Compositions and methods for skipping exon 45 in Duchenne muscular dystrophy

Abstract

Described herein in various embodiments is a composition comprising: (a) a cell-penetrating peptide (CPP) sequence; and (b) an antisense compound, wherein the antisense compound targets exon 45 of the DMD gene.

Description

[Technical field]

[0001] This application claims the benefit of priority based on the filing dates of U.S. Provisional Application No. 63 / 244,915, filed September 16, 2021, U.S. Provisional Application No. 63 / 337,574, filed May 2, 2022, U.S. Provisional Application No. 63 / 354,454, filed June 22, 2022, U.S. Provisional Application No. 63 / 239,671, filed September 1, 2021, U.S. Provisional Application No. 63 / 290,960, filed December 17, 2021, U.S. Provisional Application No. 63 / 298,565, filed January 11, 2022, and U.S. Provisional Application No. 63 / 268,577, filed February 25, 2022, the contents of which are specifically incorporated by reference in their entireties herein. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to alterations in the protein dystrophin. Genetic modifications in the gene DMD, which codes for dystrophin, cause DMD. These genetic modifications shift the DMD reading frame, leading to a non-functional truncated DMD protein. One method for treating DMD patients involves delivering to the patient a compound that restores the DMD reading frame. Antisense compounds can restore the DMD reading frame by skipping internal exons associated with the shift in the DMD reading frame that leads to a non-functional truncated DMD protein. Exon skipping generates a dystrophin protein that retains functionality that is lost in the disease state.

[0003] DMD is caused by mutations in one or more of several exons. For example, 8.1% of DMD patients have a mutation in exon 45 of DMD. Aartsma-Rus.Human Mutation,Vo.30,No.3,293-299(2009).Antisense oligonucleotide, casimersen, has been approved for exon 45 skipping, but this drug has low efficacy, possibly due to poor intracellular delivery of the therapeutic agent.

[0004] The limited ability to reach intracellular compartments is a major problem for antisense oligonucleotide therapeutics such as casimersen.Attempts have been made to improve intracellular delivery of antisense compounds, for example, by using carrier systems such as polymers, cationic liposomes, or by chemical modification of constructs, for example, by covalently binding with cholesterol molecules.However, intracellular delivery efficiency remains low, and there is a continuing need for improved delivery systems to improve the efficacy of these antisense compounds.

[0005] There is an unmet need for effective compositions for delivering antisense compounds to intracellular compartments to treat DMD. Summary of the Invention

[0006] The composition for delivering nucleic acid is described herein.In an embodiment, the nucleic acid is antisense compound (AC).In an embodiment, the antisense compound targets exon 45 in the subject with Duchenne muscular dystrophy (DMD).

[0007] In embodiments, the compound comprises: (a) a cyclic peptide (also referred to herein as a cell penetrating peptide or "CPP"), (b) an antisense compound (AC) that is complementary to a target sequence of the DMD gene in the pre-mRNA sequence; wherein the target sequence comprises at least a portion of the 5' flanking intron of exon 45, at least a portion of exon 45, at least a portion of the 3' flanking intron of exon 45, or a combination thereof.

[0008] In embodiments, the AC comprises at least one modified nucleotide or nucleic acid selected from phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino (PMO) nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNAs), nucleotides containing 2'-O-methyl (2'-OMe) modified backbones, 2'O-methoxy-ethyl (2'-MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides, and 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA). In embodiments, the AC comprises at least one PMO (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 PMOs, including all ranges contained therein). In embodiments, each nucleotide in the AC is a PMO.

[0009] In an embodiment, the AC comprises the sequence: 5'-ATGCCATCCTGGAGTTCCTGTA-3'. In an embodiment, the AC comprises the sequence: 5'-CCCAATGCCATCCTGGAGTTCCT-3'.

[0010] In an embodiment, the cyclic peptide is FGFGRGRQ. In an embodiment, the cyclic peptide is GfFGrGrQ. In an embodiment, the cyclic peptide is FfΦGRGRQ.

[0011] In an embodiment, the EEV is Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH.

[0012] In embodiments, provided herein are pharmaceutical compositions comprising the compounds described herein.

[0013] In embodiments, provided herein are cells comprising a compound described herein.

[0014] In embodiments, provided herein is a method of treating DMD, comprising administering to a patient in need thereof a compound described herein. [Brief description of the drawings]

