Exon-skipping oligomer conjugates for muscular dystrophies

Abstract

Antisense oligomers complementary to selected target sites in the human dystrophin gene for inducing exon 50 skipping are described. In various embodiments, the antisense oligomers are described according to formula (I): TIFF2022511055000202.tif13687 or a pharmaceutically acceptable salt thereof, 100 is defined herein.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 779,028, filed December 13, 2018. The entire teachings of the above applications are incorporated by reference in their entireties.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (name: 8171_50_WO00_SL.txt, size: 10,080 bytes, creation date: November 6, 2019) filed concurrently with this specification are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to novel antisense oligomers suitable for exon 50 skipping in the human dystrophin gene and pharmaceutical compositions thereof. The present disclosure also provides methods for inducing exon 50 skipping using the novel antisense oligomers, methods for producing dystrophin in subjects with a mutation in the dystrophin gene that is amenable to exon 50 skipping, and methods for treating subjects with a mutation in the dystrophin gene that is amenable to exon 50 skipping. [Background technology]

[0004] Duchenne muscular dystrophy (DMD) is caused by defective expression of the protein dystrophin. The gene that codes for the protein contains 79 exons spread across more than 2 million nucleotides of DNA. Any mutation in an 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(s) or the duplication of one or more exons, can prevent the production of functional dystrophin and result in DMD.

[0005] 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 prevent premature termination of translation of the mRNA into protein. If the joining of upstream and downstream exons in the processing of the mutant 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 Becker phenotype.

[0006] There is a need for antisense oligomers suitable for exon 50 skipping and corresponding pharmaceutical compositions that are useful in therapeutic methods for producing dystrophin and treating DMD. Summary of the Invention [Means for solving the problem]

[0007] The antisense oligomer, or a pharma- ceutically acceptable salt thereof, is capable of binding to a selected target and inducing exon skipping in the human dystrophin gene, the antisense oligomer comprising a sequence of bases that is complementary to an exon 50 target region of a dystrophin pre-mRNA designated as an annealing site, the base sequence and the annealing site being selected from: [Table 9]

[0008] T is thymine or uracil. In one embodiment, each T is thymine.

[0009] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in SEQ ID NO:3.

[0010] In one embodiment, the antisense oligomer contains a T moiety attached to the 5' end of the antisense oligomer, wherein the T moiety is selected from the following: [ka]

[0011] In certain embodiments, the antisense oligomer is conjugated to one or more cell membrane permeable peptides (referred to herein as "CPP"). In certain embodiments, one or more CPPs are attached to the ends of the antisense oligomer. In certain embodiments, at least one CPP is attached to the 5' end of the antisense oligomer. In certain embodiments, at least one CPP is attached to the 3' end of the antisense oligomer. In certain embodiments, a first CPP is attached to the 5' end of the antisense oligomer, and a second CPP is attached to the 3' end of the antisense oligomer.

[0012] In some embodiments, the CPP is an arginine-rich peptide. The term "arginine-rich" refers to a CPP having at least two, preferably 2, 3, 4, 5, 6, 7, or 8 arginine residues, each optionally separated by one or more uncharged hydrophobic residues, and optionally containing about 6 to 14 amino acid residues. As explained below, the CPP is preferably linked at its carboxy terminus to the 3' and / or 5' end of the antisense oligonucleotide via a linker, which may also be one or more amino acids and is preferably selected from H, acyl, acetyl, benzoyl, or stearoyl.a Substituent R containing a In some embodiments, R a is acetyl.

[0013] As seen in the table below, non-limiting examples of CPPs for use herein include -(RXR)4-R a (SEQ ID NO: 15), -R-(FFR)3-R a (SEQ ID NO: 16), -BX-(RXR)4-R a (SEQ ID NO: 17), -BXR-(FFR)3-R a (SEQ ID NO: 18), -GLY-R-(FFR)3-R a (SEQ ID NO: 19), -GLY-R5-R a (SEQ ID NO: 20), -R5-R a (SEQ ID NO: 21), -GLY-R6-R a (SEQ ID NO: 11), and -R6-R a (SEQ ID NO: 10), in which R a is selected from H, acyl, acetyl, benzoyl, and stearoyl, R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine, and GLY (or G) is glycine. CPP "R5 (SEQ ID NO:21)" is meant to represent a peptide of five arginine residues linked together via amide bonds (a single substituent, e.g., R 5 (SEQ ID NO:21). CPP "R6 (SEQ ID NO:10)" is meant to represent a peptide of six arginine residues linked together via amide bonds (not a single substituent, e.g., R 6 In some embodiments, R a is acetyl.

[0014] Exemplary CPPs are shown in Table 1 (SEQ ID NOs: 10, 11, and 15-21). [Table 1-1]

[0015] CPPs, their synthesis, and methods of conjugating to oligomers are further described in U.S. Application Publication No. US2012 / 0289457, and International Patent Application Publication Nos. WO2004 / 097017, WO2009 / 005793, and WO2012 / 150960, the disclosures of which are incorporated by reference in their entireties.

[0016] In some embodiments, the antisense oligonucleotide comprises a substituent "Z" defined as a combination of a CPP and a linker. The linker bridges the CPP at its carboxy terminus to the 3' terminus and / or 5' terminus of the oligonucleotide. In various embodiments, the antisense oligonucleotide may comprise only one CPP linked to the 3' terminus of the oligomer. In other embodiments, the antisense oligonucleotide may comprise only one CPP linked to the 5' terminus of the oligomer.

[0017] The linker in Z can comprise, for example, 1, 2, 3, 4, or 5 amino acids.

[0018] In certain embodiments, Z is selected from: -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CHNH-CPP, and the following formula: [ka] wherein the CPP is attached to the linker moiety via an amide bond at the CPP carboxy terminus.

[0019] In various embodiments, the CPP is an arginine-rich peptide as described herein and shown in Table 1. In various embodiments, the arginine-rich CPP is -R-R a(i.e., five arginine residues, SEQ ID NO:21), where R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. a is acetyl. In various embodiments, the CPP is SEQ ID NO:21 and the linker is -C(O)(CH2)5NH-, -C(O)(CH2)2NH-, -C(O)(CH2)2NHC(O)(CH2)5NH-, -C(O)CH2NH-, and [ka] is selected from the group consisting of:

[0020] In some embodiments, the linker comprises 1, 2, 3, 4, or 5 amino acids.

[0021] In some embodiments, the CPP is SEQ ID NO:21 and the linker is Gly. In some embodiments, the CPP is SEQ ID NO:20.

[0022] In certain embodiments, the arginine-rich CPP is -R a (i.e., 6 arginine residues, SEQ ID NO:10), where R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. a is acetyl. In various embodiments, the CPP is selected from SEQ ID NO: 10, 15, or 16, and the linker is -C(O)(CH2)5NH-, -C(O)(CH2)2NH-, -C(O)(CH2)2NHC(O)(CH2)5NH-, -C(O)CHNH-, and [ka] is selected from the group consisting of:

[0023] In some embodiments, the linker comprises 1, 2, 3, 4, or 5 amino acids.

[0024] In some embodiments, the CPP is SEQ ID NO:10 and the linker is Gly. In some embodiments, the CPP is SEQ ID NO:11.

[0025] In certain embodiments, Z is —C(O)CHNH—R—R covalently attached to the antisense oligomer of the disclosure at the 5′ and / or 3′ end of the oligomer. a ("R6" is disclosed as SEQ ID NO:10), where R a is H, acyl, acetyl, benzoyl, or stearoyl and caps the amino terminus of R6 (SEQ ID NO:10). a In these non-limiting examples, the CPP is -R-R a (SEQ ID NO:10), and the linker is -C(O)CH2NH- (i.e., GLY). Z = -C(O)CH2NH-R6-R a ("R6" is disclosed as SEQ ID NO:10) is also exemplified by the following structure: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. a is acetyl.

[0026] In various embodiments, the CPP is -R-R a (SEQ ID NO:10), also exemplified by the formula: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. In certain embodiments, the CPP is SEQ ID NO:11. In some embodiments, R a is acetyl.

[0027] In some embodiments, the CPP is -(RXR)-R a (SEQ ID NO:15) and is also exemplified by the formula: [ka]

[0028] In various embodiments, the CPP is -R-(FFR)3-R a (SEQ ID NO:16) and is also exemplified by the formula: [ka]

[0029] In various embodiments, Z is selected from: -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CHNH-CPP, and the following formula: [ka] wherein the CPP is attached to a linker moiety by an amide bond at the CPP carboxy terminus, and the CPP has the following structure: [ka] (-R-(FFR)3-R a ) (SEQ ID NO: 16), [ka] (-(RXR)4-R a ) (SEQ ID NO: 15), [ka] and (-R 6- R a ) (SEQ ID NO: 10) In some embodiments, R a is acetyl.

[0030] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO:3.

[0031] In some embodiments, the nucleobases of the modified antisense oligomer are linked to morpholino ring structures which are joined by phosphorus-containing intersubunit linkages connecting the morpholino nitrogen of one ring structure to the 5' exocyclic carbon of an adjacent ring structure.

[0032] In some embodiments, the nucleobases of the antisense oligomer are linked to peptide nucleic acids (PNAs), in which the phosphate-sugar polynucleotide backbone is replaced with a flexible pseudo-peptide polymer to which the nucleobases are linked.

[0033] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a locked nucleic acid (LNA), a locked nucleic acid structure being a chemically modified nucleotide analog in which a ribose moiety has an extra bridge connecting the 2' oxygen and the 4' carbon.

[0034] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a bridged nucleic acid (BNA), where the sugar conformation is restricted or locked by the introduction of an additional bridge structure to the furanose backbone.

[0035] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a 2'-O,4'-C-ethylene-bridged nucleic acid (ENA).

[0036] In some embodiments, modified antisense oligomers may contain unlocked nucleic acid (UNA) subunits. UNA and UNA oligomers are analogs of RNA in which the C2'-C3' bond of the subunit has been truncated.

[0037] In some embodiments, modified antisense oligomers contain one or more phosphorothioates (or S-oligos) in which one of the non-bridging oxygens is replaced with sulfur. In some embodiments, modified antisense oligomers contain one or more 2'O-methyl, 2'O-MOE, MCE, and 2'-F in which the 2'-OH of the ribose is replaced with a methyl, methoxyethyl, 2-(N-methylcarbamoyl)ethyl, or fluoro group, respectively.

[0038] In some embodiments, the modified antisense oligomer is tricyclo-DNA (tc-DNA), a constrained DNA analog in which each nucleotide is modified by the introduction of a cyclopropane ring to limit the conformational flexibility of the backbone and optimize the backbone geometry for torsion angle γ.

[0039] In various embodiments, the present disclosure provides an antisense oligomer according to formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase which together form a targeting sequence, T is [ka] wherein the distal -OH or -NH2 of the T moiety is optionally linked to a cell membrane penetrating peptide; R 100 is hydrogen or a cell membrane-permeable peptide, Each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 10] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0040] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO:3.

[0041] In another aspect, the present disclosure provides an antisense oligomer of formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu of 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 11] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0042] In some embodiments, the distal --OH of formula (II) is linked to a cell membrane penetrating peptide.

[0043] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO:3.

[0044] In another aspect, the present disclosure provides an antisense oligomer of formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu of 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 12] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0045] In some embodiments, the distal --OH of formula (III) is linked to a cell membrane penetrating peptide.

[0046] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO:3.

[0047] In another aspect, the disclosure provides an antisense oligomer of formula (IV): [ka] wherein each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 13] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0048] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO:3.

[0049] In some embodiments of formula (IV), the antisense oligomer is according to formula (IVa). [ka]

[0050] In another aspect, the disclosure provides an antisense oligomer of formula (V): [ka] wherein each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 14] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO:3.

[0051] In another aspect, the disclosure provides a method for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the subject having a mutation in the dystrophin gene that is amenable to exon 50 skipping, the method comprising administering to the subject an antisense oligomer of the disclosure. The disclosure also addresses the use of an antisense oligomer of the disclosure for the manufacture of a medicament for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the subject having a mutation in the dystrophin gene that is amenable to exon 50 skipping.

[0052] In another embodiment, the present disclosure provides a method of restoring an mRNA reading frame to induce dystrophin production in a subject having a mutation in the dystrophin gene that is amenable to exon 50 skipping, the method comprising administering an antisense oligomer of the present disclosure to the subject. In another embodiment, the present disclosure provides a method of excluding exon 50 from a dystrophin pre-mRNA during mRNA processing in a subject having a mutation in the dystrophin gene that is amenable to exon 50 skipping, the method comprising administering an antisense oligomer of the present disclosure to the subject. In another embodiment, the present disclosure provides a method of binding exon 50, intron 49, and / or intron 50 of a dystrophin pre-mRNA in a subject having a mutation in the dystrophin gene that is amenable to exon 50 skipping, the method comprising administering an antisense oligomer of the present disclosure to the subject.

[0053] In another aspect, the present disclosure provides herein an antisense oligomer of the present disclosure for use in therapy.In certain embodiments, the present disclosure provides an antisense oligomer of the present disclosure for use in the treatment of Duchenne muscular dystrophy.In certain embodiments, the present disclosure provides an antisense oligomer of the present disclosure for use in the manufacture of a medicament for use in therapy.In certain embodiments, the present disclosure provides an antisense oligomer of the present disclosure for use in the manufacture of a medicament for the treatment of Duchenne muscular dystrophy.

[0054] In another aspect, the disclosure also provides a kit for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the subject having a mutation in the dystrophin gene amenable to exon 50 skipping, the kit comprising at least an antisense oligomer of the disclosure packaged in a suitable container and instructions for its use. In certain embodiments, for example, the following are provided: (Item 1) An antisense oligomer capable of binding to a selected target and inducing exon skipping in a human dystrophin gene, or a pharma- ceutically acceptable salt thereof, wherein the antisense oligomer comprises a sequence of bases that is complementary to an exon 50 target region of a dystrophin pre-mRNA designated as an annealing site, the sequence of bases and the annealing site being selected from the following:

Table 30

change

change

change

Table 31

change

change

change

change

change

change

change

change

Table 32

change

change

change

change

change

Table 33

change

[0055] The embodiments of the present disclosure generally relate to an improved antisense oligomer and its method of use, which is specifically designed to induce exon skipping in human dystrophin gene.Dystrophin plays an important role in muscle function, and various muscle-related diseases are characterized by the mutant form of this gene.Therefore, in certain embodiments, the improved antisense oligomer described herein induces exon skipping in the mutant form of human dystrophin gene, such as the mutant dystrophin gene found in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0056] Due to the abnormal mRNA splicing events caused by mutation, these mutant human dystrophin genes either express defective dystrophin protein or do not express any measurable dystrophin, which is a condition that causes various forms of muscular dystrophy.To treat this condition, the antisense oligomer of the present disclosure hybridizes to the selected region of the pre-processed mRNA of mutant human dystrophin gene, and induces exon skipping and differential splicing in otherwise abnormally spliced ​​dystrophin mRNA, thereby allowing muscle cells to produce the mRNA transcript that codes for functional dystrophin protein.In certain embodiments, the resulting dystrophin protein is not necessarily the "wild type" form of dystrophin, but rather a truncated but functional form of dystrophin.

[0057] By increasing the levels of functional dystrophin protein in muscle cells, these and related embodiments are useful for the prevention and treatment of muscular dystrophies, particularly those forms of muscular dystrophies, such as DMD and BMD, which are characterized by defective expression of dystrophin protein due to aberrant mRNA splicing. Certain antisense oligomers described herein further provide improved dystrophin-exon specific targeting over other oligomers, thereby offering significant and practical advantages over alternative methods of treating related forms of muscular dystrophies.

[0058] Accordingly, the present disclosure relates to an antisense oligomer or a pharma- ceutically acceptable salt thereof capable of binding to a selected target and inducing exon skipping in a human dystrophin gene, the antisense oligomer comprising a sequence of bases that is complementary to an exon 50 target region of a dystrophin pre-mRNA designated as an annealing site, the base sequence and the annealing site being selected from: [Table 15] T is thymine or uracil.

