods provided herein, antisense oligomer conjugates for use according to the present disclosure may be formulated for administration in any convenient manner for use in human or veterinary medicine, by analogy with other pharmaceuticals. Antisense oligomer conjugates, and their corresponding formulations, may be administered alone or in combination with other therapeutic strategies in the treatment of muscular dystrophies, such as myoblast transplantation, stem cell therapy, administration of aminoglycoside antibiotics, proteasome inhibitors, and upregulation therapy (e.g., upregulation of utrophin, the autosomal paralog of dystrophin).

[0231] In some embodiments, an additional therapeutic agent may be administered prior to, simultaneously with, or after administration of an antisense oligomer conjugate of the present disclosure. For example, an antisense oligomer conjugate may be administered in combination with a steroid and / or an antibiotic. In certain embodiments, an antisense oligomer conjugate is administered to a patient receiving background steroid therapy (e.g., intermittent or chronic / continuous background steroid therapy). For example, in some embodiments, the patient has been treated with a corticosteroid prior to administration of the antisense oligomer and continues to receive steroid therapy. In some embodiments, the steroid is a glucocorticoid or prednisone.

[0232] The described routes of administration are intended as a guide only, as one of skill in the art can readily determine the optimum route of administration, and any dosage, for any particular animal and condition.Several approaches have been attempted to introduce functional novel genetic material into cells, both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include incorporation of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.: 5074S-5079S), into nonretroviral vectors (e.g., adeno-associated virus vectors) (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434), or delivery of the transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; ​​Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. (19 ... al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209, and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855), binding to ligand-specific, cation transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624), or using naked DNA, expression vectors (Nabel et al. (1990), supra; Wolff et al. (1990) Science, 247:1465-1468).Direct injection of transgenes into tissues results in only local expression (Rosenfeld (1992) supra; Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra). Brigham et al.'s group (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) have only reported in vivo transfection of mouse lungs after either intravenous or intratracheal administration of DNA-liposome complexes. An example of a review article on human gene therapy procedures is Anderson, Science (1992) 256:808-813.

[0233] In further embodiments, the pharmaceutical compositions of the present disclosure may additionally comprise carbohydrates, as provided in Han et al., Nat. Comms. 7, 10981 (2016), the entirety of which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions of the present disclosure may comprise 5% hexose carbohydrates. For example, the pharmaceutical compositions of the present disclosure may comprise 5% glucose, 5% fructose, or 5% mannose. In certain embodiments, the pharmaceutical compositions of the present disclosure may comprise 2.5% glucose and 2.5% fructose. In some embodiments, a pharmaceutical composition of the present disclosure may comprise carbohydrates selected from arabinose present in an amount of 5% by volume, glucose present in an amount of 5% by volume, sorbitol present in an amount of 5% by volume, galactose present in an amount of 5% by volume, fructose present in an amount of 5% by volume, xylitol present in an amount of 5% by volume, mannose present in an amount of 5% by volume, a combination of glucose and fructose, each present in an amount of 2.5% by volume, and a combination of glucose present in an amount of 5.7% by volume, fructose present in an amount of 2.86% by volume, and xylitol present in an amount of 1.4% by volume.

[0234] In certain embodiments, the antisense oligomer conjugates described herein are administered in a liquid pharmaceutical formulation, wherein the concentration of the conjugate is about 50 mg / ml.

[0235] Regardless of the route of administration selected, the antisense oligomer conjugates of the present disclosure, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present disclosure, may be formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those skilled in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being unacceptably toxic to the patient.

[0236] How to use Treatment with the antisense oligomer conjugates described herein can be administered to patients in need thereof using the dosing regimens described in this disclosure.

[0237] In one aspect, the disclosure provides a method for treating DMD in a subject in need thereof, the subject having a mutation in the dystrophin gene that is amenable to exon skipping, the method comprising administering to the subject an antisense oligomer conjugate described herein. In some aspects, the exon is exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of the human dystrophin gene.

[0238] In another aspect, the disclosure provides a method of restoring the mRNA reading frame and inducing dystrophin production in a subject having a mutation in the dystrophin gene that is amenable to exon skipping (e.g., exon 44, exon 45, exon 50, exon 51, exon 52, exon 53 skipping), the method comprising administering to the subject an antisense oligomer conjugate described herein.

[0239] In another aspect, the present disclosure provides a method of excluding an exon (e.g., exon 44, exon 45, exon 50, exon 51, exon 52, exon 53) from a dystrophin pre-mRNA during mRNA processing in a subject having a mutation in the dystrophin gene that is amenable to exon skipping, the method comprising administering to the subject an antisense oligomer conjugate described herein. In another aspect, the present disclosure provides a method of binding an exon (e.g., exon 44, exon 45, exon 50, exon 51, exon 52, exon 53) of a dystrophin pre-mRNA in a subject having a mutation in the dystrophin gene that is amenable to exon skipping (e.g., exon 44, exon 45, exon 50, exon 51, exon 52, exon 53 skipping), the method comprising administering to the subject an antisense oligomer conjugate described herein.

[0240] The term "restoration" with respect to dystrophin synthesis or production generally refers to the production of dystrophin protein, including truncated forms of dystrophin, in a patient with muscular dystrophy following treatment with an antisense oligomer conjugate described herein. In some embodiments, treatment results in an increase in new dystrophin production in the patient of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween). In some embodiments, treatment increases the number of dystrophin-positive fibers in a subject to at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%-100% of normal. In other embodiments, the treatment increases the number of dystrophin-positive fibers in the subject to about 20% to about 60%, or about 30% to about 50% of normal. The percentage of dystrophin-positive fibers in a patient after treatment can be determined by muscle biopsy using known techniques. For example, a muscle biopsy can be collected from a suitable muscle, such as the patient's biceps.