[0015] [Figure 1] A and B show conjugation chemistries for linking antisense compounds (AC) to cell penetrating peptides (CPPs). [Diagram 2] A and B are [ka] 1 shows conjugation chemistry for linking a cell penetrating peptide (CPP) shown as (A) to an antisense compound (AC), where the CPP contains a PEG4 linker and the AC is shown without (A) and with (B) a linker containing a polyethylene glycol (PEG2 or miniPEG) moiety. "R" in the diagram represents a palmitoyl group. [Diagram 3] An example of an endosomal escape vehicle (EEV) design using a representative CPP is shown. It is understood that the CPP can include any of the CPPs disclosed herein. [Figure 4] 1 shows the exon 45 skipping efficiency at 5 μM and 10 μM of ACs selected from Table 8. [Diagram 5] A-D show exon skipping assessment in triceps (A), tibialis anterior (B), diaphragm (C) and heart (D) 1 week after (intravenous) treatment with EEV-PMO-MDX23-1 assessed by 2-step RT-PCR. [Figure 6]A-D show duration of effect (1 week, 2 weeks, 4 weeks, and 8 weeks) in triceps (A), tibialis anterior (B), diaphragm (C), and heart (D) following intravenous administration of 80 mpk EEV-PMO-MDX23-1. [Figure 7] Results are shown following intravenous administration of 40mpk EEV-PMO-MDX23-1 (4 weekly doses). [Figure 8] Wire hang data for D2.mdx. Animals treated with 80mpk EEV-PMO-MDX23-1 once every two weeks had wire hang times that were statistically indistinguishable from wild type animals after 12 weeks of treatment. DBA wild type Vehicle (saline); D2.mdx Vehicle (saline); D2.mdx EEV-PMO-MDX23-1; D2.mdx EEV-PMO-MDX23-2; D2.mdx PMO-MDX23 (5'-GGCCAAACCTCGGCTTACCTGAAAT-3'). [Figure 9] AB show creatine kinase activity in D2 MDX mice before treatment (A) and 4 weeks after treatment (B). [Figure 10] AB show creatine kinase activity in serum of D2 MDX mice 8 weeks (A) and 12 weeks (B) after administration. [Figure 11] AB show grip strength of D2MDX treated with EEV-PMO-MDX23-3 before administration (A) and 12 weeks after administration (B). [Figure 12] A-D show exon skipping efficiency in the diaphragm (A), heart (B), biceps (C) and tibialis anterior (D) of hDMD mice injected with 40, 60 or 80 mpk of positive control. [Figure 13] AC show exon skipping in tibialis anterior muscle (A), diaphragm (B), and heart (C) detected by one-step RT-PCR. [Figure 14]AC show exon skipping in tibialis anterior muscle (A), diaphragm (B) and heart (C) of hDMD mice one week after injection with 60 mpk of positive control (EEV-PMO-DMD45-1) or candidate PMO conjugated to EEV-2. [Figure 15A] 1 shows an assessment of DMD45 skipping in DMDΔ46-48 iPSC-derived myoblasts treated with 30 μM casimersen conjugated to EEV-2 (EEV-PMO-DMD45-1) or one of ten EEV-PMO compounds. [Figure 15B] 1 shows an assessment of DMD45 skipping in DMDΔ46-48 iPSC-derived myoblasts treated with 30 μM casimersen conjugated to EEV-2 (EEV-PMO-DMD45-1) or one of ten EEV-PMO compounds. [Figure 15C] 1 shows the assessment of DMD45 skipping in human cardiac cells treated with three EEV-PMO compounds. [Figure 15D] 1 shows the assessment of DMD45 skipping in human cardiac cells treated with three EEV-PMO compounds. [Figure 15E] 1 shows the assessment of DMD45 skipping in human cardiac cells treated with three EEV-PMO compounds. [Figure 16] CTGlo cell viability assay results for EEV PMOs EEV-PMO-DMD45-2, EEV-PMO-DMD45-3, EEV-PMO-DMD45-4, and EEV-PMO-DMD45-5 normalized to melittin positive control. [Figure 17] CTGlo cell viability assay for EEV PMOs EEV-PMO-DMD45-6, EEV-PMO-DMD45-7, EEV-PMO-DMD45-8, EEV-PMO-DMD45-9, normalized to melittin positive control. [Figure 18]CTGlo cell viability assay results for EEV PMOs EEV-PMO-DMD45-10, EEV-PMO-DMD45-11, and positive control, normalized to melittin positive control. [Figure 19] The structure of EEV-PMO-DMD-45-5 is shown. [Figure 20] The structure of EEV-PMO-DMD-45-7 is shown. [Figure 21A] 1 shows a synthetic scheme for an exemplary EEV-PMO. [Figure 21B] 1 shows a synthetic scheme for an exemplary EEV-PMO. [Figure 21C] 1 shows a synthetic scheme for an exemplary EEV-PMO. [Figure 21D] 1 shows a synthetic scheme for an exemplary EEV-PMO. [Figure 22A] Contrasting localization in THP cells for PMO, EEV-PMO and EEV-NLS-PMO as determined by LC-MS / MS: Total cellular uptake. [Figure 22B] Contrasting localization in THP cells for PMO, EEV-PMO and EEV-NLS-PMO as determined by LC-MS / MS: Subcellular localization. [Figure 22C] Contrasting localization in THP cells for PMO, EEV-PMO and EEV-NLS-PMO as determined by LC-MS / MS: Nuclear uptake. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] compound In various embodiments, disclosed herein are compounds for treating Duchenne muscular dystrophy (DMD). In embodiments, DMD is caused by a mutation in exon 45. In embodiments, the compounds are designed to deliver antisense compounds (ACs) that are complementary to a target sequence of the DMD gene in the pre-mRNA sequence, the target sequence comprising at least a portion of the 5' adjacent intron of exon 45, at least a portion of exon 45, at least a portion of the 3' adjacent intron of exon 45, or a combination thereof. In embodiments, the compounds are delivered intracellularly to a subject in need thereof. In embodiments, the compounds deliver ACs that are complementary to a target sequence comprising an intron-exon junction of exon 45 of the DMD gene. In embodiments, the compounds deliver ACs that are complementary to a target sequence comprising an intronic nucleotide sequence upstream (or 5') of exon 45 of the DMD gene.

[0017] In embodiments, the compound alters the splicing pattern of a target pre-mRNA bound by the AC, resulting in the formation of a re-spliced ​​target protein, in embodiments, the re-spliced ​​target protein has increased function compared to a target protein produced by splicing of the target pre-mRNA in the absence of the AC. In embodiments, the re-spliced ​​target protein increases target protein function by about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, or more, including all values ​​and ranges in between, as compared to the function of the target protein produced by splicing. In embodiments, the re-spliced ​​target protein restores function to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% (including all values ​​and ranges therebetween) of the function of the wild-type target protein.

[0018] In embodiments, the compounds disclosed herein comprise an AC moiety and a cyclic peptide moiety (also referred to as a cell penetrating peptide (CPP) moiety) that facilitates intracellular delivery of the AC. In embodiments, the CPP moiety is cyclic (referred to herein as a cyclic peptide). In embodiments, the compounds can cross cell membranes in vivo and bind to target pre-mRNA. In embodiments, the compounds comprise a) at least one cyclic peptide and b) at least one AC, where the cyclic peptide is directly or indirectly coupled to the AC. In embodiments, the compounds comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more AC moieties. In embodiments, the compounds comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cyclic peptides. In embodiments, the compounds comprise one AC moiety. In embodiments, the compounds comprise two AC moieties. As used herein, "coupled" can refer to a covalent or non-covalent association between a cyclic peptide and an AC, including fusing the cyclic peptide to the AC and chemically conjugating the cyclic peptide to the AC. A non-limiting example of a means for non-covalently binding a cyclic peptide to an AC is by streptavidin / biotin interaction, for example, by conjugating biotin to the cyclic peptide and fusing streptavidin to the AC. In the resulting compound, the CPP is coupled to the AC by a non-covalent association between biotin and streptavidin.