[0059] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in SEQ ID NO:3.

[0060] In one embodiment, the antisense oligomer contains a T moiety attached to the 5' end of the antisense oligomer, wherein the T moiety is selected from the following: [ka]

[0061] In certain embodiments, the antisense oligomer is conjugated to one or more cell membrane permeable peptides (referred to herein as "CPP"). In certain embodiments, one or more CPPs are attached to the ends of the antisense oligomer. In certain embodiments, at least one CPP is attached to the 5' end of the antisense oligomer. In certain embodiments, at least one CPP is attached to the 3' end of the antisense oligomer. In certain embodiments, a first CPP is attached to the 5' end of the antisense oligomer, and a second CPP is attached to the 3' end of the antisense oligomer.

[0062] In some embodiments, the CPP is an arginine-rich peptide. The term "arginine-rich" refers to a CPP having at least two, preferably 2, 3, 4, 5, 6, 7, or 8 arginine residues, each optionally separated by one or more uncharged hydrophobic residues, and optionally containing about 6 to 14 amino acid residues. As explained below, the CPP is preferably linked at its carboxy terminus to the 3' and / or 5' end of the antisense oligonucleotide via a linker, which may also be one or more amino acids and is preferably selected from H, acyl, acetyl, benzoyl, or stearoyl. a Substituent R containing a In some embodiments, R a is acetyl.

[0063] As seen in the table below, non-limiting examples of CPPs for use herein include -(RXR)4-R a (SEQ ID NO: 15), R-(FFR)3-R a (SEQ ID NO: 16), -BX-(RXR)4-R a (SEQ ID NO: 17), -BXR-(FFR)3-R a (SEQ ID NO: 18), -GLY-R-(FFR)3-R a (SEQ ID NO: 19), -GLY-R5-Ra (SEQ ID NO: 20), -R5-R a (SEQ ID NO: 21), -GLY-R6-R a (SEQ ID NO: 11), and -R6-R a (SEQ ID NO: 10), in which R a is selected from H, acyl, benzoyl, and stearoyl, R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine, and GLY (or G) is glycine. CPP "R5 (SEQ ID NO:21)" is meant to represent a peptide of five arginine residues linked together via amide bonds (a single substituent, e.g., R 5 (SEQ ID NO:21). CPP "R6 (SEQ ID NO:10)" is meant to represent a peptide of six arginine residues linked together via amide bonds (not a single substituent, e.g., R 6 (not SEQ ID NO: 10). In some embodiments, R a is acetyl.

[0064] Exemplary CPPs are shown in Table 1 (SEQ ID NOs: 10, 11, and 15-21). [Table 1-2]

[0065] CPPs, their synthesis, and methods of conjugating to oligomers are further described in U.S. Application Publication No. US2012 / 0289457, and International Patent Application Publication Nos. WO2004 / 097017, WO2009 / 005793, and WO2012 / 150960, the disclosures of which are incorporated by reference in their entireties.

[0066] In some embodiments, the antisense oligonucleotide comprises a substituent "Z" defined as a combination of a CPP and a linker. The linker bridges the CPP at its carboxy terminus to the 3' terminus and / or 5' terminus of the oligonucleotide. In various embodiments, the antisense oligonucleotide may comprise only one CPP linked to the 3' terminus of the oligomer. In other embodiments, the antisense oligonucleotide may comprise only one CPP linked to the 5' terminus of the oligomer.

[0067] The linker in Z can comprise, for example, 1, 2, 3, 4, or 5 amino acids. In certain embodiments, Z is selected from: -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CHNH-CPP, and the following formula: [ka] wherein the CPP is attached to the linker moiety via an amide bond at the CPP carboxy terminus.

[0068] In various embodiments, the CPP is an arginine-rich peptide as described herein and shown in Table 1. In certain embodiments, the arginine-rich CPP is -R a (i.e., five arginine residues, SEQ ID NO:21), where R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. a is acetyl. In various embodiments, the CPP is selected from SEQ ID NO: 15, 16, or 21, and the linker is -C(O)(CH2)5NH-, -C(O)(CH2)2NH-, -C(O)(CH2)2NHC(O)(CH2)5NH-, -C(O)CHNH-, and [ka] In some embodiments, the linker comprises 1, 2, 3, 4, or 5 amino acids.

[0069] In some embodiments, the CPP is SEQ ID NO:21 and the linker is Gly. In some embodiments, the CPP is SEQ ID NO:20.

[0070] In certain embodiments, the arginine-rich CPP is -R a (i.e., 6 arginine residues, SEQ ID NO:10), where R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. a is acetyl. In various embodiments, the CPP is selected from SEQ ID NO: 10, 15, or 16, and the linker is -C(O)(CH2)5NH-, -C(O)(CH2)2NH-, -C(O)(CH2)2NHC(O)(CH2)5NH-, -C(O)CHNH-, and [ka] In some embodiments, the linker comprises 1, 2, 3, 4, or 5 amino acids.

[0071] In some embodiments, the CPP is SEQ ID NO:10 and the linker is Gly. In some embodiments, the CPP is SEQ ID NO:11.

[0072] In certain embodiments, Z is —C(O)CHNH—R—R covalently attached to the antisense oligomer of the disclosure at the 5′ and / or 3′ end of the oligomer. a ("R6" is disclosed as SEQ ID NO:10), where R ais H, acyl, acetyl, benzoyl, or stearoyl and caps the amino terminus of R6 (SEQ ID NO:10). a In these non-limiting examples, the CPP is -R-R a (SEQ ID NO:10), and the linker is -C(O)CH2NH- (i.e., GLY). Z = -C(O)CH2NH-R6-R a ("R6" is disclosed as SEQ ID NO:10) is also exemplified by the following structure: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl.

[0073] In various embodiments, the CPP is -R-R a (SEQ ID NO:10), also exemplified by the formula: [ka] In the formula, R a is selected from H, acyl, acetyl, benzoyl, and stearoyl. In certain embodiments, the CPP is SEQ ID NO:11. In some embodiments, R a is acetyl.

[0074] In some embodiments, the CPP is -(RXR)-R a (SEQ ID NO:15) and is also exemplified by the formula: [ka]

[0075] In various embodiments, the CPP is -R-(FFR)3-R a (SEQ ID NO:16) and is also exemplified by the formula: [ka]

[0076] In various embodiments, Z is selected from: -C(O)(CH2)5NH-CPP, -C(O)(CH2)2NH-CPP, -C(O)(CH2)2NHC(O)(CH2)5NH-CPP, -C(O)CHNH-CPP, and the following formula: [ka] wherein the CPP is attached to a linker moiety by an amide bond at the CPP carboxy terminus, and the CPP has the following structure: [ka] (-R-(FFR)3-R a ) (SEQ ID NO: 16), [ka] (-(RXR)4-R a ) (SEQ ID NO: 15), [ka] or (-R 6- R a ) (SEQ ID NO: 10) In some embodiments, R a is acetyl.

[0077] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO:3.

[0078] In some embodiments, the nucleobases of the modified antisense oligomer are linked to morpholino ring structures that are joined by phosphorus-containing intersubunit linkages connecting the morpholino nitrogen of one ring structure to the 5' exocyclic carbon of an adjacent ring structure. In such embodiments, the T in each of SEQ ID NOs:1-9 is preferably thymine.

[0079] In some embodiments, the nucleobases of the antisense oligomer are linked to peptide nucleic acids (PNAs), in which the phosphate-sugar polynucleotide backbone is replaced with a flexible pseudo-peptide polymer to which the nucleobases are linked.

[0080] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a locked nucleic acid (LNA), a locked nucleic acid structure that is a chemically modified nucleotide analog in which a ribose moiety has an extra bridge connecting the 2' oxygen and the 4' carbon. In such embodiments, each nucleobase linked to the LNA of each of SEQ ID NOs:1-9 contains a 5-methyl group.

[0081] In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a bridged nucleic acid (BNA), and the sugar conformation is restricted or locked by the introduction of an additional bridge structure to the furanose backbone. In some embodiments, at least one of the nucleobases of the antisense oligomer is linked to a 2'-O,4'-C-ethylene bridged nucleic acid (ENA). In such embodiments, each nucleobase linked to the BNA or ENA of each of SEQ ID NOs: 1-9 contains a 5-methyl group.

[0082] In some embodiments, where the backbone chemistry permits, each thymine in SEQ ID NOs:1-9 is a uracil.

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described.For the purpose of this disclosure, the following terms are defined below.

[0084] I. Definition As used herein, the term "alkyl" refers to a saturated straight chain or branched 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. 10 and alkyl. In certain embodiments, the alkyl group comprises 1-6 carbon atoms, i.e., C1-C6 alkyl. 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 the 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 skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, which is incorporated herein by reference. 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.

[0085] "Suitable for exon 50 skipping", as used herein with respect to a subject or patient, is intended to include subjects and patients with one or more mutations in the dystrophin gene, in which the absence of skipping of exon 50 of dystrophin pre-mRNA results in out-of-frame reading frame, thereby preventing translation of pre-mRNA and making the subject or patient unable to produce functional or semi-functional dystrophin.It is well within the understanding of those skilled 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).

[0086] As used herein, the term "oligomer" refers to a sequence of subunits linked by intersubunit linkages. 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 RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence, provided that either the subunits, the intersubunit linkages, or both, are not naturally occurring. In certain embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO). In other embodiments, the antisense oligomer is a 2'-O-methyl phosphorothioate. In other embodiments, the antisense oligomers of the present disclosure are peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or bridged nucleic acids (BNAs), such as 2'-O,4'-C-ethylene bridged nucleic acids (ENAs). Additional exemplary embodiments are described herein.

[0087] 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 can 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 can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Alternatively, continuing the example, there can be "complete" or "perfect" (100%) complementarity between a given nucleobase sequence and another nucleobase sequence. The degree of complementarity between nucleobase sequences has a significant effect on the efficiency and strength of hybridization between the sequences.

[0088] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to the amount of a therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, that is effective in producing the desired therapeutic effect.For antisense oligomers, this effect is typically produced by inhibiting the translation or natural splice processing of a selected target sequence, or by producing a clinically significant amount of dystrophin (statistical significance).

[0089] In some embodiments, the effective amount is about 1 mg / kg to about 200 mg / kg of a composition comprising an antisense oligomer over a period of time to treat a subject. In some embodiments, the effective amount is about 1 mg / kg to about 200 mg / kg of a composition comprising an antisense oligomer to increase the number of dystrophin-positive fibers in a subject. In certain embodiments, the effective amount is about 1 mg / kg to about 200 mg / kg of a composition comprising an antisense oligomer to stabilize, maintain, or improve a patient's walking distance compared to a healthy counterpart, for example, in a 6-minute walk test (6MWT).

[0090] "Enhance" or "enhancing", or "increase" or "increasing", or "stimulate" or "stimulating" generally refers to the ability of one or more of the foregoing antisense oligomers or pharmaceutical compositions to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject compared to the response caused by either no antisense oligomer 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 that are apparent from the understanding in the art and the description herein.

[0091] As used herein, terms such as "function" and "functionality" refer to biological, enzymatic, or therapeutic functions.

[0092] 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 the altered or "defective" form of dystrophin protein typically present in certain subjects with DMD or BMD.As an example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibril organization (or disintegration), contractile activity, and spontaneous clustering of acetylcholine receptors (see, for example, Brown et al., Journal of Cell Science. 112:209-216, 1999).In addition, animal models are valuable resources for studying disease pathogenesis and provide a means to test dystrophin-related activity. Two of the most widely used animal models for DMD research are mdx mice and golden retriever muscular dystrophy (GRMD) dogs, both of which are dystrophin negative (see, for example, Collins & Morgan, Int J Exp Pathol 84:165-172, 2003). These and other animal models can be used to measure the functional activity of various dystrophin proteins. Included are truncated forms of dystrophin, such as those produced after administration of certain exon skipping antisense oligomers of the present disclosure.

[0093] The term "mismatch" refers to one or more nucleobases (whether contiguous or separated) in the oligomer nucleobase sequence that do not match the target pre-mRNA according to base pairing rules. While perfect 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 oligomers of the present disclosure include nucleobase sequence variations near the ends, internal variations, and when present, typically within about 6, 5, 4, 3, 2, or 1 subunit at the 5' and / or 3' ends. In certain embodiments, one, two, or three nucleobases can be removed and still provide binding on the target.

[0094] 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.

[0095] In certain embodiments, the morpholinos are conjugated to a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include: [ka] The distal -OH or -NH2 of the T moiety is optionally linked to a cell membrane penetrating peptide.

[0096] Of the exemplary tail moieties above, "TEG" or "EG3" refers to the following tail moieties: [ka]

[0097] Of the exemplary tail moieties above, "GT" refers to the following tail moieties: [ka]

[0098] As used herein, the terms "-G-R5 (SEQ ID NO:20)" and "-G-R5-Ac (SEQ ID NO:20)" are used interchangeably and refer to peptide moieties conjugated to the antisense oligomers of the present disclosure. In various embodiments, "G" represents a glycine residue conjugated to "R5 (SEQ ID NO:21)" by an amide bond, and each "R" represents an arginine residue conjugated together by an amide bond, whereby "R5 (SEQ ID NO:21)" refers to five arginine residues conjugated together by amide bonds. The arginine residues can have any configuration, for example, the arginine residues can be L-arginine residues, D-arginine residues, or a mixture of D-arginine and L-arginine residues. In certain embodiments, "-G-R5 (SEQ ID NO:20)" or "-G-R5-Ac (SEQ ID NO:20)" is linked to the distal -OH or NH2 of the "tail" portion. In certain embodiments, "-G-R5 (SEQ ID NO:20)" or "-G-R5-Ac (SEQ ID NO:20)" is conjugated to the morpholine ring nitrogen of the 3'-most morpholino subunit of a PMO antisense oligomer of the present disclosure. In some embodiments, "-G-R5 (SEQ ID NO:20)" or "-G-R5-Ac (SEQ ID NO:20)" is conjugated to the 3' end of an antisense oligomer of the present disclosure and is of the formula: [ka] or a pharma- ceutically acceptable salt thereof, or [ka]

[0099] As used herein, the terms "-G-R6 (SEQ ID NO: 11)" and "-G-R6-Ac (SEQ ID NO: 11)" and "R6G (SEQ ID NO: 11)" are used interchangeably and refer to peptide moieties conjugated to the antisense oligomers of the present disclosure. In various embodiments, "G" represents a glycine residue conjugated to "R6 (SEQ ID NO: 10)" by an amide bond, and each "R" represents an arginine residue conjugated together by an amide bond, whereby "R6 (SEQ ID NO: 10)" refers to six arginine residues conjugated together by amide bonds. The arginine residues can have any configuration, for example, the arginine residues can be L-arginine residues, D-arginine residues, or a mixture of D-arginine residues and L-arginine residues. In certain embodiments, "-G-R6 (SEQ ID NO:11)" or "-G-R6-Ac (SEQ ID NO:11)" is an -OH radical at the distal end of the "tail" portion or -NH2. In certain embodiments, "-G-R6 (SEQ ID NO:11)" or "-G-R6-Ac (SEQ ID NO:11)" is conjugated to the morpholine ring nitrogen of the 3'-most morpholino subunit of a PMO antisense oligomer of the present disclosure. In some embodiments, "-G-R6 (SEQ ID NO:11)" or "-G-R6-Ac (SEQ ID NO:11)" is conjugated to the 3'-terminus of an antisense oligomer of the present disclosure and is of the formula: [ka]

[0100] The terms "nucleobase" (Nu), "base-pairing moiety," or "base" are used interchangeably and refer to the purine or pyrimidine bases 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 the 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.

[0101] 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 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.

[0102] 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. Biol., 2006, 10, 622-627, the contents of which are incorporated herein by reference, are contemplated as useful in the antisense oligomers described herein. Examples of extended size nucleobases include those shown below, as well as tautomers thereof. [ka]

[0103] 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.