[0241] Analysis of the percentage of positive dystrophin fibers can be performed before and / or after treatment, or at time points throughout the course of treatment. In some embodiments, the post-treatment biopsy is collected from the muscle contralateral to the pre-treatment biopsy. Pre-treatment and post-treatment dystrophin expression analysis can be performed using any suitable assay for dystrophin. In some embodiments, immunohistochemical detection is performed on tissue sections from muscle biopsies using an antibody that is a marker for dystrophin, such as a monoclonal or polyclonal antibody. For example, the MANDYS106 antibody, which is a sensitive marker for dystrophin, can be used. Any suitable secondary antibody can be used.

[0242] In some embodiments, the percentage of dystrophin-positive fibers is calculated by dividing the number of positive fibers by the total number of fibers counted. A normal muscle sample has 100% dystrophin-positive fibers. Thus, the percentage of dystrophin-positive fibers can be expressed as a percentage of normal. When counting dystrophin-positive fibers in post-treatment muscle, a baseline can be set using a pre-treatment muscle section from the patient to control for the presence of trace levels of dystrophin in pre-treatment muscle and revertant fibers. This can be used as a threshold for counting dystrophin-positive fibers in the post-treatment muscle sections of that patient. In other embodiments, antibody-stained tissue sections can also be used for dystrophin quantification using Bioquant image analysis software (Bioquant Image Analysis Corporation, Nashville, TN). Total dystrophin fluorescent signal intensity can be reported as a percentage of normal. Additionally, Western blot analysis using monoclonal or polyclonal anti-dystrophin antibodies can be used to determine the percentage of dystrophin-positive fibers. For example, the anti-dystrophin antibody NCL-Dysl, from Leica Biosystems, can be used. The percentage of dystrophin-positive fibers can also be analyzed by determining the expression of components of the sarcoglycan complex (β, γ) and / or neuronal NOS.

[0243] In some embodiments, treatment with the antisense oligomer conjugates of the present disclosure slows or reduces the progressive respiratory muscle dysfunction and / or failure in DMD patients that would be expected without treatment. In some embodiments, treatment with the antisense oligomer conjugates of the present disclosure can reduce or eliminate the need for ventilatory support that would be expected without treatment. In some embodiments, measurements of respiratory function to track disease progression and evaluate potential therapeutic interventions include maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and forced vital capacity (FVC). MIP and MEP measure the pressure levels a person can generate during inhalation and exhalation, respectively, and are sensitive measures of respiratory muscle strength. MIP is a measure of diaphragm muscle weakness.

[0244] In some embodiments, MEP may decline before changes in other pulmonary function tests, including MIP and FVC. In certain embodiments, MEP may be an early indicator of respiratory dysfunction. In certain embodiments, FVC may be used to measure the total volume of air expelled during forced expiration after maximal inspiration. In DMD patients, FVC increases in parallel with physical growth until the early teens. However, as growth slows or becomes poorer with disease progression and muscle weakness progresses, vital capacity enters a decline phase, declining at an average rate of about 8-8.5 percent per year after age 10-12. In certain embodiments, percent predicted MIP (MIP adjusted for weight), percent predicted MEP (MEP adjusted for age), and percent predicted FVC (FVC adjusted for age and height) are supportive analyses.

[0245] As used herein, the terms "subject" and "patient" include any animal that exhibits or is at risk of exhibiting a condition that can be treated with the antisense oligomer conjugates of the present disclosure, such as a subject (or patient) that has or is at risk of having DMD or BMD, or any of the symptoms associated with these conditions (e.g., muscle fiber loss). Suitable subjects (or patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and household animals, or pets (such as cats or dogs). Non-human primates, and preferably human patients (or subjects) are included. Also included are methods of producing dystrophin in subjects (or patients) that have a mutation in the dystrophin gene that is amenable to exon skipping (e.g., exon 44, exon 45, exon 50, exon 51, exon 52, exon 53 skipping).

[0246] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to the administration of a compound, drug, or other substance directly into the central nervous system, thereby entering the patient's system and therefore being subject to metabolism and other similar processes, such as, for example, subcutaneous administration.

[0247] The phrase "target sequence" refers to a sequence of nucleobases of an oligomer that is complementary to a sequence of nucleotides in a target pre-mRNA. In some embodiments of the present disclosure, the sequence of nucleotides in the target pre-mRNA is an exon 51 annealing site in a dystrophin pre-mRNA designated as H51A (+66 + 95). In some embodiments of the present disclosure, the sequence of nucleotides in the target pre-mRNA is an exon 45 annealing site in a dystrophin pre-mRNA designated as H45A (-03 + 19). In some embodiments, the sequence of nucleotides in the target pre-mRNA is an exon 53 annealing site in a dystrophin pre-mRNA designated as H53A (+36 + 60).

[0248] "Treatment" of a subject (e.g., a mammal, such as a human) is any type of intervention used in an attempt to change the natural course of the subject. Treatment includes, but is not limited to, administration of an antisense oligomer conjugate, or a pharmaceutical composition thereof, and can be performed either prophylactically or after the onset of a pathological event or contact with a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition associated with a dystrophin protein, such as in certain forms of muscular dystrophy, and can include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. Also included is "prophylactic" treatment, which can be aimed at slowing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate a complete eradication, cure, or prevention of the disease or condition, or its associated symptoms.