[0019] In embodiments, cyclic peptide is directly or indirectly conjugated to AC, thereby forming cyclic peptide-AC conjugate.Conjugation of AC to CPP can occur at any suitable site of these moieties.For example, 5' or 3' end of AC can be conjugated to C-terminus, N-terminus or side chain of amino acid in CPP.

[0020] In an embodiment, the AC is covalently bonded to the cyclic peptide. Covalent bond, as used herein, refers to a construct in which the CPP moiety is covalently bonded to the 5' and / or 3' end of the AC moiety. Such a conjugate may alternatively be described as having a cell-penetrating moiety and an oligonucleotide moiety. Methods of covalent bonding are well known in the art. A covalently bonded AC-cyclic peptide conjugate, according to certain embodiments of the present disclosure, comprises an AC moiety and a cyclic peptide moiety associated with each other by a linker.

[0021] In embodiments, the AC may be chemically conjugated to the cyclic peptide by a moiety on the 5' or 3' end of the AC. In yet other embodiments, the AC may be conjugated to the cyclic peptide by a side chain of an amino acid on the cyclic peptide. Any amino acid side chain on the cyclic peptide that is capable of forming or can be modified to form a covalent bond can be used to link the AC to the cyclic peptide. The amino acid on the cyclic peptide may be a natural or unnatural amino acid. In embodiments, the amino acid on the cyclic peptide used to conjugate the AC is aspartic acid, glutamic acid, glutamine, asparagine, lysine, ornithine, 2,3-diaminopropionic acid, or an analog thereof, the side chain of which is replaced with a bond to the AC or a linker. In embodiments, the amino acid is lysine or an analog thereof. In embodiments, the amino acid is glutamic acid or an analog thereof. In embodiments, the amino acid is aspartic acid or an analog thereof.

[0022] Endosomal escape vehicles (EEVs) Provided herein is an endosomal escape vehicle (EEV) that can be used to transport AC across a cell membrane, for example, to deliver AC to the cytosol or nucleus of a cell. The EEV can include a cell-penetrating peptide (CPP), for example, a cyclic cell-penetrating peptide (cCPP) conjugated to an exocyclic peptide (EP). The EP may be interchangeably referred to as a regulatory peptide (MP). The EP can include a sequence of a nuclear localization signal (NLS). The EP can be bound to the AC. The EP can be bound to the cCPP. The EP can be bound to the AC and the cCPP. The coupling between the EP, AC, cCPP, or combinations thereof can be non-covalent or covalent. The EP can be bound to the N-terminus of the cCPP via a peptide bond. The EP can be bound to the C-terminus of the cCPP via a peptide bond. The EP can be bound to the cCPP via a side chain of an amino acid in the cCPP. The EP can be bound to the cCPP via a side chain of a lysine that can be conjugated to a side chain of a glutamine in the cCPP. The EP can be conjugated to the 5' or 3' end of the AC. The EP can be attached to a linker. The exocyclic peptide can be conjugated to the amino group of the linker. The EP can be coupled to the linker by the C-terminus of the EP and to the cCPP via the side chain on the cCPP and / or on the EP. For example, the EP can contain a terminal lysine, which can then be coupled to a cCPP containing glutamine via an amide bond. If the EP contains a terminal lysine and can be used to bind the cCPP using the side chain of the lysine, the C-terminus or N-terminus can be attached to the linker on the AC.

[0023] Exocyclic peptides The exocyclic peptide (EP) may contain from 2 to 10 amino acid residues, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues (including all ranges and values ​​therebetween). The EP may contain from 6 to 9 amino acid residues. The EP may contain from 4 to 8 amino acid residues.

[0024] Each amino acid in the exocyclic peptide may be a natural or unnatural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homologue of a natural amino acid in that it has a structure similar to the natural amino acid so as to mimic the structure and reactivity of the natural amino acid. An unnatural amino acid may be a modified amino acid and / or an amino acid analog that is not one of the 20 common natural amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. An unnatural amino acid may be a D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids, along with their abbreviations used herein, are listed in Table 1. For example, an amino acid can be A, G, P, K, R, V, F, H, NaI, or citrulline.

[0025] The EP may contain at least one positively charged amino acid residue, for example, at least one lysine residue, and / or at least one amino acid residue containing a side chain containing a guanidine group or a protonated form thereof. The EP may contain one or two amino acid residues containing a side chain containing a guanidine group or a protonated form thereof. The amino acid residue containing a side chain containing a guanidine group may be an arginine residue. The protonated form may refer to a salt thereof throughout this disclosure.

[0026] The EP may include at least two, at least three, or at least four or more lysine residues. The EP may include two, three, or four lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group, such as, for example, a trifluoroacetyl (-COCF3) group, an allyloxycarbonyl (Alloc) group, a 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) group, or a (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) ​​group. The amino group on the side chain of each lysine residue may be substituted with a trifluoroacetyl (-COCF3) group. The protecting group may be included to allow amide conjugation. The protecting group may be removed after the EP is conjugated to the cCPP.

[0027] The EP may comprise at least two amino acid residues having a hydrophobic side chain. The amino acid residues having a hydrophobic side chain may be selected from valine, proline, alanine, leucine, isoleucine, and methionine. The amino acid residue having a hydrophobic side chain may be valine or proline.

[0028] The EP may contain at least one positively charged amino acid residue, e.g., at least one lysine residue and / or at least one arginine residue. The EP may contain at least two, at least three, or at least four or more lysine and / or arginine residues.

[0029] EP is KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH, KHKK, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, K KKKR, KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, where B is beta alanine. The amino acids in EP may have D or L stereochemistry.

[0030] EP may include KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP may include PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

[0031] EP can be comprised of KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP can be comprised of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP can have D or L stereochemistry.