[0104] 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 iterations 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 iterations of the structural feature between the brackets is indicated by a variable shown outside the brackets, such as "Z".

[0105] 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 and / or mixtures thereof. Examples of such diagrams are shown below. [ka]

[0106] The phrase "pharmacologically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the subject being treated therewith.

[0107] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or any type of formulation auxiliary. 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 carboxymethyl cellulose, ethyl cellulose, 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 ... leaven oil, corn oil, and soybean oil; glycols, such as propylene glycol esters, for example, ethyl oleate and ethyl laurate; agar; buffers, for example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; non-toxic compatible lubricants, for example, sodium lauryl sulfate and magnesium stearate; colorants; release agents; coating agents; sweetening agents; flavoring agents; fragrances; preservatives; and antioxidants.

[0108] The term "restoration" with respect to dystrophin synthesis or production generally refers to the production of dystrophin protein, including truncated forms of dystrophin, in muscular dystrophy patients after treatment with antisense oligomers as described herein.The proportion of dystrophin-positive fibers in patients after treatment can be determined by muscle biopsy using known techniques.For example, muscle biopsy can be taken from a suitable muscle, such as the biceps brachii muscle, of a patient.

[0109] 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, post-treatment biopsy is taken 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 biopsy using an antibody that is a marker for dystrophin, such as a monoclonal or polyclonal antibody. For example, MANDYS106 antibody, which is a highly sensitive marker for dystrophin, can be used. Any suitable secondary antibody can be used.

[0110] 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 pre-treatment muscle sections from the patient to control for the presence of trace levels of dystrophin in pre-treatment muscle as well as revertant fibers. This can be used as a threshold for counting dystrophin-positive fibers in 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, anti-dystrophin antibody NCL-Dys1 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.

[0111] In some embodiments, treatment with the antisense oligomer 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 of the present disclosure can reduce or eliminate the need for ventilatory support that would be expected without treatment.In some embodiments, the measurement of respiratory function to track the course of disease and evaluate possible therapeutic interventions includes maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and forced vital capacity (FVC).MIP and MEP measure the pressure level that 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.

[0112] 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.

[0113] As used herein, the terms "subject" and "patient" include any animal that exhibits or is at risk of exhibiting symptoms that can be treated with the antisense oligomer 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 is a method of producing dystrophin in a subject (or patient) that has a mutation in the dystrophin gene that is suitable for exon 50 skipping.

[0114] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to administration of a compound, drug, or other material other than directly into the central nervous system, thereby entering the patient's system and thus being subject to metabolic and other similar processes, such as, for example, subcutaneous administration.

[0115] The phrase "targeting sequence" or "base sequence" refers to the sequence of the nucleic acid base of the oligomer that is complementary to the sequence of the nucleotide in the target pre-mRNA.In some embodiments of the present disclosure, the sequence of the nucleotide in the target pre-mRNA is the exon 50, intron 49, and / or intron 50 annealing site in the dystrophin pre-mRNA designated as H50D(+04-18), H50D(+07-18), H50D(+07-16), H50D(+07-17), H50A(-19+07), H50D(+07-15), H50A(-02+23), H50D(+06-18), or H50D(+07-20).In one embodiment, the annealing site targeted by the antisense oligomer described herein is H50D(+07-16).

[0116] "Treatment" of a subject (e.g., a mammal, such as a human) or cell is any type of intervention used in an attempt to change the natural course of the subject or cell. Treatment includes, but is not limited to, administration of an oligomer or pharmaceutical composition thereof, and can be performed either prophylactically or after the onset of a pathological event or after contact with a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition associated with dystrophin protein, such as 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 are "prophylactic" treatments, 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.

[0117] In some embodiments, treatment with the antisense oligomers of the present disclosure increases new dystrophin production, delays disease progression, delays or reduces lameness, reduces muscle inflammation, reduces muscle damage, improves muscle function, reduces loss of lung function, and / or enhances muscle regeneration that would be expected without treatment. In some embodiments, treatment maintains, delays, 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 the 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-stair climbing 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.

[0118] In some embodiments, treatment with the antisense oligomers of the present disclosure increases new dystrophin production and delays or reduces the lameness predicted without treatment. For example, treatment may stabilize, maintain, improve, or increase the walking ability of the subject (e.g., stabilization of walking). In some embodiments, treatment maintains or increases the patient's stable walking distance, for example, as measured by the 6-minute walk test (6MWT) described by McDonald et al. (Muscle Nerve, 2010; 42: 966-74, incorporated herein by reference). Changes in 6-minute walk distance (6MWD) can be expressed as absolute values, percentage changes, or % predicted changes. The performance of DMD patients in the 6MWT compared to the typical performance of healthy counterparts can be determined by calculating % predicted values. For example, % predicted 6MWD can be calculated for men using the following equation: 196.72 + (39.81 x age) - (1.36 x 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 (meters)) (Henricson et al. PLoS Curr., 2012, version 2, incorporated herein by reference).

[0119] 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 6MWT over the course of about one 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 equation based on age and height 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 or 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).

[0120] 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. H#A / D(x:y)

[0121] 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 will be 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.

[0122] II. Antisense Oligomers A. Antisense oligomers designed to induce exon 50 skipping In certain embodiments, the antisense oligomers of the present disclosure are complementary to exon 50, intron 49, and / or intron 50 target regions of the dystrophin gene and induce exon 50 skipping. In particular, the present disclosure relates to antisense oligomers complementary to exon 50, intron 49, and / or intron 50 target regions of the dystrophin pre-mRNA designated as annealing sites. In some embodiments, the annealing sites are selected from H50D(+04-18), H50D(+07-18), H50D(+07-16), H50D(+07-17), H50A(-19+07), H50D(+07-15), H50A(-02+23), H50D(+06-18), or H50D(+07-20). In some embodiments, the annealing site is H50D(+07-16).

[0123] The antisense oligomers of the present disclosure target dystrophin pre-mRNA and induce the skipping of exon 50, so that it is excluded or skipped from the mature spliced ​​mRNA transcript. By skipping exon 50, the disrupted reading frame is restored to an in-frame mutation. Although DMD consists of various genetic subtypes, the antisense oligomers of the present disclosure are specifically designed to skip exon 50 of dystrophin pre-mRNA. DMD mutations that are suitable for skipping exon 50 include a subgroup (4%) of DMD patients.

[0124] The nucleobase sequence of an antisense oligomer that induces exon 50 skipping is designed to be complementary to a specific target sequence within exon 50, intron 49, and / or intron 50 of the dystrophin pre-mRNA. In some embodiments, the antisense oligomer is a PMO, and each morpholino ring of the PMO is linked to a nucleobase, including, for example, a nucleobase found in DNA (adenine, cytosine, guanine, and thymine).

[0125] B. Characteristics of Oligomeric Chemical Substances The antisense oligomer of the present disclosure can use various antisense oligomer chemistries. Examples of oligomer chemistries include, but are not limited to, morpholino oligomers, phosphorothioate modified oligomers, 2'-O-methyl modified oligomers, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate oligomers, 2'-O-MOE modified oligomers, 2'-fluoro modified oligomers, 2'-O,4'C-ethylene bridged nucleic acid (ENA), tricyclo-DNA, tricyclo-DNA phosphorothioate subunits, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified oligomers (including any combination of the above). Phosphorothioate and 2'-O-Me modified chemistries can be combined to generate 2'-O-Me-phosphorothioate backbones. See, for example, PCT Publication Nos. WO / 2013 / 112053 and WO / 2009 / 008725, which are incorporated herein by reference in their entireties. Where permitted by the chemistry utilized, each T in any of SEQ ID NOs:1-9 may be uracil. Where permitted by the chemistry utilized, the relevant nucleobase in any of SEQ ID NOs:1-9 may include a 5-methyl group. Exemplary embodiments of oligomeric chemistries of the present disclosure are further described below.

[0126] 1. Peptide Nucleic Acid (PNA) Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone is structurally homomorphic with a deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligomers according to Watson-Crick base pairing rules, mimicking DNA in terms of base pair recognition. The backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well suited for antisense applications (see structure below). The backbone is uncharged, resulting in PNA / DNA or PNA / RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases. Non-limiting examples of PNAs are shown below. [ka]

[0127] Despite the radical structural change relative to the natural structure, PNAs are capable of sequence-specific binding to DNA or RNA in a helical form. PNA features include high binding affinity to complementary DNA or RNA, destabilizing effects caused by single base mismatches, resistance to nucleases and proteases, salt-independent hybridization with DNA or RNA, and triplex formation with homopurine DNA. PANAGENE™ has developed its proprietary Bts PNA monomer (Bts, benzothiazole-2-sulfonyl group) and proprietary oligomerization process. PNA oligomerization using Bts PNA monomer consists of repeated cycles of deprotection, coupling, and capping. PNAs can be produced synthetically using any technique known in the art. See, for example, U.S. Patent Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. Also, for the preparation of PNAs, see U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the above is incorporated by reference in its entirety.

[0128] 2. Locked Nucleic Acid (LNA) Antisense oligomers may also contain "locked nucleic acid" subunits (LNAs). "LNAs" are members of a class of modifications called bridged nucleic acids (BNAs). BNAs are characterized by a covalent bond that locks the conformation of the ribose ring to a C30-endo (northern) sugar pucker. In the case of LNAs, the bridge is composed of a methylene between the 2'-O and 4'-C positions. LNAs enhance backbone preorganization and base stacking to enhance hybridization and thermal stability.

[0129] The structure of LNA can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998) 54: 3607; Jesper Wengel, Accounts of Chem. Research (1999) 32: 301; Obika, et al., Tetrahedron Letters (1997) 38: 8735; Obika, et al., Tetrahedron Letters (1998) 39: 5401; and Obika, et al., Bioorganic Medicinal Chemistry (2008) 16: 9230, which are incorporated herein by reference in their entirety. Non-limiting examples of LNA are shown below. [ka]

[0130] The antisense oligomer of the present disclosure may incorporate one or more LNAs. In some cases, the antisense oligomer may be completely composed of LNA. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligomers are described, for example, in U.S. Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated by reference in its entirety. Exemplary intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers may be used. Further embodiments include LNA-containing antisense oligomers, in which each LNA subunit is separated by a DNA subunit. Certain antisense oligomers are composed of alternating LNA and DNA subunits, where the intersubunit linker is phosphorothioate.

[0131] 2'O,4'C-Ethylene-bridged Nucleic Acids (ENAs) are another member of the BNA class. Non-limiting examples are provided below. [ka]

[0132] ENA oligomers and their preparation are described in Obika et al., Tetrahedron Lett (1997) 38(50):8735, which is incorporated herein by reference in its entirety. Antisense oligomers of the present disclosure may incorporate one or more ENA subunits.

[0133] 3. Unlocked Nucleic Acid (UNA) Antisense oligomers can also contain unlocked nucleic acid (UNA) subunits. UNA and UNA oligomers are analogs of RNA in which the C2'-C3' bond of the subunit is broken. LNA is conformationally restricted (relative to DNA and RNA), whereas UNA is highly flexible. UNA is disclosed, for example, in WO2016 / 070166. Non-limiting examples of UNA are shown below. [ka]

[0134] Exemplary intersubunit linkers include phosphodiester and phosphorothioate moieties. Alternatively, non-phosphorus-containing linkers can be used.

[0135] 4. Phosphorothioates "Phosphorothioates" (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens is replaced with a sulfur. Non-limiting examples of phosphorothioates are shown below. [ka]

[0136] Sulfurization of internucleotide bonds reduces the action of endonucleases and exonucleases, including 5' to 3' and 3' to 5' DNA POL1 exonuclease, nucleases S1 and P1, RNase, serum nuclease, and snake venom phosphodiesterase. Phosphorothioates are made by two main routes: the action of a solution of elemental sulfur in carbon disulfide on hydrogen phosphonates, or sulfurization of phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, for example, Iyer et al., J. Org. Chem. 55, 4693-4699, 1990, which is incorporated herein by reference in its entirety). The latter method avoids the problems of the insolubility of elemental sulfur in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also result in phosphorothioates of higher purity.

[0137] 5. Tricyclo-DNA and tricyclo-phosphorothioate subunits Tricyclo-DNA (tc-DNA) is a class of constrained DNA analogues in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry of the torsion angle γ. Homobasic adenine and thymine-containing tc-DNA forms highly stable AT base pairs with complementary RNA. Tricyclo-DNA and its synthesis are described in International Patent Application Publication No. WO2010 / 115993, which is incorporated herein by reference in its entirety. The antisense oligomer of the present disclosure may incorporate one or more tricyclo-DNA subunits. In some cases, the antisense oligomer may be completely composed of tricyclo-DNA subunits.

[0138] Tricyclo-phosphorothioate subunits are tricyclo-DNA subunits with phosphorothioate intersubunit linkages.Tricyclo-phosphorothioate subunits and their synthesis are described in International Patent Application Publication No. WO2013 / 053928, which is incorporated herein by reference in its entirety.Antisense oligomers of the present disclosure may incorporate one or more tricyclo-DNA subunits.In some cases, antisense oligomers may be entirely composed of tricyclo-DNA subunits.Non-limiting examples of tricyclo-DNA / tricyclo-phosphorothioate subunits are shown below. [ka]

[0139] 6. 2'-O-Methyl, 2'-O-MOE, and 2'-F Oligomers "2'-O-Me oligomer" molecules have a methyl group at the 2'-OH residue of the ribose molecule. 2'-O-Me-RNA behaves the same (or similar) as DNA, but is protected from nuclease degradation. 2'-O-Me-RNA can also be combined with phosphorothioate oligomers (PTO) for further stabilization. 2'O-Me oligomers (phosphodiester or phosphorothioate) can be synthesized according to conventional techniques in the art (see, for example, Yoo et al., Nucleic Acids Res. 32:2008-16, 2004, which is incorporated herein by reference in its entirety). Non-limiting examples of 2'O-Me oligomers are shown below. [ka]

[0140] 2'-O-Methoxyethyl oligomers (2'-O-MOE) have a methoxyethyl group at the 2'-OH residue of the ribose molecule and are discussed in Martin et al., Helv. Chim. Acta, 78, 486-504, 1995, which is incorporated herein by reference in its entirety. Non-limiting examples of 2'-O-MOE subunits are shown below. [ka]

[0141] 2'-Fluoro (2'-F) oligomers have a fluorescent radical at the 2' position instead of 2'-OH. Non-limiting examples of 2'-F oligomers are shown below. [ka]

[0142] 2'-Fluoro oligomers are further described in WO2004 / 043977, which is incorporated herein by reference in its entirety.

[0143] The 2'-O-Methyl, 2'-O-MOE, and 2'-F oligomers may also contain one or more phosphorothioate (PS) linkages, as shown below. [ka]

[0144] Additionally, 2'-O-methyl, 2'-O-MOE, and 2'-F oligomers may contain PS intersubunit linkages throughout the oligomer, such as, for example, the 2'-O-methyl PS oligomer drisapersen shown below. [ka]

[0145] Alternatively, the 2'-O-methyl, 2'-O-MOE, and / or 2'-F oligomers may contain a PS linkage at the terminus of the oligomer, as shown below. [ka]

[0146] During the ceremony, R is CH2CH2OCH3 (methoxyethyl or MOE); X, Y, and Z represent the number of nucleotides contained within the designated 5'-wing, central gap, and 3'-wing regions, respectively.

[0147] The antisense oligomer of the present disclosure may incorporate one or more 2'-O-methyl, 2'-O-MOE, and 2'-F subunits and may utilize any of the intersubunit linkages described herein.In some cases, the antisense oligomer of the present disclosure may be completely composed of 2'-O-methyl, 2'-O-MOE, or 2'-F subunits.One embodiment of the antisense oligomer of the present disclosure is completely composed of 2'-O-methyl subunits.