[0249] In some embodiments, treatment with an antisense oligomer of the present disclosure increases new dystrophin production, slows disease progression that would be expected without treatment, slows or reduces lameness, reduces muscle inflammation, reduces muscle damage, improves muscle function, reduces loss of lung function, and / or enhances muscle regeneration. In some embodiments, treatment maintains, slows, or slows disease progression. In some embodiments, treatment maintains ambulation or reduces lameness. In some embodiments, treatment maintains lung function or reduces loss of lung function. In some embodiments, treatment maintains or increases a patient's stable walking distance, for example, as measured by the 6-minute walk test (6MWT). In some embodiments, treatment maintains or reduces the time to walk / run 10 meters (i.e., 10-meter walk / run test). In some embodiments, treatment maintains or reduces the time to stand up from a supine position (i.e., time to stand test). In some embodiments, the treatment maintains or reduces the time to climb four standard stairs (i.e., the four-step climb test). In some embodiments, the treatment maintains or reduces inflammation in the patient's muscles, for example, as measured by MRI (e.g., MRI of leg muscles). In some embodiments, the MRI measures T2 and / or fat fraction to identify muscle degeneration. MRI can identify changes in muscle structure and composition caused by inflammation, edema, muscle damage, and fat infiltration.

[0250] In some embodiments, treatment with the antisense oligomer conjugates of the present disclosure increases new dystrophin production and delays or reduces the loss of ambulation that would be expected without treatment. For example, treatment may stabilize, maintain, improve, or increase the walking ability of a subject (e.g., stabilization of ambulation). In some embodiments, treatment maintains or increases the patient's stable walking distance as measured by the 6-minute walk test (6MWT), for example, as described by McDonald, et al. (Muscle Nerve, 2010; 42:966-74, incorporated herein by reference). The change in 6-minute walk distance (6MWD) may be expressed as an absolute value, a percentage change, or a change in % predicted value. In some embodiments, treatment maintains or improves stable walking distance in the 6MWT from a 20% deficit in the subject compared to healthy counterparts. The performance of DMD patients in the 6MWT compared to the typical performance of healthy counterparts may be determined by calculating the % predicted value. For example, the % predicted 6MWD for men may be calculated using the following equation: 196.72+(39.81×age)−(1.36×age) 2 ) + (132.28 × height in meters). For women, the % predicted 6MWD can be calculated using the following equation: 188.61 + (51.50 × age) - (1.86 × age 2 ) + (86.10 × height in meters) (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference). In some embodiments, treatment with an antisense oligomer increases stable walking distance in a patient from baseline to greater than 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 50 meters (including all integers therebetween).

[0251] Loss of muscle function in DMD patients may occur against the background of normal childhood growth and development. Indeed, children with DMD may show an increase in walking distance during the 6MWT over the course of about a year, despite progressive muscle impairment. In some embodiments, 6MWD from DMD patients is compared to existing normative data from typically developing control subjects and age- and sex-matched subjects. In some embodiments, normal growth and development may be accounted for using an age- and height-based equation fitted to normative data. Using such an equation, 6MWD in subjects with DMD may be converted to percent predicted (% predicted) values. In certain embodiments, analysis of % predicted 6MWD data represents a way to account for normal growth and development, and may show that increases in function at younger ages (e.g., 7 years and younger) represent a stabilization rather than an improvement in performance in DMD patients (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference).

[0252] In order to distinguish between different antisense molecules, a nomenclature system for antisense molecules has been proposed and published (see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature system has become particularly suitable when testing several slightly different antisense molecules all directed against the same target region, as shown below. [number] H#A / D(x:y)

[0253] The first letter indicates the species (e.g., H: human, M: mouse, C: dog). "#" indicates the target dystrophin exon number. "A / D" indicates the acceptor or donor splice site at the beginning and end of the exon, respectively. (xy) represents the annealing coordinates, and "-" or "+" indicates the intron or exon sequence, respectively. For example, A(-6+18) indicates the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The closest splice site is the acceptor, so these coordinates are preceded by "A". The description of the annealing coordinates at the donor splice site can be D(+2-18), where the last 2 exon bases and the first 18 intron bases correspond to the annealing site of the antisense molecule. The entire exon annealing coordinate is represented by A(+65+85), ie, the site from 65th to 85th nucleotides from the start of the exon.

[0254] Restoring the dystrophin reading frame using exon skipping Potential therapeutic approach to treat DMD caused by out-of-frame mutation of dystrophin gene is suggested by milder form of dystrophinopathy known as BMD caused by in-frame mutation.The ability to convert out-of-frame mutation into in-frame mutation hypothetically keeps mRNA reading frame and produces internally truncated but functional dystrophin protein.The antisense oligomer conjugate of the present disclosure is designed to achieve this.

[0255] Clinical outcomes to analyze the effect of antisense oligomer conjugates complementary to a target region of human dystrophin pre-mRNA and inducing exon skipping include percentage of dystrophin positive fibers (PDPF), 6 minute walk test (6MWT), loss of ambulation (LOA), North Star Ambulation Assessment (NSAA), pulmonary function tests (PFTs), ability to stand up (from supine position) without external support, de novo dystrophin production, and other functional measures.