[0032] The EP may comprise an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP may consist of an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP may comprise an NLS comprising the amino acid sequence PKKKRKV. The EP may consist of an NLS comprising the amino acid sequence PKKKRKV. The EP may comprise an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK. The EP may consist of an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK.

[0033] All exocyclic sequences may also contain an N-terminal acetyl group. Thus, for example, the EP may have the structure: Ac-PKKKRKV.

[0034] Cell-penetrating peptides (CPPs) The cell penetrating peptide (CPP) may comprise 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). The cCPP can penetrate the cell membrane. An exocyclic peptide (EP) can be conjugated to the cCPP, and the resulting construct can be called an endosomal escape vehicle (EEV). The cCPP can guide the AC to penetrate the cell membrane. The cCPP can deliver the AC to the cytosol of the cell. The cCPP can deliver the AC to the cell location where the target (e.g., pre-mRNA) is located. To conjugate the cCPP to the AC, at least one bond or lone pair on the cCPP can be replaced.

[0035] The total number of amino acid residues in a cCPP can range from 6 to 20 amino acid residues, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, including all ranges and subranges therebetween. A cCPP can contain 6 to 13 amino acid residues. A cCPP disclosed herein can contain 6 to 10 amino acids. By way of example, a cCPP containing 6-10 amino acid residues can be represented by Formulas IA to IE: [ka] wherein AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and AA 10 is an amino acid residue.

[0036] The cCPP may contain 6 to 8 amino acids. The cCPP may contain 8 amino acids.

[0037] Each amino acid in a cCPP may be a natural or unnatural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homologue of a natural amino acid in that it has a structure similar to the natural amino acid so as to mimic the structure and reactivity of the natural amino acid. An unnatural amino acid may be a modified amino acid and / or an amino acid analog that is not one of the 20 common natural amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. An unnatural amino acid may be a D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids, along with their abbreviations used herein, are listed in Table 1. [Table 1-1] [Table 1-2]

[0038] A cCPP can contain from 4 to 20 amino acids, where (i) at least one amino acid has a side chain that includes a guanidine group or a protonated form thereof, and (ii) at least one amino acid has no side chain, or [ka] or a protonated form thereof, and (iii) at least two amino acids have side chains that, independently, contain an aromatic or heteroaromatic group.

[0039] At least two of the amino acids may have no side chains, or [ka] or a protonated form thereof. As used herein, when no side chain is present, an amino acid has two hydrogen atoms on the carbon atom(s) connecting the amine and the carboxylic acid (e.g., -CH2-).

[0040] The amino acid having no side chain can be glycine or β-alanine.

[0041] The cCPP may comprise 6 to 20 amino acid residues forming the cCPP, where (i) at least one amino acid may be a glycine, β-alanine, or 4-aminobutyric acid residue, (ii) at least one amino acid may have a side chain that includes an aryl or heteroaryl group, (iii) at least one amino acid may have a guanidine group, [ka] or having a side chain containing the protonated form thereof.

[0042] The cCPP can comprise 6 to 20 amino acid residues forming the cCPP, where (i) at least two amino acids can be independently glycine, β-alanine, or 4-aminobutyric acid residues, (ii) at least one amino acid can have a side chain that includes an aryl or heteroaryl group, (iii) at least one amino acid can have a guanidine group, [ka] or having a side chain containing the protonated form thereof.

[0043] The cCPP may comprise from 6 to 20 amino acid residues forming the cCPP, where (i) at least three amino acids may be, independently, glycine, β-alanine, or 4-aminobutyric acid residues, (ii) at least one amino acid may have a side chain that includes an aromatic or heteroaromatic group, (iii) at least one amino acid may have a guanidine group, [ka] or a side chain containing the protonated form thereof.

[0044] Glycine and related amino acid residues The cCPP may include (i) 1, 2, 3, 4, 5 or 6 glycine β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 2 glycine β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) three or four glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof.

[0045] The cCPP may include (i) 1, 2, 3, 4, 5, or 6 glycine residues. The cCPP may include (i) 2 glycine residues. The cCPP may include (i) 3 glycine residues. The cCPP may include (i) 4 glycine residues. The cCPP may include (i) 5 glycine residues. The cCPP may include (i) 6 glycine residues. The cCPP may include (i) 3, 4, or 5 glycine residues. The cCPP may include (i) 3 or 4 glycine residues. The cCPP may include (i) 2 or 3 glycine residues. The cCPP may include (i) 1 or 2 glycine residues.

[0046] The cCPP may include (i) 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof.

[0047] The cCPP may include at least three glycine residues. The cCPP may include (i) three, four, five, or six glycine residues. The cCPP may include (i) three glycine residues. The cCPP may include (i) four glycine residues. The cCPP may include (i) five glycine residues. The cCPP may include (i) six glycine residues. The cCPP may include (i) three, four, or five glycine residues. The cCPP may include (i) three or four glycine residues.

[0048] In embodiments, none of the glycine, β-alanine, or 4-aminobutyric acid residues in the cCPP are adjacent. Two or three glycine, β-alanine, 4- or aminobutyric acid residues may be adjacent. Two glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent.

[0049] In embodiments, none of the glycine residues in the cCPP are adjacent. Each glycine residue in the cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be adjacent. Two glycine residues may be adjacent.

[0050] Amino acid side chains containing aromatic or heteroaromatic groups The cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 2 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 3 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 4 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 5 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 6 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 2, 3 or 4 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. A cCPP can contain (ii) two or three amino acid residues that independently have side chains that contain an aromatic or heteroaromatic group.

[0051] The cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 2 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 3 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 4 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 5 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 6 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 2, 3 or 4 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 2 or 3 amino acid residues that independently have a side chain that comprises an aromatic group.

[0052] The aromatic group can be 6- to 14-membered aryl. The aryl can be phenyl, naphthyl, or anthracenyl, each of which is optionally substituted. The aryl can be phenyl or naphthyl, each of which is optionally substituted. The heteroaromatic group can be 6- to 14-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S. The heteroaryl can be pyridyl, quinolyl, or isoquinolyl.