[0148] 7. 2'-O-[2-(N-methylcarbamoyl)ethyl] oligomer (MCE) MCE is another example of a 2'-O modified ribonucleoside useful in the antisense oligomers of the present disclosure, where the 2'-OH is derivatized to a 2-(N-methylcarbamoyl)ethyl moiety to enhance nuclease resistance. A non-limiting example of an MCE oligomer is shown below. [ka]

[0149] MCE and its synthesis are described in Yamada et al., J. Org. Chem. (2011) 76(9):3042-53, which is incorporated by reference in its entirety. Antisense oligomers of the present disclosure may incorporate one or more MCE subunits.

[0150] 8. Stereospecific Oligomers Stereospecific oligomers are those in which the stereochemistry of each phosphorus-containing linkage is fixed by synthetic methods such that substantially stereopure oligomers are produced. Non-limiting examples of stereospecific oligomers are shown below. [ka]

[0151] In the above example, each phosphorus of oligomer has the same stereochemistry.Additional examples include oligomers described herein.For example, LNA, ENA, tricyclo-DNA, MCE, 2'-O-methyl, 2'-O-MOE, 2'-F and morpholino-based oligomers can be prepared using stereospecific phosphorus-containing internucleoside linkages, such as phosphorothioate, phosphodiester, phosphoramidate, phosphorodiamidate or other phosphorus-containing internucleoside linkages. Stereospecific oligomers, methods of preparation, chiral controlled synthesis, chiral designs, and chiral auxiliaries for use in the preparation of such oligomers are described, for example, in WO2017 / 192664, WO2017 / 192679, WO2017 / 062862, WO2017 / 015575, WO2017 / 015555, WO2015 / 107425, WO2015 / 108048, WO No. 2015 / 108046, WO2015 / 108047, WO2012 / 039448, WO2010 / 064146, WO2011 / 034072, WO2014 / 010250, WO2014 / 012081, WO2013 / 0127858, and WO2011 / 005761, each of which is incorporated herein by reference in its entirety.

[0152] The stereospecific oligomer is R P or S P The oligomer may have phosphorus-containing internucleoside linkages in any configuration. Chiral phosphorus-containing linkages in which the configuration of the linkage is controlled are referred to as "stereo-pure" and chiral phosphorus-containing linkages in which the configuration of the linkage is not controlled are referred to as "stereoirregular". In certain embodiments, the oligomers of the present disclosure include a plurality of stereo-pure and stereoirregular linkages, such that the resulting oligomer has stereo-pure subunits at pre-specified positions of the oligomer. Examples of the positions of stereo-pure subunits are provided in International Patent Application Publication No. WO2017 / 062862(A2) in Figures 7A and 7B. In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereoirregular. In one embodiment, all chiral phosphorus-containing linkages in the oligomer are stereo-pure.

[0153] In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), all n chiral phosphorus-containing linkages in the oligomer are stereoirregular. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), all n chiral phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 10% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 20% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 30% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 40% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 50% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 60% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n (n is an integer equal to or greater than 1) chiral phosphorus-containing linkages, at least 70% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 80% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure. In one embodiment of an oligomer having n chiral phosphorus-containing linkages (n is an integer equal to or greater than 1), at least 90% (rounded to the nearest whole number) of the n phosphorus-containing linkages in the oligomer are stereopure.

[0154] In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomers have the same stereo-orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least two consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least three consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least four consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least five consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least six consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 7 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 8 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., SP or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 9 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 10 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 11 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 12 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 13 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 14 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 15 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R PIn one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 16 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 17 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 18 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer has at least 19 consecutive stereopure phosphorus-containing linkages of the same stereo orientation (i.e., S P or R P The compound contains at least 20 consecutive stereopure phosphorus-containing linkages of the formula (I) or (II).

[0155] In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomers have the same stereo-orientation (i.e., S P or R P (either S P At least two consecutive stereopure phosphorus-containing linkages of the orientation R P and at least two consecutive stereopure phosphorus-containing linkages in the oriented direction.

[0156] In one embodiment of an oligomer having n chiral phosphorus-containing linkages, where n is an integer equal to or greater than 1, the oligomer contains at least two consecutive stereopure phosphorus-containing linkages of the same stereoorientation in an alternating pattern. For example, the oligomer may contain, in order, the following: two or more R P , 2 or more S P , and two or more R P etc.

[0157] 9. Morpholino Oligomers Exemplary embodiments of the present disclosure include compounds having 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). The morpholino described herein is intended to include all stereoisomers and tautomers of the above general structure. The synthesis, structure and binding properties of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476 and 8,299,206, all of which are incorporated herein by reference.

[0158] In certain embodiments, the morpholinos are conjugated to a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include: [ka] Including, The distal --OH or --NH2 of the "tail" portion is optionally linked to a cell membrane penetrating peptide.

[0159] In various embodiments, the present disclosure provides an antisense oligomer according to formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: each Nu is a nucleobase which together form a targeting sequence, T is [ka] wherein the distal -OH or -NH2 of the T moiety is optionally linked to a cell membrane penetrating peptide; R 100 is hydrogen or a cell membrane-permeable peptide, Each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 16] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0160] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO: 3.

[0161] In various embodiments, T is [ka] and the distal --OH of the T moiety is optionally linked to a cell membrane penetrating peptide.

[0162] In various embodiments, R 100 is hydrogen. In various other embodiments, R 100 is a cell membrane penetrating peptide. In various embodiments, R 100 -R 5 (SEQ ID NO:21). In various other embodiments, R 100 -GR 5 (SEQ ID NO: 20). In various embodiments, R 100 -R 6 (SEQ ID NO: 10). In various other embodiments, R 100 -GR 6 (Sequence number: 11).

[0163] In some embodiments, the antisense oligomer of formula (I) is in free base form. In some embodiments, the antisense oligomer of formula (I) is its pharma- ceutically acceptable salt. In some embodiments, the antisense oligomer of formula (I) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is 1HCl, 2HCl, 3HCl, 4HCl, 5HCl, or 6HCl salt. In certain embodiments, the HCl salt is 6HCl salt.

[0164] In various embodiments, T is [ka] and the distal -OH of the T moiety is optionally linked to a cell membrane penetrating peptide, R 100 is a cell membrane penetrating peptide.

[0165] In various embodiments, T is [ka] and R 100 is a cell membrane penetrating peptide.

[0166] In various embodiments, T is [ka] and R 100 -GR 5 (Sequence number: 20).

[0167] In various embodiments, T is [ka] and R 100 -GR 6 (Sequence number: 11).

[0168] In some embodiments, the antisense oligomer of the present disclosure is according to formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 17] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0169] In some embodiments, the distal --OH of formula (II) is linked to a cell membrane penetrating peptide.

[0170] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO: 3.

[0171] In some embodiments, the antisense oligomer of formula (II) is in free base form. In some embodiments, the antisense oligomer of formula (II) is in its pharma- ceutically acceptable salt form. In some embodiments, the antisense oligomer of formula (II) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is 1HCl, 2HCl, 3HCl, 4HCl, 5HCl, or 6HCl salt. In certain embodiments, the HCl salt is 6HCl salt.

[0172] In some embodiments, the antisense oligomer of the disclosure is according to formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 18] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0173] In some embodiments, the distal --OH of formula (III) is linked to a cell membrane penetrating peptide.

[0174] In some embodiments, the distal --OH of formula (III) is optionally linked to a cell membrane penetrating peptide.

[0175] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO: 3.

[0176] In some embodiments, the antisense oligomer of formula (III) is in free base form. In some embodiments, the antisense oligomer of formula (III) is its pharma- ceutically acceptable salt. In some embodiments, the antisense oligomer of formula (III) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is 6HCl salt.

[0177] In some embodiments, the antisense oligomer of the disclosure is according to formula (IV): [ka] wherein each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 19] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0178] In some embodiments, the distal --OH of formula (IV) is optionally linked to a cell membrane penetrating peptide.

[0179] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO: 3.

[0180] For example, in some embodiments, including some embodiments of formula (IV), the antisense oligomer of the disclosure conforms to formula (IVa). [ka]

[0181] In some embodiments, the antisense oligomer of the disclosure is according to formula (V): [ka] or a pharma- ceutically acceptable salt thereof, wherein each Nu, 1 to n, corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 20] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0182] In some embodiments, the distal --OH of formula (IV) is linked to a cell membrane penetrating peptide.

[0183] In some embodiments, the distal --OH of formula (V) is optionally linked to a cell membrane penetrating peptide.

[0184] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO: 3.

[0185] In some embodiments, the antisense oligomer of formula (V) is in free base form. In some embodiments, the antisense oligomer of formula (V) is its pharma- ceutically acceptable salt. In some embodiments, the antisense oligomer of formula (V) is its HCl (hydrochloric acid) salt. In certain embodiments, the HCl salt is a 5-HCl salt.

[0186] In some embodiments, the antisense oligomer of the disclosure is according to formula (VI): [ka] wherein each Nu from 1 to n corresponds, from 5' to 3', to a nucleobase in one of the following: [Table 21] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0187] In some embodiments, the distal --OH of formula (VI) is optionally linked to a cell membrane penetrating peptide.

[0188] In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, each Nu from 1 to n corresponds, from 5' to 3', to SEQ ID NO: 3.

[0189] 10. Nucleobase Modifications and Substitutions In certain embodiments, the antisense oligomer of the present disclosure is composed of RNA nucleobases and DNA nucleobases (often referred to simply as "bases" in the art). 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 of the present disclosure is composed of cytosine (C), guanine (G), thymine (T), adenine (A), 5-methylcytosine (5mC), uracil (U), and hypoxanthine (I).

[0190] 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 replaced 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.

[0191] Purine bases contain a pyrimidine ring fused to an imidazole ring, as described by the general formula below: [ka]

[0192] 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.

[0193] The pyrimidine base contains a six-membered pyrimidine ring, as described by the general formula: [ka]

[0194] Cytosine, uracil and thymine are the most commonly found pyrimidine bases in nucleic acid.Other naturally occurring pyrimidines include, but are not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil and 4-thiouracil.In one embodiment, the oligomer described herein contains thymine base instead of uracil.

[0195] Other suitable bases include 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamps and derivatives thereof, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; 2 -Cyclopentylguanine (cPent-G), N 2 -cyclopentyl-2-aminopurine (cPent-AP), and N 22-propyl-2-aminopurine (Pr-AP), pseudouracil, or derivatives thereof; and degenerate or universal bases such as 2,6-difluorotoluene, or non-existent bases such as abasic sites (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. Patent 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 normal N-glycoside in uridine. Pseudouridine-containing synthetic mRNAs may have an improved safety profile compared to uridine-containing mPvNAs (WO2009 / 127230, incorporated herein by reference in its entirety).

[0196] Certain nucleobases are particularly useful for increasing the binding affinity of the antisense oligomers 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.

[0197] 11. Pharmaceutically acceptable salts of antisense oligomers Certain embodiments of the antisense oligomers described herein may contain basic functional groups such as amino or alkylamino, and thus can form pharmaceutically acceptable salts with pharmaceutically acceptable acids.The term "pharmaceutically acceptable salts" in this respect refers to the relatively non-toxic inorganic and organic acid addition salts of the antisense oligomers of the present disclosure.These salts can be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting the purified antisense oligomers of the present disclosure in their free base form with suitable organic or inorganic acid, and then isolating the salts thus formed during subsequent 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).

[0198] Pharmaceutically acceptable salts of the subject antisense oligomers include conventional non-toxic salts or quaternary ammonium salts of the antisense oligomers, for example, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; as well as salts prepared from organic acids, such as 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 (salicyclic acid), sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid, and the like.

[0199] In certain embodiments, the antisense oligomers of the present disclosure may contain one or more acidic functional groups, and thus may 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 oligomers of the present disclosure. These salts may also be prepared in situ during the administration vehicle or dosage form manufacturing process, or may be prepared by separately reacting the purified antisense oligomer in its free acid form with a suitable base, such as a 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 forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (See, for example, Berge et al., supra).

[0200] III. Formulations and Modes of Administration In certain embodiments, the present disclosure provides a formulation or pharmaceutical composition suitable for therapeutic delivery of the antisense oligomers described herein.Thus, in certain embodiments, the present disclosure provides a pharma- ceutically acceptable composition comprising a therapeutically effective amount of one or more of the antisense oligomers described herein, formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents.Although it is possible to administer the antisense oligomers of the present disclosure alone, it is preferred to administer the antisense oligomers as a pharmaceutical formulation (composition).In one embodiment, the antisense oligomer of the formulation is according to formula (III) or a pharma- ceutically acceptable salt thereof.

[0201] Methods for delivery of nucleic acid molecules that may be applicable to the antisense oligomers 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 the delivery of virtually any nucleic acid molecule, including the antisense oligomers of the present disclosure.

[0202] 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 an immersion (aqueous or non-aqueous solution or suspension), tablet (for buccal, sublingual, or systemic absorption), bolus, powder, granule, paste for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained release formulation; (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.

[0203] Some examples of materials that can serve as pharma- ceutically acceptable carriers include: (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; (10) glycols, such as protease, glycerol, sorbitol ... Examples of suitable non-toxic and non-toxic organic solvents include, but are not limited to, 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 distilled water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffers, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic and compatible substances used in pharmaceutical formulations.

[0204] Additional non-limiting examples of agents suitable for formulation with the antisense oligomers of the present disclosure include PEG-conjugated nucleic acids; phospholipid-conjugated nucleic acids; nucleic acids containing lipophilic moieties; phosphorothioates; P-glycoprotein inhibitors (such as Pluronic P85) that 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).

[0205] The present disclosure also features the use of compositions comprising surface-modified liposomes containing poly(ethylene glycol) ("PEG") lipids (PEG-modified, branched, and 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, possibly by extravasation and entrapment in vascularized 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 compared to conventional cationic liposomes, which are known to accumulate in tissues, particularly in MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO96 / 10391; Ansell et al., International PCT Publication No. WO96 / 10390; Holland et al., International PCT 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.

[0206] In further embodiments, the present disclosure includes the antisense oligomer pharmaceutical composition prepared for delivery, as described in US Patent Nos. 6,692,911, 7,163,695, and 7,070,807.In this regard, in one embodiment, the present disclosure provides the antisense oligomer of the present disclosure in a composition comprising a copolymer of lysine and histidine (HK) (described in US Patent Nos. 7,163,695, 7,070,807, and 6,692,911), either 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 the antisense oligomer in a pharmaceutical composition comprising gluconate-modified polyhistidine or gluconyl-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.

[0207] Wetting agents, emulsifying agents, and lubricants (such as 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.

[0208] 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.

[0209] The formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will generally be that amount of active ingredient that produces a therapeutic effect. Generally, this amount will range from about 0.1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0210] In certain embodiments, the formulation of the present disclosure comprises an excipient selected from cyclodextrin, cellulose, liposome, micelle-forming agent, such as bile acid, and polymer carrier, such as polyester and polyanhydride, and an antisense oligomer of the present disclosure.In one embodiment, the antisense oligomer of the formulation is according to formula (IV).In one embodiment, the antisense oligomer of the formulation is according to formula (IVa).In certain embodiments, the above formulation makes the antisense oligomer of the present disclosure orally bioavailable.

[0211] The methods of preparing these formulations or pharmaceutical compositions include the step of bringing into association the antisense oligomer of the present disclosure with a carrier, and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the antisense oligomer of the present disclosure with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0212] 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 liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (with an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of an antisense oligomer of the present disclosure as an active ingredient. The antisense oligomer of the present disclosure may also be administered as a bolus, electuary, or paste.