[0256] In some embodiments, the present disclosure provides a method for producing dystrophin in a subject having a mutation in the dystrophin gene that is amenable to exon skipping (e.g., exons 44, 45, 50, 51, 52, 53), the method comprising administering to the subject an antisense oligomer conjugate, or a pharma- ceutically acceptable salt thereof, as described herein. In certain embodiments, the present disclosure provides a method for restoring the mRNA reading frame to induce dystrophin protein production in a subject having Duchenne muscular dystrophy (DMD) having a mutation in the dystrophin gene that is amenable to exon skipping (e.g., exons 44, 45, 50, 51, 52, 53). Protein production can be measured by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, or immunohistochemistry (IHC).

[0257] In some embodiments, the disclosure provides a method of treating DMD in a subject in need thereof, the subject having a mutation in the dystrophin gene that is amenable to exon skipping (e.g., exons 44, 45, 50, 51, 52, 53), the method comprising administering to the subject an antisense oligomer conjugate, or a pharma- ceutically acceptable salt thereof, as described herein. In various embodiments, the treatment of the subject is measured by delaying disease progression. In some embodiments, the treatment of the subject is measured by maintaining ambulation in the subject, or reducing ambulation in the subject. In some embodiments, ambulation is measured using the 6-minute walk test (6MWT). In certain embodiments, ambulation is measured using the North Start Ambulation Assessment (NSAA).

[0258] In various embodiments, the present disclosure provides a method for maintaining lung function or reducing loss of lung function in a subject with DMD, the subject having a mutation in the DMD gene that is amenable to exon skipping (e.g., exons 44, 45, 50, 51, 52, 53), the method comprising administering to the subject an antisense oligomer conjugate, or a pharma- ceutically acceptable salt thereof, as described herein. In some embodiments, the lung function is measured as maximum expiratory pressure (MEP). In certain embodiments, the lung function is measured as maximum inspiratory pressure (MIP). In some embodiments, the lung function is measured as forced vital capacity (FVC).

[0259] In certain aspects, the methods of the disclosure comprise administering to a subject having DMD a pharmaceutical formulation comprising an antisense oligomer conjugate described herein, or a pharma- ceutically acceptable salt thereof, wherein the concentration of the conjugate in the formulation is about 50 mg / ml.

[0260] In certain embodiments, the antisense oligomer conjugate described herein is described for use in therapy.In certain embodiments, the antisense oligomer conjugate described herein is described for use in the treatment of Duchenne muscular dystrophy.In certain embodiments, the antisense oligomer conjugate described herein is described for use in the manufacture of a medicament for use in therapy.In certain embodiments, the antisense oligomer conjugate described herein is described for use in the manufacture of a medicament for the treatment of Duchenne muscular dystrophy. EXAMPLES

[0261] Although the above disclosure has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be readily apparent to one skilled in the art, in light of the teachings of the present disclosure, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce substantially similar results.

[0262] Example 1. In vitro evaluation of exon 51 skipping activity by PPMO of formula (VIC) The exon 51 skipping activity of all six PPMOs, which conform to the structure of the following formula (VIC), was evaluated: [ka] wherein m is 0, 1, 2, 3, 4, or 5, and the target sequence is 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO:1).

[0263] The six PPMOs tested were synthesized and characterized in-house according to the protocols described, for example, in U.S. Pat. No. 10,888,578 and are listed in Table 1 (G is glycine and R is arginine). [Table 1] Table 1

[0264] Exon skipping analysis The in vitro exon skipping activity of six PPMOs was measured in a cellular model of DMD using donor-derived immortalized myoblasts carrying an exon 51 skipping-optimized deletion in exon 52 of the DMD gene. For the assay, myoblasts were differentiated into myotubes and exon skipping activity was measured by ddPCR after 96 hours of treatment with PPMOs at concentrations ranging from 0.1 to 100 μM. The potency and maximum exon skipping activity of the tested PPMOs measured at 100 μM are shown in Table 2 below. Maximum exon skipping activity is the mean and standard deviation of the percentage of skipped copies of four technical replicates measured at the concentration that showed maximum activity. [Table 2]

[0265] EC of PPMO-11 and PPMO-10 50 Values ​​could not be determined precisely due to incomplete concentration-response curves.

[0266] The results show that all of the PPMOs tested are pharmacologically active and exhibit exon skipping activity. All PPMOs tested had measurable, concentration-dependent exon skipping activity in the assay.

[0267] Materials and Methods Cell lines and culture conditions Myoblasts isolated from adrenal muscle of a 16-year-old healthy donor (reference AB1190C16PV) and a 16-year-old DMD patient with an exon 52 deletion (reference KM1328DMD16PV) and immortalized by the Institute of Myology by ectopic expression of hTERT and CDK4 as previously described (Mamchaoui, K. et al., Skeletal Muscle 1(1):34, 2011) were used for this assay. Cells were plated at 50 μl / cm in growth medium containing Medium 199, 1 volume Dulbecco's Modified Eagle Medium (DMEM), 4 volumes 20% fetal bovine serum, 50 μg / ml gentamicin, 25 μg / ml fetuin, 0.5 μg / ml bFGF, 5 ng / ml EGF, 0.2 μg / ml dexamethasone, 5 μg / ml insulin, 1% collagen I, and 0.5% MaxGel (Sigma-Aldrich E0282)-coated tissue culture plates. 2 The mixture was incubated at 37°C for 3 hours.

[0268] Compound Testing Immediately prior to testing, all compounds were dissolved in sterile water and concentrations were confirmed by spectrophotometer. Myoblasts were seeded in 96-well clear-bottom imaging plates (Perkin Elmer #6055300) coated with 1% collagen I and 0.5% MaxGel (Sigma-Aldrich E0282) at 50 μl / well in growth medium for 3 hours at 37°C, at 6000 cells / well. 24 hours after seeding, cultures were switched to differentiation medium containing DMEM, 2% heat-inactivated FBS, 50 μg / ml gentamicin, and 10 μg / ml insulin. 48 hours after switching to differentiation medium, PPMO was added and cultures were incubated for an additional 4 days before analysis, for a total of approximately 96 hours of continuous compound exposure.