[0053] The amino acid residues having a side chain containing an aromatic or heteroaromatic group may each independently be bis(homonapthylalanine), homonaphthylalanine, naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, 3-(3-benzothienyl)-alanine or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic or heteroaromatic group may each independently be [ka] wherein H on the N-terminus and / or H on the C-terminus is replaced by a peptide bond.

[0054] Amino acid residues having a side chain containing an aromatic or heteroaromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, or homophenylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, homophenylalanine, homophenylalanine, bis(homonaphthylalanine), or bis(homonaphthylalanine), each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of phenylalanine or naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine. At least two amino acid residues having a side chain containing an aromatic group may be a residue of phenylalanine. Each amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine.

[0055] In embodiments, none of the amino acids having a side chain containing an aromatic or heteroaromatic group are adjacent. Two amino acids having a side chain containing an aromatic or heteroaromatic group may be adjacent. Two adjacent amino acids may have opposite stereochemistry. Two adjacent amino acids may have the same stereochemistry. Three amino acids having a side chain containing an aromatic or heteroaromatic group may be adjacent. Three adjacent amino acids may have the same stereochemistry. Three adjacent amino acids may have alternate stereochemistry.

[0056] The amino acid residues containing aromatic or heteroaromatic groups may be L-amino acids. The amino acid residues containing aromatic or heteroaromatic groups may be D-amino acids. The amino acid residues containing aromatic or heteroaromatic groups may be a mixture of D- and L-amino acids.

[0057] An optional substituent may be, for example, any atom or group that does not significantly (e.g., more than 50%) reduce the cytoplasmic delivery efficiency of the cCPP compared to an otherwise identical sequence without the substituent. An optional substituent may be a hydrophobic or hydrophilic substituent. An optional substituent may be a hydrophobic substituent. A substituent may increase the solvent accessible surface area (as defined herein) of a hydrophobic amino acid. A substituent may be halogen, alkyl, alkenyl, alkynylene, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio. A substituent may be halogen.

[0058] Without wishing to be bound by theory, it is believed that amino acids with aromatic or heteroaromatic groups with higher hydrophobicity values ​​(i.e., amino acids with side chains containing aromatic or heteroaromatic groups) can improve the cytoplasmic delivery efficiency of cCPPs compared to amino acids with lower hydrophobicity values. Each hydrophobic amino acid can independently have a hydrophobicity value greater than that of glycine. Each hydrophobic amino acid can independently have a hydrophobicity value greater than that of alanine. Each hydrophobic amino acid can independently have a hydrophobicity value equal to or greater than that of phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 2 lists the hydrophobicity values ​​for various amino acids reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. USA 1984; 81(1): 140-144), Engleman et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986; 1986(15): 321-53), Kyte and Doolittle (J. Mol. Biol. 1982; 157(1): 105-132), Hoop and Woods (Proc. Natl. Acad. Sci. USA 1981; 78(6): 3824-3828), and Janin (Nature. 1979; 277(5696): 491-492), each of which is incorporated herein by reference in its entirety. Hydrophobicity can be measured using the hydrophobicity scale reported in Engleman et al. [Table 2]

[0059] The size of the aromatic or heteroaromatic group can be selected to improve the cytoplasmic delivery efficiency of the cCPP. Without wishing to be bound by theory, it is believed that a larger aromatic or heteroaromatic group on the side chain of an amino acid may improve the cytoplasmic delivery efficiency compared to an otherwise identical sequence having a smaller hydrophobic amino acid. The size of the hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, the steric effect of the hydrophobic amino acid, the solvent accessible surface area (SASA) of the side chain, or a combination thereof. The size of the hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, with larger hydrophobic amino acids having side chains with molecular weights of at least about 90 g / mol, or at least about 130 g / mol, or at least about 141 g / mol. The size of the amino acid can be measured in terms of the SASA of the hydrophobic side chain. The hydrophobic amino acid can have a side chain with a SASA equal to or greater than alanine, or equal to or greater than glycine. The larger hydrophobic amino acid can have a side chain with a SASA greater than alanine, or greater than glycine. The hydrophobic amino acid may have an aromatic or heteroaromatic group with a SASA of about piperidine-2-carboxylic acid or greater, about tryptophan or greater, about phenylalanine or greater, or about naphthylalanine or greater. H1 ) is at least about 200 Å 2 , at least about 210 Å 2 , at least about 220 Å 2 , at least about 240 Å 2 , at least about 250 Å 2 , at least about 260 Å 2 , at least about 270 Å 2 , at least about 280 Å 2 , at least about 290 Å 2 , at least about 300 Å 2 , at least about 310 Å 2 , at least about 320 Å 2 , or at least about 330 Å 2 A second hydrophobic amino acid (AA H2 ) is at least about 200 Å 2 , at least about 210 Å2 , at least about 220 Å 2 , at least about 240 Å 2 , at least about 250 Å 2 , at least about 260 Å 2 , at least about 270 Å 2 , at least about 280 Å 2 , at least about 290 Å 2 , at least about 300 Å 2 , at least about 310 Å 2 , at least about 320 Å 2 , or at least about 330 Å 2 The side chain may have a SASA of AA H1 and A.A. H2 The side chains of 2 , at least about 360 Å 2 , at least about 370 Å 2 , at least about 380 Å 2 , at least about 390 Å 2 , at least about 400 Å 2 , at least about 410 Å 2 , at least about 420 Å 2 , at least about 430 Å 2 , at least about 440 Å 2 , at least about 450 Å 2 , at least about 460 Å 2 , at least about 470 Å 2 , at least about 480 Å 2 , at least about 490 Å 2 , about 500Å 2 More than, at least about 510 Å 2 , at least about 520 Å 2 , at least about 530 Å 2 , at least about 540 Å 2 , at least about 550 Å 2 , at least about 560 Å 2 , at least about 570 Å 2 , at least about 580 Å 2 , at least about 590 Å 2 , at least about 600 Å 2 , at least about 610 Å 2 , at least about 620 Å 2, at least about 630 Å 2 , at least about 640 Å 2 , about 650 Å 2 More than, at least about 660 Å 2 , at least about 670 Å 2 , at least about 680 Å 2 , at least about 690 Å 2 , or at least about 700 Å 2 AA H2 AA H1 The hydrophobic amino acid residue may have a side chain with a SASA that is equal to or less than the SASA of the hydrophobic side chain of. By way of example, and not by way of limitation, a cCPP having a Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a Phe-Arg motif. A cCPP having a Phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a Nal-Phe-Arg motif, and a phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a nal-Phe-Arg motif.