[0213] In solid dosage forms of the present disclosure for oral administration (capsules, tablets, pills, dragees, powders, granules, trouches, and the like), the active ingredient may be combined with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid, (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia, (3) humectants, such as glycerol, (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) disintegrants, such as glycerol, (6) disintegrants, such as glycerol, (7) disintegrants, such as glycerol, (8) disintegrants, such as glycerol, (9) disintegrants, such as glycerol, (10) disintegrants, such as glycerol, (11) disintegrants, such as glycerol, (12) disintegrants, such as glycerol, (13) disintegrants, such as glycerol, (14) disintegrants, such as glycerol, (15) disintegrants, such as glycerol, (16) disintegrants, such as glycerol, (17) disintegrants, such as glycerol, (18) disintegrants, such as glycerol, (19) disintegrants, such as glycerol, (20) disintegrants, such as glycerol, (21) disintegrants, such as glycerol, (22) disintegrants, such as glycerol, (23) disintegrants, such as glycerol, (24) disintegrants, such as glycer (5) dissolution retarders, such as paraffin, (6) absorption enhancers, such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate, (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants, (8) absorbents, such as kaolin and bentonite clay, (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof, (10) coloring agents, and (11) release control agents, such as crospovidone or ethylcellulose. In the case of capsules, tablets, and pills, the pharmaceutical composition may also include buffering agents. Similar types of solid pharmaceutical compositions may also be used as fillers in soft and hard shell gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0214] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents or dispersants. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0215] Tablets and other solid dosage forms of the pharmaceutical composition of the present disclosure, such as dragees, capsules, pills, and granules, can be obtained or prepared with optional coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide slow or controlled release of the active ingredient therein, using, for example, hydroxypropylmethylcellulose in various proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They can be formulated for rapid release, such as, for example, lyophilization. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid pharmaceutical composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These pharmaceutical compositions can also optionally contain opacifying agents, and can be compositions that release the active ingredient(s) only or preferentially in certain parts of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0216] The liquid dosage form for oral administration of the antisense oligomer of the present disclosure includes pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and mixtures thereof.

[0217] Besides inert diluents, the oral pharmaceutical compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

[0218] In addition to the active compounds, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0219] Formulations for rectal or vaginal administration may be presented as suppositories, which can be prepared by mixing one or more compounds of the present disclosure with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

[0220] The preparation or dosage form for topical or transdermal administration of the oligomer provided herein includes powder, spray, ointment, paste, cream, lotion, gel, solution, patch and inhalant.The active oligomer conjugate can be mixed with a pharmaceutically acceptable carrier under sterile conditions and with any preservative, buffer or propellant that may be required.The ointment, paste, cream and gel can contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof, in addition to the active compound of the present disclosure.

[0221] Powders and sprays can contain, in addition to the antisense oligomers of the present disclosure, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can further contain customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0222] Transdermal patch has the additional advantage of providing controlled delivery of the antisense oligomer of the present disclosure into the body.Such dosage forms can be made by dissolving the oligomer in a suitable medium or dispersing it in a suitable medium.Absorption enhancers can also be used to increase the flux of drug across the skin.The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the drug in a polymer matrix or gel, among other methods known in the art.

[0223] 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 or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can 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 can 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 of the pharmaceutical composition is according to formula (IV). In one embodiment, the antisense oligomer of the pharmaceutical composition is according to formula (IVa).

[0224] These pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action on the subject oligomeric conjugates may be ensured by including various antibacterial and antifungal agents, for example, parabens, 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 including agents that delay absorption, such as aluminum monostearate and gelatin.

[0225] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injection in order to prolong the effect of drugs.This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility, among other methods known in the art.The absorption rate of drugs then depends on their dissolution rate, which in turn depends on crystal size and crystalline form.Alternatively, the delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drugs in oil vehicles.

[0226] 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.

[0227] When the antisense oligomers of the present disclosure are administered as pharmaceuticals to humans and animals, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1-99% (more preferably 10-30%) of the antisense oligomer in combination with a pharma- ceutically acceptable carrier.

[0228] The formulation or preparation 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, by lotion or ointment topically or by suppository rectally.

[0229] Regardless of the route of administration selected, the antisense oligomers 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. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure may vary 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.

[0230] The selected dosage level will depend on a variety of factors, including the activity of the particular antisense oligomer 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.

[0231] 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 start the dosage of the antisense oligomer 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 of the present disclosure is the amount of the antisense oligomer 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 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 effect.

[0232] In some embodiments, the antisense oligomers of the present disclosure are generally administered at a dose of about 1 to about 200 mg / kg, hi some embodiments, the dose for intravenous administration is about 0.5 mg to 200 mg / kg.

[0233] In some embodiments, the antisense oligomer of formula (I) is generally administered at a dose of about 1 to about 200 mg / kg. In some embodiments, the dose of the antisense oligomer of formula (I) for intravenous administration is about 0.5 mg to about 200 mg / kg. In some embodiments, the antisense oligomer of formula (II) is generally administered at a dose of about 1 to about 200 mg / kg. In some embodiments, the dose of the antisense oligomer of formula (II) for intravenous administration is about 0.5 mg to about 200 mg / kg. In some embodiments, the antisense oligomer of formula (III) is generally administered at a dose of about 1 to about 200 mg / kg. In some embodiments, the dose of the antisense oligomer of formula (III) for intravenous administration is about 0.5 mg to about 200 mg / kg. In some embodiments, the antisense oligomer of formula (IV) is generally administered at a dose of about 1 to about 200 mg / kg. In some embodiments, the dose of the antisense oligomer of formula (IV) for intravenous administration is about 0.5 mg to about 200 mg / kg. In some embodiments, the antisense oligomer of formula (IVa) is generally administered at a dose of about 1 to about 200 mg / kg. In some embodiments, the dose of the antisense oligomer of formula (IVa) for intravenous administration is about 0.5 mg to about 200 mg / kg. In some embodiments, the antisense oligomer of formula (V) is generally administered at a dose of about 1 to about 200 mg / kg. In some embodiments, the dose of the antisense oligomer of formula (V) for intravenous administration is about 0.5 mg to about 200 mg / kg. In some embodiments, the antisense oligomer of formula (VI) is generally administered at a dose of about 1 to about 200 mg / kg. In some embodiments, the dose of the antisense oligomer of Formula (VI) for intravenous administration is from about 0.5 mg to about 200 mg / kg.

[0234] As is understood in the art, weekly, biweekly, triweekly, or monthly administration may be one or more administrations or sub-doses as discussed herein.

[0235] The nucleic acid molecules and antisense oligomers described herein can be administered to cells by various 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, microemulsification technology can be utilized to improve the bioavailability of lipophilic (water-insoluble) pharmaceutical agents.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 compounds in the hepatobiliary circulation.

[0236] 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.

[0237] Although all suitable amphiphilic carriers are contemplated, currently preferred carriers are generally those that have a generally recognized as safe (GRAS) status and can both solubilize the antisense oligomers of the present disclosure and microemulsify at a later stage when the solution comes into contact with a complex aqueous phase (such as that found in the human gastrointestinal tract). Amphiphilic components that meet these requirements typically have an HLB (hydrophilic-lipophilic balance) value of 2-20, and their structures contain straight-chain aliphatic radicals ranging from C-6 to C-20. Examples include polyethylene glycolated fatty acid glycerides and polyethylene glycols.

[0238] Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various fully or partially hydrogenated vegetable oils. Such oils may advantageously consist of tri-, di-, and mono-fatty acid glycerides, as well as 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).

[0239] Commercially available amphiphilic carriers, 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- 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), may be particularly useful.

[0240] In certain embodiments, delivery may occur by using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., to introduce the pharmaceutical composition of the present disclosure into suitable host cells. In particular, the pharmaceutical composition of the present disclosure may be formulated for delivery by being encapsulated in either lipid particles, liposomes, vesicles, nanospheres, nanoparticles, or the like. The formulation and use of such delivery vehicles may be carried out using known and conventional techniques.

[0241] Suitable hydrophilic polymers 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 having a weight average molecular weight of 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.

[0242] 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.

[0243] 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.

[0244] 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).

[0245] 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). In addition, 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.

[0246] 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 U.S. Pat. No. 3,453,257]. Additionally, sulfoalkyl ether cyclodextrin derivatives have been described by Stella et al. (US Pat. No. 5,134,127).

[0247] 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 from 0.02 to 0.05 μm in diameter, while large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large oligolamellar and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm.

[0248] Liposomes with several non-concentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are called multivesicular vesicles. One aspect of the present disclosure relates to a formulation that includes liposomes containing antisense oligomers of the present disclosure, and the liposomal membrane is formulated to provide liposomes with increased loading capacity. Alternatively, or in addition, the antisense oligomers of the present disclosure can be contained within or adsorbed onto the liposomal bilayer of the liposome. The antisense oligomers of the present disclosure can be aggregated with lipid surfactants and loaded within the interior space of the liposome. In these cases, the liposomal membrane is formulated to resist the disruptive effect of active agent-surfactant aggregates.

[0249] According to one embodiment of the present disclosure, the lipid bilayer of the liposome contains lipids derivatized with poly(ethylene glycol) (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space encapsulated by the liposome and extend from the exterior of the lipid bilayer into the surrounding environment.

[0250] The active agent contained within the liposomes of the present disclosure is in solubilized form. Aggregates of surfactant and active agent (such as emulsions or micelles containing the active agent of interest) may be entrapped within the interior space of liposomes according to the present disclosure. The surfactant acts to disperse and solubilize the active agent and may be selected from any suitable aliphatic, alicyclic, or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholines (LPGs) 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, aiding in micelle formation. Surfactants with CMOs in the micromolar range are preferred, and higher CMC surfactants may be utilized to prepare micelles entrapped within the liposomes of the present disclosure.

[0251] 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 lipids derivatized with hydrophilic polymers into preformed liposomes, e.g., by exposing preformed liposomes to micelles composed of lipid-grafted polymers, at lipid concentrations corresponding to the final molar percentage of desired derivatized lipids in the liposomes. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques, as known in the art.

[0252] In another exemplary formulation procedure, active agent is first dispersed by sonication in lysophosphatidylcholine or other low CMC surfactants (including polymer-grafted lipids) that easily solubilize hydrophobic molecules.Then, the resulting micelle suspension of active agent is used to rehydrate the dried lipid sample that contains suitable mole percent of polymer-grafted lipids or cholesterol.Then, the lipid and active agent suspension is 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.

[0253] In one aspect of the present disclosure, liposomes are prepared to have a substantially uniform size in a selected size range.One effective size determination 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 maximum 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.

[0254] 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, for example, using a pH-sensitive coating that releases only at low pH, as found in the stomach, or high pH, ​​as found in the intestine. Enteric coatings 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 of the matrix 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 add microstructure to the matrix are added as microparticles. The range is typically 1 to 30 percent (w / w polymer).

[0255] Uptake can also be manipulated by changing the residence time of the particle in the intestine.This can be achieved, for example, by coating the particle with a mucoadhesive polymer or by selecting it as the encapsulation material.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 cyanoacrylate and methacrylate).

[0256] Antisense oligomers can be formulated to be contained in or adapted to be released by surgical or medical devices or implants.In certain embodiments, implants can be coated or otherwise treated with antisense oligomers.For example, hydrogels or other polymers such as biocompatible and / or biodegradable polymers can be used to coat implants with pharmaceutical compositions of the present disclosure (i.e., the compositions can be adapted to be used with medical devices by using hydrogels or other 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.

[0257] In addition to the methods provided herein, the antisense oligomers for use according to the present disclosure can be formulated for administration in any convenient manner for use in human or veterinary medicine, by analogy with other pharmaceuticals.Antisense oligomers and their corresponding formulations can be administered alone or in combination with other therapeutic strategies in the treatment of muscular dystrophy, 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).

[0258] In some embodiments, additional therapeutic agents may be administered prior to, simultaneously with, or after administration of the antisense oligomer of the present disclosure. For example, the antisense oligomer may be administered in combination with steroids and / or antibiotics. In certain embodiments, the antisense oligomer is administered to a patient undergoing background steroid therapy (e.g., intermittent or long-term / 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 undergo steroid therapy. In some embodiments, the steroid is a glucocorticoid or prednisone.

[0259] The described routes of administration are intended as a guide only, as one skilled in the art can easily determine the optimal route of administration and any dosage for any particular animal and condition.Several approaches have been attempted to introduce functional new 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 non-retroviral 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. al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209, and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855), ligand-specific, binding to 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 a transgene into a tissue 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).The group of Brigham et al. (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) has 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.

[0260] In further embodiments, the pharmaceutical composition 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 composition of the present disclosure may comprise 5% hexose carbohydrates. For example, the pharmaceutical composition of the present disclosure may comprise 5% glucose, 5% fructose, or 5% mannose. In certain embodiments, the pharmaceutical composition 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.

[0261] IV.How to use Restoring the dystrophin reading frame using exon skipping Potential therapeutic approach for the treatment of 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 maintains mRNA reading frame and produces internally truncated but still functional dystrophin protein.The antisense oligomer of the present disclosure is designed to achieve this.

[0262] Hybridization of an antisense oligomer of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (IVa), Formula (V), Formula (VI) with a targeted pre-mRNA sequence inhibits formation of the pre-mRNA splicing complex and deletes exon 50 from the mature mRNA. The structure and conformation of the antisense oligomers of the present disclosure allow for sequence-specific base pairing to the complementary sequence.

[0263] Normal dystrophin mRNA containing all 79 exons produces normal dystrophin protein. The shape of each exon indicates how the codon is split between exons. Notably, a codon consists of three nucleotides. Rectangular exons begin and end with a complete codon. Arrow-shaped exons begin with a complete codon but end with a split codon that contains only nucleotide #1 of the codon. Nucleotides #2 and #3 of this codon are contained in the subsequent exon that begins with a chevron shape.

[0264] Clinical outcomes to analyze the effect of antisense oligomers complementary to target regions in exon 50, intron 49, and / or intron 50 of human dystrophin pre-mRNA and inducing exon 50 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 rise (from supine position) without external support, de novo dystrophin production, and other functional measures.

[0265] In some embodiments, the present disclosure provides a method for producing dystrophin in a subject having a mutation in the dystrophin gene that is suitable for exon 50 skipping, the method comprising administering to the subject an antisense oligomer 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 suitable for exon 50 skipping. Protein production can be measured by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, or immunohistochemistry (IHC).

[0266] In some embodiments, the present 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 50 skipping, the method comprising administering to the subject an antisense oligomer as described herein or a pharma- ceutically acceptable salt thereof. 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).

[0267] In some embodiments, the present disclosure provides a method for maintaining or reducing the loss of lung function in a subject with DMD, the subject has a mutation in the DMD gene that is suitable for skipping exon 50, and the method comprises administering to the subject an antisense oligomer 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).

[0268] In further embodiments, the pharmaceutical composition of the present disclosure may be co-administered with carbohydrates in the methods of the present disclosure, either in the same formulation or in a separate formulation, 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 composition of the present disclosure may be co-administered with 5% hexose carbohydrates. For example, the pharmaceutical composition of the present disclosure may be co-administered with 5% glucose, 5% fructose, or 5% mannose. In certain embodiments, the pharmaceutical composition of the present disclosure may be co-administered with 2.5% glucose and 2.5% fructose. In some embodiments, the pharmaceutical compositions of the present disclosure may be co-administered with a carbohydrate 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.

[0269] In various embodiments, the antisense oligomer of the present disclosure is co-administered with a therapeutically effective amount of a nonsteroidal anti-inflammatory compound. In some embodiments, the nonsteroidal anti-inflammatory compound is an NF-kB inhibitor. For example, in some embodiments, the NF-kB inhibitor can be CAT-1004 or a pharmaceutically acceptable salt thereof. In various embodiments, the NF-kB inhibitor can be a conjugate of salicylate and DHA. In some embodiments, the NF-kB inhibitor is CAT-1041 or a pharmaceutically acceptable salt thereof. In certain embodiments, the NF-kB inhibitor is a conjugate of salicylate and EPA. In various embodiments, the NF-kB inhibitor is [ka] or a pharma- ceutically acceptable salt thereof.

[0270] In some embodiments, the nonsteroidal anti-inflammatory compound is a TGF-b inhibitor. For example, in certain embodiments, the TGF-b inhibitor is HT-100.

[0271] In certain embodiments, the antisense oligomer described herein is described for use in therapy.In certain embodiments, the antisense oligomer described herein is described for use in the treatment of Duchenne muscular dystrophy.In certain embodiments, the antisense oligomer described herein is described for use in the manufacture of a medicament for use in therapy.In certain embodiments, the antisense oligomer described herein is described for use in the manufacture of a medicament for the treatment of Duchenne muscular dystrophy.