[0269] ddPCR analysis of human DMD Exon51 skipping RNA was isolated using RN easy Micro-column (QIAGEN Catalog No. 74004) with DNAase treatment according to the manufacturer's recommendations. 30 ng of isolated RNA was first denatured at 70°C for 2 minutes and mixed with reagents from One-step RT-ddPCR Advanced Kit for Probes (BioRad Catalog No. 1864021) and PNP mix according to Table 3 below. Droplets were generated from the prepared RNA sample mix using an automated droplet generator. After droplet generation, the plate was sealed and run on a C 1000 thermocycler (BioRad) according to the thermocycler program in Table 4 below. The copy numbers of FAM and HEX positive droplets are determined by a QX200 droplet reader. The percentage of exon skipping is determined as copy number of FAM positive droplets / (copy number of FAM positive droplets+copy number of HEX positive droplets)*100. All data were analyzed using GraphPad Prism 8 and EC50s were determined based on four-parameter logistic curve fitting. [Table 3] [Table 4]

[0270] Example 2. In vivo studies of exposure of PPMO of formula (VIC) in non-human primates (NHP) and mdx mice after administration of PPMO-1 1. NHP Study. Assessment of plasma exposure of PPMO of formula (VIC) following intravenous (IV) administration of PPMO-1 to cynomolgus monkeys. Non-human primates (NHPs) received 1-hour IV infusions of PPMO-1 at dose levels of 30 or 60 mg / kg once every 4 weeks, i.e., on days 1, 29, 57, and 85. Blood samples were collected pre-dose on day 1, and 1, 2, 4, 8, 12, 16, and 24 hours after the start of each infusion. Blood was processed to plasma for analysis of PPMO-1 and its metabolite concentrations by liquid chromatography mass spectrometry (LC / MS / MS). PPMO-1 is an antisense oligomer conjugate having the following structure: [ka]

[0271] 2. Test in mdx mice. 14 Assessment of the distribution of PPMO of formula (VIC) after a single intravenous (IV) administration of C-PPMO-1 Male mdx mice received an average dose of 51.6 mg / kg. 14 They received a single intravenous injection of C-PPMO-1. 14 C-PPMO-1 was formulated as an aqueous solution in 0.9% (w / v) sodium chloride for injection at 10 mg / mL and administered at a radioactivity level of 220 μCi / kg animal weight. Blood samples were collected from each mouse by cardiac puncture at approximately 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing. Plasma samples obtained from a second group of male mice at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing were pooled by time point to generate nine pooled samples, each containing 0.3 g of each sample. The nine pools were then pooled to generate a single 0.083-24 hour AUC representative pooled sample, containing each time point pool between 2.84 and 306 μL, as determined using a time-weighted pooling method (Hop et al., 1998). Urine samples collected from male mice at 0-24 and 24-48 hours post-dose were pooled across all animals, with 0.3g-0.5g of each sample. Samples were pooled using a constant percentage (10%) of sample weight.

[0272] Fecal samples collected from male mice at 0–24, 24–48, and 48–72 hours post-dose were pooled across all animals as appropriate, with 1.1–1.6 g of each sample. Samples were pooled using a constant percentage (5%) of sample weight.

[0273] For quantitative analysis of PPMO-1 and its metabolites, the radioactivity of each pooled sample was determined by liquid scintillation counting (LSC) and LC / MS.

[0274] result: PPMO-1 was identified as the major analyte in NHP plasma, and PPMO-10 and PPMO-11 were administered PPMO-1 at 30 mg / kg and 60 mg / kg, respectively, and the AUC last The major metabolites were identified in 10.5%, 6.7%, 3.7%, and 3.1% of the total exposure (AUC last ) were 257±138 h*ug / mL, 20±7 h*ug / mL, and 8±4 h*ug / mL for PPMO-1, PPMO-10, and PPMO-11, respectively. Five other metabolites (PPMO-12, PPMO-13, PPMO-14, PPMO-15, and PMO) were detected and identified at lower levels than PPMO-10 and PPMO-11. Note that the observed PPMO-15 data is at least partially due to leakage of the LC-MS / MS signal due to the similar retention time of PPMO-1, and therefore PPMO-15 levels may be overestimated.

[0275] In mdx mice, PPMO-10 and PPMO-11 metabolites were identified in all matrices (plasma, urine, and feces). Radiochemical and LC-MS analysis of plasma extracts demonstrated that SRP-5051 was 14 C-PPMO-1 was identified as the most abundant plasma component after intravenous administration. Under initial LC-MS conditions (gradient 1), AUC pooled plasma 14 The concentration of C-PPMO-1 was 1510 ng equivalents. 14C-PPMO-1 / g (ng equivalents / g), or 66.6% of the sample radioactivity. The concentrations of coeluting PPMO-11 and PPMO-10 were 547 ng equivalents / g, or 24.1% of the sample radioactivity. M3, which was not identified by LC-MS, had a plasma concentration of 69.7 ng equivalents / g, or 3.1% of the sample radioactivity. Under the revised LC-MS conditions (gradient 2), the concentrations of PPMO-1, PPMO-11, and PPMO-10 were 1370, 251, and 258 ng equivalents / g, respectively, or 60.5%, 11.1%, and 11.4% of the sample radioactivity, respectively. Unidentified M4 had a plasma concentration of 138 ng / g, or 6.1% of the plasma radioactivity.