[0060] As used herein, "hydrophobic surface area" or "SASA" refers to the solvent accessible surface area of ​​an amino acid side chain in square angstroms (Å 2 ) and for example, SASA can be calculated using the "rolling ball" algorithm developed by Shrake & Rupley (J Mol Biol. 79(2):351-71), which is incorporated by reference in its entirety for all purposes. This algorithm uses a solvent "sphere" of a specific radius to probe the surface of the molecule. A typical value for the sphere is 1.4 Å, which approximates the radius of a water molecule.

[0061] The SASA values ​​of certain side chains are shown in Table 3 below. The SASA values ​​described herein are based on the theoretical values ​​listed in Table 3 below, as reported by Tien et al. (PLOS ONE 8(11):e80635. https: / / doi.org / 10.1371 / journal.pone.0080635), which is incorporated herein by reference in its entirety for all purposes. [Table 3]

[0062] Amino acid residues having a side chain containing a guanidine group, a guanidine substituent, or the protonated form thereof As used herein, guanidine has the structure: [ka] Refers to...

[0063] As used herein, the protonated form of guanidine has the structure: [ka] Refers to...

[0064] A guanidine substituent refers to a functional group on the side chain of an amino acid that is positively charged at or above physiological pH, or a functional group that can reproduce the hydrogen bond donating and accepting activity of a guanidinium group.

[0065] The guanidine substituents facilitate cell penetration and delivery of therapeutic agents while reducing toxicity associated with the guanidine group or its protonated form. The cCPP may include at least one amino acid having a side chain that includes a guanidine or guanidinium substituent. The cCPP may include at least two amino acids having side chains that include a guanidine or guanidinium substituent. The cCPP may include at least three amino acids having side chains that include a guanidine or guanidinium substituent.

[0066] The guanidine or guanidinium group may be an isostere of guanidine or guanidinium. The guanidine or guanidinium substituent may be less basic than guanidine.

[0067] As used herein, a guanidine substituent is: [ka] or its protonated form.

[0068] The present disclosure provides cCPPs comprising 4 to 20 amino acid residues, wherein (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof, and (ii) at least one amino acid residue has no side chain, or [ka] or a protonated form thereof, and (iii) at least two amino acid residues have side chains that, independently, contain an aromatic or heteroaromatic group.

[0069] At least two of the amino acid residues may have no side chains, or [ka] or a protonated form thereof. As used herein, when no side chain is present, the amino acid residue has two hydrogen atoms on the carbon atom connecting the amine and carboxylic acid (e.g., -CH2-).

[0070] cCPP consists of the following parts: [ka] or at least one amino acid having a side chain that includes one of its protonated forms.

[0071] The cCPP can include at least two amino acids, each amino acid independently comprising the moiety: [ka] or one of its protonated forms. At least two of the amino acids are [ka] or a protonated form thereof. At least one amino acid may have a side chain comprising the same moiety selected from [ka] or a protonated form thereof. At least two of the amino acids may have side chains containing [ka] or its protonated form. [ka] or its protonated form. [ka] or its protonated form. [ka] or a side chain containing the protonated form thereof. [ka] or its protonated form can be attached to the terminus of an amino acid side chain. [ka] can be attached to the terminus of an amino acid side chain.

[0072] The cCPP may comprise 2, 3, 4, 5 or 6 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 2 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 3 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 4 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 5 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 6 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) 2, 3, 4, or 5 amino acid residues that independently have a side chain that includes a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) 2, 3, or 4 amino acid residues that independently have a side chain that includes a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) 2 or 3 amino acid residues that independently have a side chain that includes a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) at least one amino acid residue that has a side chain that includes a guanidine group or a protonated form thereof. The cCPP may comprise (iii) 2 amino acid residues that have a side chain that includes a guanidine group or a protonated form thereof. The cCPP may comprise (iii) 3 amino acid residues that have a side chain that includes a guanidine group or a protonated form thereof.

[0073] The amino acid residues may independently have side chains that contain non-adjacent guanidine groups, guanidine substituents, or their protonated forms. Two amino acid residues may independently have side chains that contain guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Three amino acid residues may independently have side chains that contain guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Four amino acid residues may independently have side chains that contain guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Adjacent amino acid residues may have the same stereochemistry. Adjacent amino acids may have alternating stereochemistry.

[0074] The amino acid residues that independently have side chains that include a guanidine group, a guanidine substituent, or a protonated form thereof can be L-amino acids. The amino acid residues that independently have side chains that include a guanidine group, a guanidine substituent, or a protonated form thereof can be D-amino acids. The amino acid residues that independently have side chains that include a guanidine group, a guanidine substituent, or a protonated form thereof can be a mixture of L or D amino acids.

[0075] Each amino acid residue having a side chain containing a guanidine group or a protonated form thereof may independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof. Each amino acid residue having a side chain containing a guanidine group or a protonated form thereof may independently be a residue of arginine or a protonated form thereof.

[0076] Each amino acid having a side chain containing a guanidine substituent or a protonated form thereof can independently be [ka] or a protonated form thereof.

[0077] Without wishing to be bound by theory, it is hypothesized that the guanidine substituents have reduced basicity compared to arginine and, in some cases, are uncharged (e.g., -N(H)C(O)) at physiological pH and can maintain bidentate hydrogen bonding interactions with phospholipids on the plasma membrane that are believed to facilitate effective membrane binding and subsequent internalization. Removal of the positive charge is also believed to reduce the toxicity of cCPPs.