[0272] V.Kit The present disclosure also provides a kit for the treatment of patients with genetic diseases, the kit comprising at least an antisense molecule (e.g., an antisense oligomer comprising a base sequence set forth in any of SEQ ID NOs: 1-9) packaged in a suitable container, together with instructions for its use. The kit may also contain peripheral reagents such as buffers, stabilizers, etc. Those skilled in the art should appreciate that the use of the above method has broad application for identifying antisense molecules suitable for use in the treatment of many other diseases. In one embodiment, the kit comprises an antisense oligomer according to any of formulas (I)-(VI). EXAMPLES

[0273] Although the above disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of the present disclosure, certain changes and modifications can 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 are not limiting. 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.

[0274] material and method Preparation of morpholino subunits [ka] Referring to Scheme 1, B represents a base-pairing moiety, and the morpholino subunit may be prepared from the corresponding ribonucleoside (1) as shown. The morpholino subunit (2) may be optionally protected by reaction with a suitable protecting group precursor, e.g., trityl chloride. The 3' protecting group is generally removed during solid-state oligomer synthesis, as described in more detail below. The base-pairing moiety may be suitably protected for solid-phase oligomer synthesis. Suitable protecting groups include benzoyl for adenine and cytosine, phenylacetyl for guanine, and pivaloyloxymethyl for hypoxanthine (inosine). The pivaloyloxymethyl group may be introduced at the N1 position of the hypoxanthine heterocyclic base. Although unprotected hypoxanthine subunits may be used, the yield in the activation reaction is much better when the base is protected. Other suitable protecting groups include those disclosed in US Pat. No. 8,076,476, which is incorporated herein by reference in its entirety.

[0275] Reaction of 3 with an activated phosphorus compound 4 yields a morpholino subunit bearing the desired linking moiety 5.

[0276] Compounds of structure 4 can be prepared using any number of methods known to those of skill in the art. Coupling with the morpholino moiety then proceeds as outlined above.

[0277] Compounds of structure 5 can be used in solid phase oligomer synthesis to prepare oligomers containing intersubunit linkages. Such methods are well known in the art. Briefly, compounds of structure 5 can be modified at the 5' end to contain a linker to a solid support. Once supported, the protecting group (e.g., trityl) of 5 at the 3' end is removed and the free amine is reacted with an activated phosphorus moiety of a second compound of structure 5. This sequence is repeated until an oligo of the desired sequence is obtained. The protecting group at the 3' end of the terminal can either be removed or left in place if a 3' modification is desired. The oligo can be removed from the solid support using any number of methods, or an exemplary treatment with a base to cleave the link to the solid support.

[0278] The preparation of morpholino oligomers in general and specific morpholino oligomers of the present disclosure is described in more detail in the Examples.

[0279] Preparation of morpholino oligomers The preparation of compounds of the present disclosure can be carried out according to Scheme 2 and using the following protocol. [ka]

[0280] Preparation of trityl piperazine phenylcarbamate 35: To a cooled suspension of compound 11 in dichloromethane (6 mL / g of 11) is added a solution of potassium carbonate (3.2 equiv.) in water (4 mL / g of potassium carbonate). To this biphasic mixture is slowly added a solution of phenyl chloroformate (1.03 equiv.) in dichloromethane (2 g / g of phenyl chloroformate). The reaction mixture is warmed to 20° C. Upon reaction completion (1-2 h), the layers are separated. The organic layer is washed with water and dried over anhydrous potassium carbonate. The product 35 is isolated by crystallization from acetonitrile.

[0281] Preparation of carbamate alcohol 36: Sodium hydride (1.2 equiv.) is suspended in 1-methyl-2-pyrrolidinone (32 mL / g of sodium hydride). Triethylene glycol (10.0 equiv.) and compound 35 (1.0 equiv.) can be added to this suspension. The resulting slurry is heated to 95° C. Upon reaction completion (1-2 h), the mixture is cooled to 20° C. To this mixture is added 30% dichloromethane / methyl tert-butyl ether (v:v) and water. The product-containing organic layer is washed successively with aqueous NaOH, aqueous succinic acid, and saturated aqueous sodium chloride. The product 36 is isolated by crystallization from dichloromethane / methyl tert-butyl ether / heptane.

[0282] Preparation of tere acid 37: To a solution of compound 36 in tetrahydrofuran (7 mL / g of 36), succinic anhydride (2.0 eq.) and DMAP (0.5 eq.) are added. The mixture is heated to 50° C. Upon reaction completion (5 h), the mixture is cooled to 20° C. and adjusted to pH 8.5 with aqueous NaHCO3. Methyl tert-butyl ether is added and the product is extracted into the aqueous layer. Dichloromethane is added and the aqueous mixture is adjusted to pH 3 with aqueous citric acid. The product-containing organic layer is washed with a mixture of citrate buffer at pH=3 and saturated aqueous sodium chloride. This dichloromethane solution of 37 is used without isolation in the preparation of compound 38.

[0283] Preparation of 38: N-hydroxy-5-norbornene-2,3-dicarboxylic imide (HONB) (1.02 equiv.), 4-dimethylaminopyridine (DMAP) (0.34 equiv.), then 1-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (1.1 equiv.) are added to a solution of compound 37. The mixture is heated to 55 °C. Upon reaction completion (4-5 h), the mixture is cooled to 20 °C and washed successively with 1:1 0.2 M citric acid / brine and brine. The dichloromethane solution is solvent exchanged into acetone and then N,N-dimethylformamide, and the product is isolated by precipitation from acetone / N,N-dimethylformamide into saturated aqueous sodium chloride. The crude product is reslurried several times in water to remove residual N,N-dimethylformamide and salts.

[0284] PMO synthesis method A: Use of disulfide anchors Introduction of the activated "tail" onto the anchor-loaded resin is carried out in dimethylimidazolidinone (DMI) by the procedure used for incorporation of the subunits during solid-phase synthesis. [ka]

[0285] This procedure can be carried out in a silanized jacketed peptide vessel (ChemGlass, NJ, USA) with a coarse porosity (40-60 μm) glass frit, an overhead stirrer, and a Teflon three-way stopcock to allow N2 to be bubbled through the frit or vacuum extraction.

[0286] The resin treatment / wash step in the procedure below consists of two basic operations: resin fluidization or stirred bed reactor and solvent / solution extraction. For resin fluidization, the stopcock is positioned to allow N2 to flow upward through the frit, and the specified resin treatment / wash solution is added to the reactor, permeating and thoroughly wetting the resin. Mixing is then started, and the resin slurry is mixed for the specified time. For solvent / solution extraction, the stopcock is positioned to allow mixing and N2 flow to be stopped, the vacuum pump started, and then the resin treatment / wash solution to be drained and discarded. All resin treatment / wash solution volumes are 15 mL / g of resin unless otherwise noted.

[0287] 1-Methyl-2-pyrrolidinone (NMP; 20 mL / g resin) is added to aminomethyl polystyrene resin (100-200 mesh; approximately 1.0 mmol / g loading based on nitrogen displacement; 75 g, 1 equiv., Polymer Labs, UK, part number 1464-X799) in a silanized jacketed peptide vessel and the resin is allowed to swell with mixing for 1-2 h. After draining the swelling solvent, the resin is washed with dichloromethane (2 × 1-2 min), 5% diisopropylethylamine in 25% isopropanol / dichloromethane (2 × 3-4 min), and dichloromethane (2 × 1-2 min). After draining the final wash, the resin is treated with a solution of disulfide anchor 34 in 1-methyl-2-pyrrolidinone (0.17 M; 15 mL / g resin, approximately 2.5 equiv.) and the resin / reagent mixture is heated at 45 °C for 60 h. Upon reaction completion, discontinue heating, drain the anchor solution, and wash the resin with 1-methyl-2-pyrrolidinone (4 × 3-4 min) and dichloromethane (6 × 1-2 min). Treat the resin with a solution of 10% (v / v) diethyl dicarbonate (DEDC) in dichloromethane (16 mL / g, 2 × 5-6 min) and then wash with dichloromethane (6 × 1-2 min). Dry the resin 39 under a stream of N2 for 1-3 h and then under vacuum to constant weight (± 2%).

[0288] Determination of Aminomethylpolystyrene-Disulfide Resin Loading: The resin loading (number of potentially available reactive sites) is determined by spectrophotometric assay of the number of triphenylmethyl (trityl) groups per gram of resin.

[0289] Transfer a known weight of dry resin (25 ± 3 mg) to a silanized 25 mL volumetric flask and add approximately 5 mL of 2% (v / v) trifluoroacetic acid in dichloromethane. Mix the contents by gently swirling and allow to stand for 30 min. The volume is brought up to 25 mL with additional 2% (v / v) trifluoroacetic acid in dichloromethane and the contents are mixed thoroughly. Using a positive displacement pipette, transfer an aliquot of the trityl-containing solution (500 μL) to a 10 mL volumetric flask and bring the volume to 10 mL with methanesulfonic acid.

[0290] The trityl cation content in the final solution is measured by UV absorbance at 431.7 nm, and the resin loading is measured in μmol / g trityl groups per gram of resin using the appropriate volume, dilution, extinction coefficient (ε: 41 μmol cm), and resin weight. Assays are performed in triplicate and the average loading is calculated.

[0291] The resin loading procedure of this example provides a loading of approximately 500 μmol / g of resin. If the disulfide anchor incorporation step is performed at room temperature for 24 h, loadings of 300-400 μmol / g are obtained.

[0292] Tail loading: The tail can be introduced to the solid support using the same setup and volumes as for the preparation of the aminomethyl polystyrene-disulfide resin. The anchor-loaded resin is first deprotected under acidic conditions and the resulting material is neutralized before coupling. In the coupling step, a solution of 38 (0.2 M) in DMI containing 4-ethylmorpholine (NEM, 0.4 M) is used instead of the 1-methyl-2-pyrrolidinone of the disulfide anchor solution. After 2 h at 45 °C, the resin 39 is washed twice with 5% diisopropylethylamine in 25% isopropanol / dichloromethane and once with DCM. A solution of benzoic anhydride (0.4 M) and NEM (0.4 M) is added to the resin. After 25 min, the reactor jacket is cooled to room temperature and the resin is washed twice with 5% diisopropylethylamine in 25% isopropanol / dichloromethane and eight times with DCM. Resin 40 is filtered and dried under high vacuum. The loading of resin 40 is defined as the loading of the original aminomethylpolystyrene-disulfide resin 39 used in the tail loading.

[0293] Solid-phase synthesis: Morpholino oligomers are prepared on a custom BioAutomation 128AVB (Plano, TX) in 4 mL BioComma polypropylene reaction columns (part number CT003-BC). When the column is placed on the synthesizer, an aluminum block with channels for water flow is placed around the column. The AVB128 alternatively adds reagent / wash solutions, holds for a specified time, and empties the column using vacuum.

[0294] For oligomers ranging in length up to about 25 subunits, aminomethylpolystyrene disulfide resins with loadings approaching 500 μmol / g of resin are preferred. For larger oligomers, aminomethylpolystyrene-disulfide resins with loadings of 300-400 μmol / g of resin are preferred. If molecules with 5'-tails are desired, tail-loaded resins are selected with the same loading guidelines.

[0295] Prepare the following reagent solutions: Detritylation solution: 1% 4-cyanopyridine and trifluoroacetic acid (w / w) in a 4:1 dichloromethane / trifluoroethanol solution; Neutralization solution: 3% diisopropylethylamine in a 5:1 dichloromethane / isopropanol solution, and Coupling solution: 0.18 M (or 0.24 M for oligomers growing longer than 20 subunits) activated morpholino subunits of the desired base and linkage type in 1,3-dimethylimidazolidinone (DMI) solution and 0.4 M N-ethylmorpholine. Dichloromethane (DCM) is used as a transition wash to separate the different reagent solution washes. With the synthesizer block set at 42° C., 2 mL of 1-methyl-2-pyrrolidinone is added to each column containing 30 mg of aminomethylpolystyrene-disulfide resin (or tail resin) and left at room temperature for 30 minutes. After washing twice with 2 mL of dichloromethane, the following synthesis cycle can be used. [Table 22]

[0296] The sequence of individual oligomers is programmed into the synthesizer such that each column receives the appropriate coupling solution (A, C, G, T, or I) in the appropriate sequence. When an oligomer in a column has completed incorporation of its final subunit, the column is removed from the block and a final cycle is performed manually using a coupling solution containing 4-methoxytriphenylmethyl chloride (0.32 M in DMI) and 0.89 M 4-ethylmorpholine.

[0297] Cleavage from the resin and removal of base and backbone protecting groups: After methoxytritylation, the resin is washed 8 times with 2 mL of 1-methyl-2-pyrrolidinone. 1 mL of cleavage solution consisting of 0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in 1-methyl-2-pyrrolidinone is added, the column is capped and left at room temperature for 30 min. It is then drained into a 12 mL Wheaton vial. The heavily shrunken resin is washed twice with 300 μL of cleavage solution. To the solution, 4.0 mL of concentrated aqueous ammonia (stored at -20 °C) is added, the vial is tightly capped (with a screw cap with a Teflon liner) and the mixture is swirled to mix the solution. The vial is placed in a 45 °C oven for 16-24 h, resulting in cleavage of the base and backbone protecting groups.

[0298] Crude product purification: Remove the vial of ammonolysis solution from the oven and allow to cool to room temperature. Dilute the solution with 20 mL of 0.28% aqueous ammonia and pass it through a 2.5 x 10 cm column containing Macroprep HQ resin (BioRad). Elute the methoxytrityl-protected oligomers using a salt gradient (A: 0.28% ammonia, B: 1 M sodium chloride in 0.28% ammonia, 0 to 100% B in 60 min). Pool the combined fractions and process further depending on the desired product.

[0299] Demethoxytritylation of morpholino oligomers: Pooled fractions from Macroprep purification are treated with 1 M H3PO4 to lower the pH to 2.5. After initial mixing, samples are allowed to stand at room temperature for 4 min, at which point they are neutralized to pH 10-11 with 2.8% ammonia / water. The product is purified by solid phase extraction (SPE).

[0300] SPE column packing and conditioning: Amberchrome CG-300M (Dow Chemicals (Rohm and Haas); Midland, MI) (3 mL) is packed into a 20 mL fritted column (BioRad Econo-Pac Chromatography Columns (732-1011)) and the resin is rinsed with 3 mL of the following: 0.28% NH4OH / 80% acetonitrile, 0.5 M NaOH / 20% ethanol, water, 50 mM H3PO4 / 80% acetonitrile, water, 0.5 NaOH / 20% ethanol, water, 0.28% NH4OH.

[0301] SPE purification: The solution from demethoxytritylation is loaded onto the column and the resin is rinsed three times with 8 mL of 0.28% aqueous ammonia. A Wheaton vial (12 mL) can be placed under the column and the product can be eluted by washing twice with 2 mL of 45% acetonitrile in 0.28% aqueous ammonia.

[0302] Product isolation: The solution is frozen in dry ice and the vial is placed in a freeze-dryer for at least 2 days to obtain a fluffy white powder. The sample is then dissolved in water and filtered through a 0.22 micron filter (Pall Life Sciences, Acrodisc 25 mm syringe filter, with 0.2 micron HT Tuffryn membrane) using a syringe, and the optical density (OD) is measured on a UV spectrophotometer to determine the OD units of oligomers present, and a sample is dispensed for analysis. The solution is then returned to a Wheaton vial for freeze-drying.

[0303] Analysis of morpholino oligomers by MALDI: MALDI-TOF mass spectrometry can be used to determine the composition of fractions in the purification and to obtain evidence of the identity (molecular weight) of the oligomers. Samples can be run after dilution using solutions of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid), 3,4,5-trihydroxyacetophenone (THAP), or alpha-cyano-4-hydroxycinnamic acid (HCCA) as matrices.