[0276] In urine, PPMO-1, PPMO10, and PPMO-11 were identified by radiochemical and LC-MS analysis. Two additional radiolabeled components (M4 and M5) were quantified by radiochemical analysis but could not be identified by LC-MS. Under the initial LC-MS conditions (gradient 1), PPMO-1 accounted for 24.3% of the radioactive dose, and the coeluting PPMO-11 and PPMO-10 accounted for 32.1% of the dose. Under revised LC-MS conditions (gradient 2), PPMO-1, PPMO11, and PPMO-10 were partially resolved and accounted for 22.0%, 14.6%, and 17.3% of the dose, respectively. Unidentified peaks M4 and M5 were ultratrace-to-trace components accounting for approximately 0.35% and 1.1% of the dose, respectively. Radiochemical and LC-MS analysis of fecal extracts quantified and identified PPMO-11 and PPMO-10 as minor metabolites. PPMO-1 was not detected in feces. M5 was quantified by radiochemical analysis but not identified by LC-MS. The coeluting (gradient 1) metabolites PPMO-11 and PPMO-10 accounted for 6.3% of the dose. Under revised LC-MS conditions (gradient 2), PPMO-11 and PPMO-10 accounted for approximately 1.6% and 4.5% of the dose, respectively, while the unidentified minor component M5 accounted for less than 0.5% of the dose.

[0277] conclusion PPMO-1 and its seven hydrolytic metabolites were detected and quantified using an LC / MS method after IV infusion of 30 and 60 mg / kg PPMO-1 into NHPs. PPMO-1 was identified as the major component with the highest levels observed at the end of the infusion and then biexponentially declining. The magnitudes of all metabolites were much lower than that of PPMO-1, especially during the first 8 h after administration. PPMO-10 was found to be the most abundant metabolite, followed by PPMO-11, accounting for 7-10% and 3-4% of the PPMO-1 AUC, respectively, while the remaining five metabolites were detected at much lower concentrations and were only partially quantifiable.

[0278] Male mdx mice 14 After a single intravenous dose of C-PPMO-1, PPMO-1 was the most abundant component in plasma and urine, accounting for 60-67% of the radioactivity in plasma samples and 22% of the urinary dose. PPMO-1 was not detectable in feces. PPMO-11 and PPMO-10 were identified as the major metabolites in plasma, urine, and feces. Three additional radiolabeled peaks were quantified by radiochemical detection but could not be identified by LC-MS.

[0279] Example 3. 14 In vivo study of plasma and tissue distribution of PPMO of formula (VIIIC) in mdx mice after administration of C-PPMO-2

[0280] Testing in mdx mice. 14 Assessment of the distribution of PPMO of formula (VIIIC) after a single intravenous (IV) administration of C-PPMO-2

[0281] Mdx mice received an average dose of 53.6 mg / kg. 14 Received a single IV bolus injection of C-PPMO-2. 14C-PPMO-2 was formulated at 10 mg / mL in 0.9 sodium chloride aqueous solution and administered at an average radioactivity level of 228 μCi / kg animal weight. Samples of whole blood and selected tissues were collected at approximately 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 144, 288, 360, and 432 hours after dosing. Plasma samples obtained from male mdx mice at 0.083, 0.25, 0.5, 1, 2, and 4 hours after dosing were pooled by time point to generate 0.083, 0.25, 0.5, 1, 2, and 4 hour pooled samples containing 0.1 g of each sample. Urine samples collected 0-24 hours and 24-72 hours after dosing were pooled to generate 0-24 hour and 24-72 hour pooled samples containing 15% by weight of each sample. Fecal samples collected at 0–24, 24–48, and 48–72 hours post-dose were pooled by collection interval to generate 0–24 hour and 24–72 hour pooled samples, which contained 6–10% (equivalent weight percent) of each sample.

[0282] Muscle and kidney samples collected from mice at 2, 24, 48, 96, 144, 288, 360, and 432 hours post-dose were pooled by collection interval to generate pooled samples containing the entire sample at 2 hours, 24-96 hours, 144-288 hours, and 360-432 hours. The radioactivity of each pooled sample was determined by LSC. The pooled samples were analyzed by LC / MS to determine the concentrations of PPMO-2 and its metabolites. PPMO-2 is an antisense oligomer conjugate having the following structure: [ka]

[0283] result 14 C- PPMO-2 is 14 After a single intravenous administration of C-PPMO-2, it underwent metabolism in male mdx mice. Five metabolites were identified by LC-MS and characterized in plasma, urine, feces, muscle, and kidney. The identified compounds have a structure according to formula (VIIIC): [ka]

[0284] wherein the target sequence is 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO:2) and m is as set forth in Table 5 below: [Table 5]

[0285] PPMO-24, PPMO-23, and PPMO-22 were present in all matrices except for feces, PPMO-21 and PPMO-20 were present in all matrices, and PPMO-2 was identified in all matrices except for feces.