[0078] One of skill in the art will understand that the N-terminus and / or C-terminus of the above non-natural aromatic hydrophobic amino acids form an amide bond upon incorporation into the peptides disclosed herein.

[0079] A cCPP may include a first amino acid having a side chain that includes an aromatic or heteroaromatic group and a second amino acid having a side chain that includes an aromatic or heteroaromatic group, where the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain that includes an aromatic or heteroaromatic group and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain that includes an aromatic or heteroaromatic group. By convention, the term "first amino acid" often refers to the N-terminal amino acid of a peptide sequence, however, as used herein, "first amino acid" is used to distinguish a reference amino acid from another amino acid (e.g., a "second amino acid") in a cCPP, and thus the term "first amino acid" may refer to the amino acid located at the N-terminus of a peptide sequence.

[0080] The cCPP can include an N-terminus of a second glycine that forms a peptide bond with an amino acid having a side chain that includes an aromatic or heteroaromatic group, and a C-terminus of the second glycine that forms a peptide bond with an amino acid having a side chain that includes a guanidine group or a protonated form thereof.

[0081] The cCPP can include a first amino acid having a side chain comprising a guanidine group or a protonated form thereof and a second amino acid having a side chain comprising a guanidine group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidine group or a protonated form thereof and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain comprising a guanidine group or a protonated form thereof.

[0082] The cCPP may comprise asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine residues. The cCPP may comprise asparagine residues. The cCPP may comprise glutamine residues.

[0083] cCPPs may contain residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.

[0084] Without wishing to be bound by theory, it is believed that the chirality of amino acids in a cCPP may affect cytoplasmic uptake efficiency. A cCPP may include at least one D amino acid. A cCPP may include 1 to 15 D amino acids. A cCPP may include 1 to 10 D amino acids. A cCPP may include 1, 2, 3, or 4 D amino acids. A cCPP may include 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids with alternating D and L chirality. A cCPP may include three adjacent amino acids with the same chirality. A cCPP may include two adjacent amino acids with the same chirality. At least two amino acids may have opposite chirality. At least two amino acids with opposite chirality may be adjacent to each other. At least three amino acids may have alternate stereochemistry with respect to each other. At least three amino acids with alternate chirality with respect to each other may be adjacent to each other. At least four amino acids have alternate stereochemistry relative to each other. At least four amino acids with alternate chirality relative to each other can be adjacent to each other. At least two amino acids can have the same chirality. At least two amino acids with the same chirality can be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids with opposite chirality can be adjacent to at least two amino acids with the same chirality. Thus, adjacent amino acids in a cCPP can have any of the following sequences: DL, LD, DLLD, LDDL, LDLLD, DLDDL, DLLDL, or LDDLD. The amino acid residues forming the cCPP can all be L-amino acids. The amino acid residues forming the cCPP can all be D-amino acids.

[0085] At least two amino acids may have different chiralities. At least two amino acids with different chiralities may be adjacent to each other. At least three amino acids may have different chiralities relative to adjacent amino acids. At least four amino acids may have different chiralities relative to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chiralities. One or more amino acid residues forming a cCPP may be achiral. A cCPP may include a motif of 3, 4, or 5 amino acids, in which two amino acids with the same chirality may be separated by an achiral amino acid. A cCPP may include the following sequences: DXD, DXDX, DXDXD, LXL, LXLX, or LXLXL, where X is an achiral amino acid. The achiral amino acid may be glycine.

[0086] [ka] or its protonated form can be adjacent to an amino acid having a side chain that includes an aromatic or heteroaromatic group. [ka] or its protonated form may be adjacent to at least one amino acid having a side chain comprising a guanidine or its protonated form. An amino acid having a side chain comprising a guanidine or its protonated form may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. [ka] or its protonated form may be adjacent to each other. Two amino acids having a side chain containing guanidine or its protonated form are adjacent to each other. A cCPP comprises at least two adjacent amino acids having side chains that may contain an aromatic or heteroaromatic group, [ka] or a protonated form thereof. A cCPP may have at least two contiguous amino acids having side chains that include an aromatic or heteroaromatic group, [ka] or at least two non-adjacent amino acids having side chains that include the protonated form thereof. The adjacent amino acids may have the same chirality. The adjacent amino acids may have opposite chiralities. Other combinations of amino acids may have any arrangement of D and L amino acids, such as any of the sequences described in the previous paragraph.

[0087] [ka] or its protonated form alternate with at least two amino acids having a side chain containing a guanidine group or its protonated form.

[0088] cCPP has the formula (A): [ka] or a protonated form thereof, During the ceremony, R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; At least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4, R5, R6, R7 are independently H or an amino acid side chain; At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidyl)alanine; AA SC is an amino acid side chain, q is 1, 2, 3 or 4.

[0089] In embodiments, at least one of R4, R5, R6, and R7 is independently an uncharged, non-aromatic side chain of an amino acid. In embodiments, at least one of R4, R5, R6, and R7 is independently H or the side chain of citrulline.

[0090] In an embodiment, a compound is provided, comprising a cyclic peptide having 6-12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, wherein at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and wherein at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids. In an embodiment, at least two amino acids of the cyclic peptide are arginine. In an embodiment, at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthalanine (3-naphth-2-yl-alanine), or a combination thereof. In an embodiment, at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, glycine, or a combination thereof. In an embodiment, the compound is a cyclic peptide having 6-12 amino acids, wherein two amino acids of the cyclic peptide are arginine, wherein at least two amino acids are aromatic hydrophobic amino acids selected from phenylalanine, naphthalanine, and combinations thereof, and wherein at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine, and combinations thereof.

[0091] In embodiments, the cyclic peptide of formula (A) is not a cyclic peptide having the following sequence: [Table 4]

[0092] cCPP has the formula (I): [ka] or a protonated form thereof, During the ceremony, R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group; At least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain, q is 1, 2, 3 or 4; Each m is independently an integer 0, 1, 2, or 3.