[0304] PMO synthesis method B: Use of nitrocarboxyphenylpropyl (NCP2) anchor NCP2 anchor synthesis: 1. Preparation of methyl 4-fluoro-3-nitrobenzoate (1) [ka] 12.7 kg of 4-fluoro-3-nitrobenzoic acid, 40 kg of methanol, and 2.82 kg of concentrated sulfuric acid can be added to a 100 L flask. The mixture is stirred at reflux (65° C.) for 36 hours. The reaction mixture is cooled to 0° C. Crystals can form at about 38° C. The mixture is held at 0° C. for 4 hours and then filtered under nitrogen. The 100 L flask is washed and the filter cake is washed with 10 kg of methanol that has been cooled to 0° C. The solid filter cake is dried on the funnel for 1 hour, transferred to a tray, and dried to constant weight in a vacuum oven at room temperature.

[0305] 2. Preparation of 3-nitro-4-(2-oxopropyl)benzoic acid A. (Z)-Methyl 4-(3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl)-3-nitrobenzoate (2) [ka] 3.98 kg of methyl 4-fluoro-3-nitrobenzoate (1) from the previous step, 9.8 kg of DMF, and 2.81 kg of methyl acetoacetate can be added to a 100 L flask. The mixture is stirred and cooled to 0° C. 3.66 kg of DBU is added over approximately 4 hours while maintaining the temperature below 5° C. The mixture is stirred for an additional hour. A solution of 8.15 kg of citric acid in 37.5 kg of purified water is added while maintaining the reaction temperature below 15° C. After the addition, the reaction mixture is stirred for an additional 30 minutes and then filtered under nitrogen. The wet filter cake is returned to the 100 L flask along with 14.8 kg of purified water. The slurry is stirred for 10 minutes and then filtered. The wet cake was returned to the 100 L flask, slurried with 14.8 kg of purified water for 10 minutes, and filtered to obtain crude (Z)-methyl 4-(3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl)-3-nitrobenzoate.

[0306] B. 3-Nitro-4-(2-oxopropyl)benzoic acid [ka] The crude (Z)-methyl 4-(3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl)-3-nitrobenzoate can be charged to a 100 L reaction flask under nitrogen. 14.2 kg of 1,4-dioxane is added and then stirred. A solution of 16.655 kg of concentrated HCl and 13.33 kg of purified water (6M HCl) is added over 2 hours while maintaining the temperature of the reaction mixture below 15° C. Once the addition is complete, the reaction mixture is heated to reflux (80° C.) for 24 hours, cooled to room temperature, and filtered under nitrogen. The solid filter cake is triturated with 14.8 kg of purified water, filtered, triturated again with 14.8 kg of purified water, and filtered. The solid is returned to the 100 L flask with 39.9 kg of DCM and refluxed with stirring for 1 hour. 1.5 kg of purified water is added to dissolve the remaining solid. The bottom organic layer is dispensed into a pre-warmed 72 L flask and then transferred back to a clean, dry 100 L flask. The solution is cooled to 0° C., held for 1 hour, and then filtered. The solid filter cake is washed twice with a solution of 9.8 kg DCM and 5 kg heptane, respectively, and then dried on the funnel. The solid is transferred to a tray and dried to a constant weight of 1.855 kg 3-nitro-4-(2-oxopropyl)benzoic acid.

[0307] 3. Preparation of N-tritylpiperazine succinate (NTP) [ka] 1.805 kg of triphenylmethyl chloride and 8.3 kg of toluene (TPC solution) can be charged to a 72 L jacketed flask under nitrogen. The mixture is stirred until the solids are dissolved. 5.61 kg of piperazine, 19.9 kg of toluene, and 3.72 kg of methanol are added to a 100 L jacketed reaction flask under nitrogen. The mixture is stirred and cooled to 0° C. The TPC solution is added slowly in portions over 4 hours while maintaining the reaction temperature at or below 10° C. The mixture is stirred at 10° C. for 1.5 hours and then allowed to warm to 14° C. 32.6 kg of purified water can be charged to the 72 L flask and then transferred to the 100 L flask while maintaining an internal batch temperature of 20+ / -5° C. The layers are allowed to split and the bottom aqueous layer is separated and saved. The organic layer is extracted three times with 32 kg of purified water each and the aqueous layer is separated and combined with the saved aqueous solution.

[0308] The remaining organic layer is cooled to 18°C ​​and a solution of 847g succinic acid in 10.87kg purified water is added slowly in portions to the organic layer. The mixture is stirred at 20+ / -5°C for 1.75 hours. The mixture is filtered and the solid is washed with 2kg TBME and 2kg acetone, then dried on the funnel. The filter cake is triturated twice with 5.7kg acetone each, filtered and washed with 1kg acetone between triturations. The solid is dried on the funnel and then transferred to a tray and dried to constant weight in a vacuum oven at room temperature.

[0309] 4. Preparation of (4-(2-hydroxypropyl)-3-nitrophenyl)(4-tritylpiperazin-1-yl)methanone A. Preparation of 1-(2-nitro-4(4-tritylpiperazine-1-carbonyl)phenyl)propan-2-one [ka] 2 kg of 3-nitro-4-(2-oxopropyl)benzoic acid (3), 18.3 kg of DCM, and 1.845 kg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl) can be charged to a 100 L jacketed flask under nitrogen. The solution is stirred until a homogenous mixture is formed. 3.048 kg of NTPs are added over 30 minutes at room temperature and stirred for 8 hours. 5.44 kg of purified water is added to the reaction mixture and stirred for 30 minutes. The layers are separated and the bottom organic layer containing the product is drained and saved. The aqueous layer is extracted twice with 5.65 kg of DCM. The combined organic layers are washed with a solution of 1.08 kg of sodium chloride in 4.08 kg of purified water. The organic layers are dried over 1.068 kg of sodium sulfate and filtered. The sodium sulfate is washed with 1.3 kg of DCM. The combined organic layers are slurried with 252 g of silica gel and filtered through a filter funnel containing a bed of 252 g of silica gel. The silica gel bed is washed with 2 kg of DCM. The combined organic layers are evaporated on a rotary evaporator, then 4.8 kg of THF is added to the residue and evaporated on a rotary evaporator until 2.5 volumes of crude 1-(2-nitro-4(4-tritylpiperazine-1-carbonyl)phenyl)propan-2-one in THF is reached.

[0310] B. Preparation of (4-(2-hydroxypropyl)-3-nitrophenyl)(4-tritylpiperazin-1-yl)methanone (5) [ka] 3600 g of 4 from the previous step and 9800 g of THF can be charged under nitrogen to a 100 L jacketed flask. The stirred solution is cooled to below 5° C. The solution is diluted with 11525 g of ethanol and 194 g of sodium borohydride is added over approximately 2 hours below 5° C. The reaction mixture is stirred for an additional 2 hours below 5° C. The reaction is quenched by slowly adding a solution of 1.1 kg of ammonium chloride in 3 kg of water to maintain the temperature below 10° C. The reaction mixture is stirred for an additional 30 minutes, filtered to remove inorganics, recharged to the 100 L jacketed flask, and extracted with 23 kg of DCM. The organic layer is separated and the aqueous layer is extracted two more times with 4.7 kg of DCM each. The combined organic layers are washed with a solution of 800 g of sodium chloride in 3 kg of water and then dried over 2.7 kg of sodium sulfate. The suspension is filtered and the filter cake is washed with 2 kg of DCM. The combined filtrates are concentrated to 2.0 volumes, diluted with approximately 360 g of ethyl acetate, and evaporated. The crude product is loaded under nitrogen onto a silica gel column of 4 kg of silica packed with DCM and eluted with 2.3 kg of ethyl acetate in 7.2 kg of DCM. The combined fractions are evaporated and the residue is taken up in 11.7 kg of toluene. The toluene solution is filtered and the filter cake is washed twice with 2 kg of toluene each. The filter cake is dried to constant weight.

[0311] 5. Preparation of 2,5-dioxopyrrolidin-1-yl (1-(2-nitro-4-(4-triphenylmethylpiperazine-1carbonyl)phenyl)propan-2-yl) carbonate (NCP2 anchor) [ka] 4.3 kg of compound 5( 1 3. 160 kg of DSC (weight adjusted based on residual toluene by H NMR; all subsequent reagents are scaled accordingly) and 12.7 kg of pyridine can be charged to a 100 L jacketed flask under nitrogen. 1To this is added 100 g of pyridine, 78.91 wt.% by H NMR, while maintaining the internal temperature below 35° C. The reaction mixture is aged at ambient room temperature for about 22 hours and then filtered. The filter cake is washed with 200 g of pyridine. In two batches, each containing ½ filtrate volume, the filtrate washes can be charged slowly to a 100 L jacketed flask containing a solution of 11 kg citric acid in 50 kg water and stirred for 30 minutes to allow solid precipitation. The solids are collected on a filter funnel, washed twice with 4.3 kg water per wash, and dried on the filter funnel under vacuum.

[0312] The combined solids are charged to a 100 L jacketed flask, dissolved in 28 kg DCM, and washed with a solution of 900 g potassium carbonate in 4.3 kg water. After 1 hour, the layers are allowed to separate and the aqueous layer is removed. The organic layer is washed with 10 kg water, separated, and dried over 3.5 kg sodium sulfate. The DCM is filtered, evaporated, and dried under vacuum to 6.16 kg NCP2 anchor.

[0313] NCP2 anchor loaded resin synthesis Approximately 52 L of NMP and 2300 g of aminomethyl polystyrene resin can be loaded into a 75 L solid phase synthesis reactor equipped with a Teflon stopcock. The resin is stirred in NMP for approximately 2 hours to swell, then drained. The resin is washed twice with 4 L of DCM per wash, then twice with 39 L of neutralization solution per wash, then twice with 39 L of DCM per wash. The NCP2 anchor solution is slowly added to the stirred resin solution, stirred at room temperature for 24 hours, then drained. The resin is washed four times with 39 L of NMP per wash and six times with 39 L of DCM per wash. The resin is treated and stirred with 1 / 2 diethyl dicarbonate (DEDC) capping solution for 30 minutes, drained, and treated and stirred with the remaining 1 / 2 DEDC capping solution for 30 minutes, drained. The resin is washed six times with 39 L of DCM per wash and then dried in an oven to a constant weight of 3573.71 g of anchor loaded resin.

[0314] Preparation of morpholino oligomers using NCP2 anchor 50L solid phase synthesis of PMO crude drug substance 1.Material [Table 2] Chemical structure of the starting material: A. Activated EG3 tail [ka] B. Activated C subunit (for preparation, see U.S. Pat. No. 8,067,571) [ka] C. Activated A Subunit (for preparation, see U.S. Pat. No. 8,067,571) [ka] D. Activated DPG Subunits (for preparation, see WO2009 / 064471) [ka] E. Activated T subunit (for preparation, see WO2013 / 082551) [ka] F. Anchor loaded resin [ka] In the formula, R 1 is the support medium. [Table 3]

[0315] 2. Synthesis of PMO crude drug substance A. Resin swelling An aliquot of 750 g of anchor-loaded resin and 10.5 L of NMP can be charged into a 50 L silanized reactor and stirred for 3 hours. The NMP is drained and the anchor-loaded resin is washed twice with 5.5 L each of DCM and twice with 5.5 L each of 30% TFE / DCM.

[0316] B. Cycle 0: EG3 tail coupling The anchor-loaded resin can be washed three times with 5.5 L each of 30% TFE / DCM, drained, washed with 5.5 L of CYTFA solution for 15 minutes, drained, washed again with 5.5 L of CYTFA solution for 15 minutes without draining, loaded with 122 mL of 1:1 NEM / DCM, and the suspension stirred for 2 minutes and drained. The resin can be washed twice with 5.5 L of neutralization solution for 5 minutes, drained, then washed twice with 5.5 L each of DCM and drained. The resin can be loaded with a solution of 706.2 g of activated EG3 tails and 234 mL of NEM in 3 L of DMI, stirred for 3 hours at room temperature, and drained. The resin can be washed twice with 5.5 L each of neutralization solution for 5 minutes, then once with 5.5 L of DCM and drained. A solution of 374.8 g benzoic anhydride and 195 mL NEM in 2680 mL NMP can be charged, stirred for 15 minutes, and drained. The resin is stirred with 5.5 L of neutralization solution for 5 minutes, then washed once with 5.5 L DCM and twice with 5.5 L each of 30% TFE / DCM. The resin is suspended in 5.5 L of 30% TFE / DCM and held for 14 hours.

[0317] C. Subunit coupling cycles 1-n [Table 4] i. Treatment before coupling Before each coupling cycle, the resin is 1) washed with 30% TFE / DCM, 2) a) treated with CYTFA solution for 15 minutes and drained, b) treated with CYTFA solution for 15 minutes to which 1:1 NEM / DCM is added, vortexed, drained, 3) vortexed three times with neutralization solution, and 4) washed twice with DCM.

[0318] ii. Post-coupling treatment After each subunit solution is drained, the resin is washed 1) with DCM and 2) twice with 30% TFE / DCM. If the resin is to be held for a period of time before the next coupling cycle, the second TFE / DCM wash is not drained and the resin is held in the TFE / DCM wash.

[0319] iii. Activation subunit coupling cycle Each coupling cycle is carried out as outlined in Table 2 for the initial C (cytosine) monomer coupling for each base-containing subunit.

[0320] iv. Final IPA wash After the final coupling step is performed, the resin is washed 8 times with 19.5 L IPA each and dried under vacuum at room temperature for about 63.5 hours to a dry weight of 5,579.8 g.

[0321] C. Cutting The above resin-bound PMO crude drug substance is split into two lots, and each lot is treated as follows: A 2,789.9 g lot of resin is 1) agitated with 10 L NMP for 2 hours, then drained of NMP, 2) washed three times with 10 L each of 30% TFE / DCM, 3) treated with 10 L CYTFA solution for 15 minutes, 4) treated with 10 L CYTFA solution for 15 minutes, then added with 130 mL of 1:1 NEM / DCM, agitated for 2 minutes, and drained. The resin is treated three times with 10 L each of neutralization solution, washed six times with 10 L DCM, and eight times with 10 L each of NMP. The resin is treated with a cleavage solution of 1530.4 g DTT and 2980 DBU in 6.96 L NMP for 2 hours to cleave the PMO crude drug substance from the resin. The cleavage solution is drained and kept in a separate tank. The reactor and resin are washed with 4.97 L of NMP combined with the cleavage solution.

[0322] D. Deprotection The combined cleavage solution and NMP wash were transferred to a pressure vessel to which was added 39.8 L of NH4OH (NH3·H2O) that had been pre-cooled in a freezer to a temperature of -10°C to -25°C. The pressure vessel was sealed and heated to 45°C for 16 hours and then allowed to cool to 25°C. This deprotection solution, containing the PMO crude drug substance, was diluted 3:1 with purified water and the pH adjusted to 3.0 with 2M phosphoric acid and then to pH 8.03 with NH4OH.

[0323] E. Purification of PMO crude drug substance The deprotected solution from Part D above containing the PMO crude drug substance is loaded onto a column of ToyoPearl Super-Q 650S anion exchange resin (Tosoh Bioscience) and eluted with a gradient of 0-35% B over 17 column volumes (Buffer A: 10 mM sodium hydroxide, Buffer B: 1 M sodium chloride in 10 mM sodium hydroxide) and fractions of acceptable purity (C18 and SCX HPLC) are pooled to yield the purified drug product solution.

[0324] The purified drug substance solution is desalted and lyophilized to provide the purified PMO drug substance. [Table 5]

[0325] CPP conjugate ("R6Gly" is disclosed as SEQ ID NO:11) [ka] Analytical procedure: Matrix-assisted laser desorption ionization time-of-flight mass spectra (MALDI-TOF-MS) can be recorded on a Bruker Autoflex™ Speed ​​using a sinapinic acid (SA) matrix. SCX-HPLC can be performed on a Thermo Dionex UltiMate 3000 system equipped with a 3000 diode array detector and a ProPac™ SCX-20 column (250×4 mm) using a flow rate of 1.0 mL / min (pH=2, column temperature 30° C.). The mobile phase can be A (25% acetonitrile in water containing 24 mM H3PO4) and B (25% acetonitrile in water containing 1 M KCl and 24 mM H3PO4). Gradient elution can be used: 0 min, 35% B; 2 min, 35% B; 22 min, 80% B; 25 min, 80% B; 25.1 min, 35% B; 30 min, 35% B.