[0286] The most abundant plasma component was PPMO-2, with a peak concentration of 206 μg equiv. 14 C-PPMO-2 / g (representing 67.5% of the total AUC determined for PPMO-2 and identified metabolites and 33.48-91.52% of the total radioactivity injected onto the HPLC column). The peak concentrations of PPMO-24, PPMO-23, PPMO-22, PPMO-21, and PPMO-20 were 5.56, 4.86, 5.87, 10.8, and 9.40 μg equivalents. 14 C-PPMO-2 / g. Based on AUC0-t, all metabolites identified and quantifiable in plasma represented less than 10% of the total AUC of identified metabolites and were considered trace. The exception was PPMO-20, which represented 14.3% of the total. PPMO-20, accounting for 29.8% of the administered dose over 0-72 hours post-dose, was the most abundant component in urine. PPMO-2 represented 3.22% of the administered dose in urine over 0-24 hours post-dose and was not detectable in urine over 24-72 hours post-dose. PPMO-20 was also the most abundant component in feces, accounting for 2.44% of the administered dose over 0-72 hours post-dose. PPMO-2 was not observed in feces.

[0287] For the tissues (biceps and kidney), PPMO-20 was again the most abundant component, with peak concentrations of 3.32 and 960 μg equiv., respectively. 14 C-PPMO-2 / g (between 12.03 and 15.28% of the total radioactivity injected onto the HPLC column for muscle, and 83.73 and 92.32% for kidney). PPMO-2 was quantifiable in biceps muscle at 2 h post-dose and in kidney up to 432 h post-dose in all sample pools, with peak concentrations of 0.863 and 33.1 μg equivalents, respectively. 14 C-PPMO-2 / g.

[0288] conclusion Male mdx mice 14 Following a single IV dose of C-PPMO-2, SRP-5045 was metabolized in male mice, producing up to 11 14 Hydrolysis of the terminal arginine (R) amino acid with loss of the N-acetyl group produced C-related peaks, five of which were identified by LC-MS. 14 C-PPMO-2 was the major biotransformation pathway. PPMO-20 and PPMO-21 metabolites were identified in all matrices. Other metabolites were identified in all matrices, except feces. PPMO-2 was present in all matrices, except feces.

[0289] PPMO-2 was identified as the major component in plasma with a peak concentration at 0.083 hours post-dose representing 91.52% of sample radioactivity. PPMO-20 was associated with a median exposure of 1.01 mg / kg / day (AUC last PPMO-20 was identified as the major metabolite, accounting for 14.3% of the total AUC. All other metabolite exposures ranged between 2.98 and 7.52% of the total AUC. PPMO-20 was identified as the major metabolite in urine, feces, biceps, and kidney, accounting for peak concentrations of 29.8% (0-24 h), 2.44% (0-24 h), and 37.5 and 87.67% of the sample radioactivity in urine and feces, and in biceps and kidney, respectively. Other metabolites were identified as minor metabolites in all matrices.

[0290] Example 4 14 In vivo study of plasma and tissue distribution of PPMO of formula (XC) in mdx mice after administration of C-PPMO-3 Testing in mdx mice Testing in male dystrophic (mdx) mice 14 Evaluation of the distribution of PPMO of formula (XC) after a single intravenous (IV) administration of C-PPMO-3.

[0291] mdx mice received an average dose of 48.7 mg / kg; 14 They received a single IV bolus injection of C-PPMO-3. PPMO-3 was formulated as an aqueous solution in 0.9% (w / v) sodium chloride at 10 mg / mL and administered at an average radioactivity level of 215 μCi / kg animal weight. Urine and feces were collected pre-dose (overnight) and at 24-h intervals up to 336 h post-dose. Samples of whole blood and selected tissues were collected at approximately 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, 72, 96, 120, and 144 h post-dose.

[0292] Plasma samples obtained from male mice at 0.083, 0.25, 0.5, 1, and 2 hours post-dose were pooled to generate pooled samples containing equal volumes of each sample at 0.083, 0.25, 0.5, 1, and 2 hours. Urine samples collected from male mice at 0-24, 24-48, 48-72, 72-96, 96-120, 120-144, 144-168, 168-192, 192-216, 216-240, 288-312, and 312-336 hours post-dose were pooled to generate pooled samples containing 10-20% by weight of each sample (evenly spaced percentages). Fecal samples collected from male mice at 0–24, 24–48, 48–72, 72–96, 96–120, 120–144, 288–312, and 312–336 h post-dose were pooled to generate 0–72, 72–144, and 288–336 h pooled samples containing 4–5% by weight of each sample (evenly spaced percentages).

[0293] Biceps muscle samples obtained from male mice at 0.25, 1, 4, 8, 24, 48, 96, 120, and 144 hours post-dose were pooled to generate pooled samples containing 100% of each sample by weight: 0.25-4, 8, 24, 48, and 96-144 hours.

[0294] Kidney samples obtained from male mice at 0.25, 1, 4, 8, 24, 48, 96, 120, and 144 h post-dose were pooled to generate pooled samples of 0.25–4, 8, 24, 48, and 96–144 h containing 100% by weight of each sample.

[0295] Samples from all matrices were analyzed by LSC to determine radioactivity and by LC / MS to quantify PPMO-3 and its metabolites.