[0326] Ac-L-Arg-L-Arg-L-Arg-L-Arg-L-Arg-L-Arg-Gly-OH (SEQ ID NO:11) hexatrifluoroacetate (614.7 mg, 0.354 mmol), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 134.4 mg, 0.354 mmol), and dimethyl sulfoxide (DMSO, 20 mL) are added to a mixture of PMO (freshly dried by lyophilization for 2 days). The mixture is stirred at room temperature for 3 min, and then N,N-diisopropylethylamine (DIPEA, 68.5 mg, 0.530 mmol) is added. After 5 min, the cloudy mixture becomes a clear solution. The reaction can be monitored by SCX-HPLC. After 2 hours, 20 mL of 10% ammonium hydroxide solution (2.8% NH3*H2O) is added. The mixture is stirred at room temperature for another 2 hours. The reaction is terminated by the addition of 400 mL of water. Trifluoroethanol (2.0 mL) is added to the solution.

[0327] The solution can be split into two portions and each portion can be purified by WCX column (10 g resin per column). Each WCX column can be first washed with 20% acetonitrile in water (v / v) to remove the PMO starting material. The washing (225 mL for each column) can be stopped when MALDI-TOF mass spectrometry analysis shows the absence of PMO signal. Each column is then washed with water (100 mL per column). The desired product can be eluted with 2.0 M guanidine HCl (140 mL for each column). The purified solution can be pooled together and then split into two portions, each of which is desalted by SPE column (10 g resin for each column).

[0328] The SPE columns can be first washed with 1.0 M aqueous NaCl (100 mL for each column) to generate the hexahydrochloride salt form. Each SPE column is then washed with water (200 mL for each column). The final desalted product can be eluted using 50% acetonitrile in water (v / v, 150 mL for each column). The acetonitrile can be removed by draining under reduced pressure. The resulting aqueous solution can be lyophilized to yield the desired product as the hexahydrochloride salt.

[0329] Example 1: PMO Using the PMO synthesis method described above, PMO#1, PMO#2, and PMO#3 were synthesized as follows. [ka] In the formula, each Nu from 1 to 22 and from 5' to 3' is H50D(+04-18) (SEQ ID NO: 1). [Table 23] [ka] In the formula, each Nu from 1 to 25 and from 5' to 3' is H50D(+07-18) (SEQ ID NO: 2). [Table 24] [ka] In the formula, each Nu from 1 to 23 and from 5' to 3' is H50D(+07-16) (SEQ ID NO: 3). [Table 25] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0330] PMO#1 (SEQ ID NO:1) resulted in a product with solubility characteristics that were too limited to allow for pharmaceutical formulation, and PMO#2 (SEQ ID NO:2) resulted in a product that could not be manufactured with sufficient yield and purity.

[0331] In contrast to the syntheses of PMO#1 and PMO#2, the synthesis of PMO#3 (which differs from PMO#2 by only two bases at the 5' end) allowed the subsequent synthesis of PPMO#3 from PMO#3 without solubility or purification issues (Example 2-below). [Table 26]

[0332] A similar process was used to synthesize PMO#4, PMO#5, PMO#6, PMO#7, PMO#8, and PMO#9.

[0333] Example 2: PPMO#3 PPMO#3 was synthesized from PMO#3 (SEQ ID NO:3) using the protocol described above. [ka] In the formula, each Nu from 1 to 23 and from 5' to 3' is SEQ ID NO:3. [Table 27] In the formula, A is [ka] and C is [ka] and G is [ka] and T is [ka] It is.

[0334] Example 3: Exon 50 skipping in vitro Two compounds targeting human dystrophin (DMD) exon 50, PMO#3 and PPMO#3 (both containing the same sequence), were evaluated for DMD exon 50 skipping in healthy human myotubes, as described in the table below. [Table 28]

[0335] Specifically, healthy human myoblasts (passage 5–6, SKB-F-SL, purchased from Zen-Bio, Inc.) were cultured to achieve 80–90% confluency in SKM-M medium before initiating differentiation by incubating in low serum medium (SKM-D, Zen-Bio, Inc.). After 5 days of differentiation, mature myotubes were incubated with the above compounds at various concentrations (i.e., 40 μm, 20 μm, 10 μm, 5 μm, 2.5 μm, and 1.25 μm). After 96 h of incubation, myotubes were washed with PBS and lysed by RLT buffer in an RNeasy Micro kit (catalog no. 74004, Qiagen) supplemented with 1% β-mercaptoethanol. Total RNA was isolated following the manufacturer's recommendations, except that 20 μL of RNase-free water was used to elute the RNA.

[0336] One-step end-point RT-PCR was performed to determine DMD exon 50 skipping by both compounds. cDNA synthesis and PCR amplification were performed using 100 ng of total RNA, gene-specific primers, and the SuperScript III One-Step RT-PCR System with Platinum Taq DNA polymerase (Cat. No. 12574-026, Invitrogen). Gene-specific primers were designed to target human DMD exons 49 and 52 (forward primer: CCA GCC ACT CAG CCA GTG AAG (SEQ ID NO: 12), reverse primer: CGA TCC GTA ATG ATT GTT CTA GCC (SEQ ID NO: 13)). cDNA synthesis and PCR amplification were performed by a BioRad CFX96 real-time thermal cycler using the program shown in Table 6. The expression of skipped and non-skipped PCR products was assessed by loading 22 μL of PCR product onto a DNA extended range LabChip of the LabChip GX system prepared with DNA 1K reagents (catalog no. 760517 and CLS760673, Perkin Elmer) according to the manufacturer's instructions. The percentage of DMD exon 50 skipping is calculated as the percentage of the molar concentration of the exon 50 skipped band compared to the total molar concentration (nmol / L) of the skipped and non-skipped bands.

[0337] A two-tailed, unpaired Student's t-test (with equal variance) was used to assess whether the means of the two groups were statistically different from each other at each dose. A P value of less than 0.05 was considered statistically significant. [Table 6]

[0338] The results showed that PPMO#3 significantly increased DMD exon 50 skipping compared to PMO#3 and are shown in the table below (as the ratio of skipping of PPMO#3 to PMO#). [Table 7]

[0339] The data in Table 7 above show that higher exon 50 skipping occurs in myotubes when cells are treated with PPMO#3 compared to PMO#3 at all concentrations. This improvement can be further demonstrated in comparative in vivo studies, such as the non-human primate (NHP) study in Example 4, where NHPs are treated with PPMO#3 or PMO#3 and exon 50 skipping is measured in various relevant muscle tissues (see Example 4 for details).

[0340] Example 4: Exon 50 skipping in NHPs To further demonstrate the efficacy of exon skipping of PPMO antisense oligomers, non-human primates are utilized: specifically, cynomolgus monkeys with intact muscle tissue are intravenously injected with PPMO#3 (Example 2), PMO#3 (Example 1), or saline.

[0341] Animals are observed throughout the study, including clinical observations (e.g., skin and fur evaluation, respiratory effects) and weight measurements. Blood and urine specimens are collected at least prior to the start of testing and 24 hours after the first and final doses (if applicable).

[0342] At each scheduled necropsy or upon euthanasia at the time of death, sections of diaphragm, duodenal smooth muscle, esophagus, and aorta, quadriceps, deltoid, biceps, and heart are harvested and snap frozen. Exon 50 skipping rates are determined using RT-PCR as described above.

[0343] Example 5: Exon 50 skipping by PMO in vitro A series of PMOs (SEQ ID NOs: 1-7, PMO#1-#7) were prepared and tested for the efficiency of exon 50 skipping. Briefly, human primary myoblasts were cultured using standard techniques. Lyophilized PMOs were resuspended in nuclease-free water, and PMO solutions were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific) to verify molarity. A range of doses of PMOs were delivered to myoblasts (e.g., 0.625, 1.25, 2.5, 5, 10, and 20 μM) using nucleoporation according to the manufacturer's instructions and the P3 kit (Lonza) and incubated overnight in a 37°C, 5% CO2 incubator prior to RNA extraction. RNA was extracted from PMO-treated cells using an RNAspin 96-well RNA isolation kit from GE Healthcare and subjected to RT-PCR using standard techniques with primers that amplified human DMD exons 49-52. Skipping was measured using a Caliper LabChip bioanalyzer, exon skipping % (i.e., band intensity of exon-skipped products compared to full-length PCR products) was calculated by the formula [exon 50 skipped products / (sum of exon 50 skipped products and exon 50 non-skipped products)×100], and EC50 values ​​were calculated based on the skipping rate induced at each concentration. As shown in Table 8, PMO oligomers of the present disclosure designed to target the splice acceptor or splice donor region of exon 50 provided exon 50 skipping with PMO#1, PMO#2, and PMO#3 providing the highest levels of exon 50 skipping activity (EC 50 <1.0 μM). [Table 8]

[0344] (1) ****=EC 50 <1.0 μM, **=EC 50 1.0-3.0 μM, *=EC 50 3.0μM or more. It should be understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be taken as limitations on the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0345] Various changes and modifications to the embodiments of the present disclosure will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the present invention, can be made without departing from the spirit or scope thereof. [Table 29]

Claims

1. An antisense oligomer capable of binding to a selected target and inducing exon skipping in a human dystrophin gene, or a pharma- ceutically acceptable salt thereof, wherein the antisense oligomer is complementary to an exon 50 target region of a dystrophin pre-mRNA designated as an annealing site, and the base sequence and annealing site of the antisense oligomer are as follows: 【Table 30】 wherein T in SEQ ID NO:3 is thymine or uracil; The nucleobase of the antisense oligomer is linked to a morpholino ring structure. An antisense oligomer, or a pharma- ceutically acceptable salt thereof.

2. The antisense oligomer comprises a moiety attached to the 5' end of the antisense oligomer, the moiety being selected from: 【Chemistry 146】 Here, R １ is CH ３ and 2. The antisense oligomer of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the antisense oligomer induces exon skipping in the human dystrophin gene.

3. The antisense oligomer described in claim 2, wherein the antisense oligomer is linked to a cell membrane-permeable peptide.

4. The antisense oligomer according to any one of claims 1 to 3, wherein a cell membrane permeable peptide is bound to the 3' end of the antisense oligomer.

5. The antisense oligomer according to claim 4 , wherein the cell membrane-permeable peptide is an arginine-rich peptide.

6. The arginine-rich peptide is -(RXR) ４ -R ａ (SEQ ID NO: 15), R-(FFR) ３ -R ａ (SEQ ID NO: 16), -B-X-(RXR) ４ -R ａ (SEQ ID NO: 17), -B-X-R-(FFR) ３ -R ａ (SEQ ID NO: 18), -GLY-R-(FFR) ３ -R ａ (SEQ ID NO: 19), -GLY-R ５ -R ａ (SEQ ID NO: 20), -R ５ -R ａ (SEQ ID NO: 21), -GLY-R ６ -R ａ (SEQ ID NO: 11), and -R ６ -R ａ (SEQ ID NO: 10), wherein R ａ The antisense oligomer of claim 5, wherein is selected from H, acyl, benzoyl, and stearoyl, R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine, and GLY (or G) is glycine.

7. An antisense oligomer according to formula (I): 【Chemistry 147】 or a pharma- ceutically acceptable salt thereof, each Nu is a nucleobase which together form a targeting sequence, E. 【Chemistry 148】 where R １ is CH ３ and R １００ is hydrogen or a cell membrane-permeable peptide; The targeting sequence is 【Table 31】 Here, A is 【Chemistry 149】 and C is 【Chemistry 150】 and G is 【Chemistry 151】 and T is 【Chemistry 152】 or a pharma- ceutically acceptable salt thereof.

8. The antisense oligomer of claim 7, wherein the distal --OH or --NH 2 of the E portion is linked to a cell membrane-permeable peptide.

9. The antisense oligomer according to claim 7 or 8, wherein the antisense oligomer contains one cell membrane-permeable peptide.

10. E is a moiety selected from the following, where R １ is CH ３ The antisense oligomer according to any one of claims 7 to 9, 【Chemistry 153】

11. E is a moiety selected from the following: 【Chemistry 154】 Here, R １ is CH ３ and R １００ The antisense oligomer according to any one of claims 7 to 10, wherein said oligonucleotide is a cell membrane-permeable peptide.

12. E is 【Chemistry 155】 and R １００ The antisense oligomer according to claim 11, wherein said peptide is a cell membrane-permeable peptide.

13. The antisense oligomer according to any one of claims 7 to 12, wherein the cell membrane-permeable peptide is an arginine-rich peptide.

14. The arginine-rich peptide is -(RXR) ４ -R ａ (SEQ ID NO: 15), R-(FFR) ３ -R ａ (SEQ ID NO: 16), -B-X-(RXR) ４ -R ａ (SEQ ID NO: 17), -B-X-R-(FFR) ３ -R ａ (SEQ ID NO: 18), -GLY-R-(FFR) ３ -R ａ (SEQ ID NO: 19), -GLY-R ５ -R ａ (SEQ ID NO: 20), -R ５ -R ａ (SEQ ID NO: 21), -GLY-R ６ -R ａ (SEQ ID NO: 11), and -R ６ -R ａ (SEQ ID NO: 10), wherein R ａ The antisense oligomer of claim 13, wherein is selected from H, acyl, benzoyl, and stearoyl, R is arginine, X is 6-aminohexanoic acid, B is β-alanine, F is phenylalanine, and GLY (or G) is glycine.

15. The antisense oligomer of any one of claims 7 to 14, wherein the antisense oligomer is in free base form.

16. The antisense oligomer according to any one of claims 7 to 14, wherein the antisense oligomer is a pharma- ceutically acceptable salt thereof.

17. An antisense oligomer according to formula (III): 【Chemistry 156】 or a pharma- ceutically acceptable salt thereof, wherein each Nu is a nucleobase and together form a targeting sequence, said targeting sequence being: 【Table 32】 Here, A is 【Chemistry 157】 and C is 【Chemistry 158】 and G is 【Chemistry 159】 and T is 【Chemistry 160】 or a pharma- ceutically acceptable salt thereof.

18. The antisense oligomer of claim 17, wherein the distal --OH of formula (III) is linked to a cell membrane-permeable peptide.

19. 19. The antisense oligomer of claim 17 or 18, wherein the antisense oligomer is in free base form.

20. 19. The antisense oligomer of claim 17 or 18, wherein the antisense oligomer is a pharma- ceutically acceptable salt thereof.

21. An antisense oligomer according to formula (IV): 【Chemistry 161】 Each Nu is a nucleobase and together form a targeting sequence, said targeting sequence being: 【Table 33】 Here, A is 【Chemistry 162】 and C is 【Chemistry 163】 and G is 【Chemistry 164】 and T is 【Chemistry 165】 , an antisense oligomer.

22. The antisense oligomer of claim 21, wherein the distal --OH of formula (IV) is linked to a cell membrane-permeable peptide.

23. 23. The antisense oligomer of claim 21 or 22, wherein the antisense oligomer conforms to the structure of formula (IVa). 【Chemistry 166】

24. A pharmaceutical composition comprising the antisense oligomer according to any one of claims 1 to 23, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

25. A composition comprising an antisense oligomer according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24 for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof.

26. 26. The composition of claim 25, wherein the subject has a mutation in the dystrophin gene that is amenable to exon 50 skipping.

27. A composition comprising the antisense oligomer of any one of claims 1 to 23 or the pharmaceutical composition of claim 24, for restoring an mRNA reading frame to induce dystrophin production in a subject.

28. 28. The composition of claim 27, wherein the subject has a mutation in the dystrophin gene that is amenable to exon 50 skipping.

Images