[0296] PPMO-3 is an antisense oligomeric conjugate having the following structure: [ka]

[0297] result 14 C- PPMO-3 is 14 After a single intravenous administration of C-PPMO-3, four metabolites were identified and characterized by LC-MS in male mice. The identified compounds have structures according to formula (XC): [ka]

[0298] wherein the target sequence is 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO:3) and m is as set forth in Table 6 below: [Table 6]

[0299] PPMO-30 and PPMO-31 were also synthesized in-house. PPMO-33 and PPMO-34 were present in plasma and urine, PPMO-31 was present in plasma, urine, and feces, and PPMO-30 was present in plasma, urine, feces, and kidney. Metabolite PPMO-30 was the most abundant metabolite identified in urine and feces, and PPMO-31 was also present at significant levels in urine. The most abundant plasma components were PPMO-3 and PPMO-33, which co-eluted with a combined peak concentration (Cmax) of 109,000 ng-eq / g. Due to the broad chromatographic peaks in the plasma samples, the retention times of PPMO-3 and PPMO-33 are similar, therefore the PPMO-33 contribution is overestimated and may be partially attributed to PPMO-3. PPMO-31 had a Cmax of 13,200 ng-eq / g. max Another component of noteworthy concentration was U5 (unspecified) with a peak concentration of 33,100 ng-eq / g. The mean Cmax of radioactivity in plasma for PPMO-3 and related compounds was observed at 0.083 hours post-dose. The highest C0 value was observed for PPMO-33 and PPMO-3 (coeluting peaks), with a plasma half-life of 0.502 hours and exposure (AUC 0-t ) was 42,000 ng-eq·h / g, and the total AUC 0-t (calculated based on the total AUC of identified metabolites and unidentified components for which pharmacokinetic parameters could be calculated). PPMO-31 was present at a significant concentration, 0-t The CO was 14,600 ng-eq / g and the exposure was 10,900 ng-eq·h / g, representing 11.34% of the total.

[0300] The most abundant component in urine was PPMO-30, accounting for 27.3% of the administered dose over 0-336 hours post-dose. PPMO-31 was also prominent, accounting for 15.3% of the administered dose over 0-336 hours post-dose. PPMO-33 accounted for 5.27% of the administered dose in urine over 0-336 hours post-dose, and PPMO-3 accounted for 1.79%. In feces, the most abundant component was PPMO-30, accounting for 4.27% of the administered dose over 0-336 hours, and PPMO-3 was not observed. In biceps muscle, the most abundant component was U6 (unidentified), with a peak concentration of 23,100 ng equivalents. の14 C-PPMO-3 / g and PPMO-3 was not observed in this tissue. PPMO-30 was the most abundant metabolite in the kidney, with a peak concentration of 419,000 ng equivalents. 14 C-PPMO-3 / g, representing 15.81% of the radioactivity in the sample. PPMO-3 was present in the kidney at the first pooled time point (0.25-4 h post-dose), quantifiable at 48 h, and also quantifiable in the pooled samples from 96-144 h post-dose, analyzed at a concentration of 152,000 ng equivalents C-PPMO-3 / g.

[0301] conclusion PPMO-3 is 14 After a single intravenous administration of C-PPMO-3, four metabolites were identified by LC-MS in male mdx mice. Hydrolysis of the terminal arginine (R) amino acid resulted in: 14 It was the major biotransformation pathway of C-PPMO-3.

[0302] Of the four metabolites identified, hydrolysis of the terminal arginine amino acid produced PPMO-33 and PPMO-34, which were present in plasma and urine, PPMO-31, which was present in plasma, urine, feces, and kidney. PPMO-3 was identified in plasma, urine, and kidney. Metabolite PPMO-30 was the most abundant metabolite identified in urine and feces, followed by PPMO-31 in plasma.

Claims

1. Antisense oligomer conjugate of formula (I): 【Chemistry 199】 or a pharmaceutically acceptable salt thereof During the ceremony, n is between 1 and 40. Each Nu is a nucleic acid base that, together, forms a target sequence complementary to the exon annealing site in dystrophin premRNA. T' is the part selected from the following: 【Chemistry 200】 During the ceremony, R １００ It is selected from the group consisting of RRRRRRG-, RRRRG-, RRRG-, RRG-, RG-, and G-, where R is arginine and G is glycine. R ２００ It is hydrogen, R １ C １ ~C ６ Alkyl antisense oligomeric conjugates or pharmaceutically acceptable salts thereof.

2. The antisense oligomer conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the target sequence is complementary to the exon 51 annealing site in dystrophin premRNA, which is designated as H51A(+66+95).

3. T' is the following part: 【Chemical Engineering 201】 In the formula, R ２００ The antisense oligomer conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein is hydrogen.

4. R １００ The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is RRRRRG-.

5. R １００ The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is RRRRG-.

6. R １００ The antisense oligomer conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R is RRRG-.

7. R １００ The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the conjugate is RRG-.

8. R １００ The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is RG-.

9. R １００ The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is G-.

10. The antisense oligomer conjugate according to claim 1, having formula (VA): 【Chemical 203】 or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5, and each Nu from 1 to 30 and 5' to 3' is as follows: Table 28 Here, A said, 【Chemical 204】 And C is, 【Chemical 205】 And G is, 【Chemical 206】 And T is, 【Chemical 207】 an antisense oligomeric conjugate or a pharmaceutically acceptable salt thereof.

11. The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 10, wherein m is 0.

12. The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 10, wherein m is 1.

13. The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 10, wherein m is 2.

14. The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 10, wherein m is 3.

15. The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 10, wherein m is 4.

16. The antisense oligomer conjugate or pharmaceutically acceptable salt thereof according to claim 10, wherein m is 5.

17. A pharmaceutical composition comprising an antisense oligomer conjugate according to any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is formulated for parenteral use.

19. A composition comprising an antisense oligomer conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, for the treatment of a patient in need of treatment having Duchenne muscular dystrophy (DMD) and having a mutation suitable for exon skipping, wherein the antisense oligomer conjugate induces exon skipping in the human dystrophin gene.

20. The composition according to claim 19, wherein the exon is selected from exons 45, 51, or 53.

21. Antisense oligomer conjugate having formula (VI) for the treatment of patients with DMD who have mutations suitable for exon 51 skipping and require treatment: 【Chemical 220】 A composition comprising a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

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