[0093] R1, R2, and R3 can each independently be H, -alkylene-aryl, or alkylene-heteroaryl. R1, R2, and R3 can each independently be H, -C 1~3 Alkylene-aryl, or C 1~3 R1, R2, and R3 may each independently be H or alkylene-aryl. R1, R2, and R3 may each independently be H or C 1~3 It can be an alkylene-aryl. 1~3The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R1, R2, and R3 are each independently selected from H, -C ... 1~3 Alkylene-Ph or C 1~3 alkylene-naphthyl. R1, R2, and R3 can each independently be H, -CH2Ph, or CH2naphthyl. R1, R2, and R3 can each independently be H or CH2Ph.

[0094] R1, R2, and R3 can each independently be the side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.

[0095] R1 can be the side chain of tyrosine. R1 can be the side chain of phenylalanine. R1 can be the side chain of 1-naphthylalanine. R1 can be the side chain of 2-naphthylalanine. R1 can be the side chain of tryptophan. R1 can be the side chain of 3-benzothienylalanine. R1 can be the side chain of 4-phenylphenylalanine. R1 can be the side chain of 3,4-difluorophenylalanine. R1 can be the side chain of 4-trifluoromethylphenylalanine. R1 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R1 can be the side chain of homophenylalanine. R1 can be the side chain of β-homophenylalanine. R1 can be the side chain of 4-tert-butyl-phenylalanine. R1 can be the side chain of 4-pyridinylalanine. R1 can be the side chain of 3-pyridinylalanine. R1 can be the side chain of 4-methylphenylalanine. R1 can be the side chain of 4-fluorophenylalanine. R1 can be the side chain of 4-chlorophenylalanine. R1 can be the side chain of 3-(9-anthryl)-alanine.

[0096] R2 can be the side chain of tyrosine. R2 can be the side chain of phenylalanine. R2 can be the side chain of 1-naphthylalanine. R1 can be the side chain of 2-naphthylalanine. R2 can be the side chain of tryptophan. R2 can be the side chain of 3-benzothienylalanine. R2 can be the side chain of 4-phenylphenylalanine. R2 can be the side chain of 3,4-difluorophenylalanine. R2 can be the side chain of 4-trifluoromethylphenylalanine. R2 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R2 can be the side chain of homophenylalanine. R2 can be the side chain of β-homophenylalanine. R2 can be the side chain of 4-tert-butyl-phenylalanine. R2 can be the side chain of 4-pyridinylalanine. R2 can be the side chain of 3-pyridinylalanine. R2 can be the side chain of 4-methylphenylalanine. R2 can be the side chain of 4-fluorophenylalanine. R2 can be the side chain of 4-chlorophenylalanine. R2 can be the side chain of 3-(9-anthryl)-alanine.

[0097] R3 may be the side chain of tyrosine. R3 may be the side chain of phenylalanine. R3 may be the side chain of 1-naphthylalanine. R3 may be the side chain of 2-naphthylalanine. R3 may be the side chain of tryptophan. R3 may be the side chain of 3-benzothienylalanine. R3 may be the side chain of 4-phenylphenylalanine. R3 may be the side chain of 3,4-difluorophenylalanine. R3 may be the side chain of 4-trifluoromethylphenylalanine. R3 may be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 may be the side chain of homophenylalanine. R3 may be the side chain of β-homophenylalanine. R3 may be the side chain of 4-tert-butyl-phenylalanine. R3 may be the side chain of 4-pyridinylalanine. R3 may be the side chain of 3-pyridinylalanine. R3 can be the side chain of 4-methylphenylalanine. R3 can be the side chain of 4-fluorophenylalanine. R3 can be the side chain of 4-chlorophenylalanine. R3 can be the side chain of 3-(9-anthryl)-alanine.

[0098] R4 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R4 can be H or alkylene-aryl. R4 can be H or C 1~3 It can be an alkylene-aryl. 1~3 The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R4 can be H, -C 1~3 Alkylene-Ph or C 1~3R4 can be H or the side chain of an amino acid of Table 1 or Table 3. R4 can be H or an amino acid residue having a side chain containing an aromatic group. R4 can be H, -CH2Ph, or CH2naphthyl. R4 can be H or CH2Ph.

[0099] R5 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R5 can be H or alkylene-aryl. R5 can be H or C 1~3 It can be an alkylene-aryl. 1~3 The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R5 can be H, -C 1~3 Alkylene-Ph or C 1~3 R5 can be H or the side chain of an amino acid of Table 1 or Table 3. R4 can be H or an amino acid residue having a side chain containing an aromatic group. R5 can be H, -CH2Ph, or CH2 naphthyl. R4 can be H or CH2Ph.

[0100] R6 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R6 can be H or alkylene-aryl. R6 can be H or C 1~3 It can be an alkylene-aryl. 1~3The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl with one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R6 can be H, -C 1~3 Alkylene-Ph or C 1~3 R6 can be alkylene-naphthyl. R6 can be H or the side chain of an amino acid of Table 1 or Table 3. R6 can be H or an amino acid residue having a side chain containing an aromatic group. R6 can be H, -CH2Ph, or CH2 naphthyl. R6 can be H or CH2Ph.

[0101] R7 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R7 can be H or alkylene-aryl. R7 can be H or C 1~3 It can be an alkylene-aryl. 1~3 The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R7 can be H, -C 1~3 Alkylene-Ph or C 1~3 R7 can be alkylene-naphthyl. R7 can be H or the side chain of an amino acid of Table 1 or Table 3. R7 can be H or an amino acid residue having a side chain containing an aromatic group. R7 can be H, -CH2Ph, or CH2 naphthyl. R7 can be H or CH2Ph.

[0102] One, two, or three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. One of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Two of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. At least one of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Up to four of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph.

[0103] One, two or three of R1, R2, R3 and R4 are -CH2Ph. One of R1, R2, R3 and R4 is -CH2Ph. Two of R1, R2, R3 and R4 are -CH2Ph. Three of R1, R2, R3 and R4 are -CH2Ph. At least one of R1, R2, R3 and R4 is -CH2Ph.

[0104] One, two or three of R1, R2, R3, R4, R5, R6, and R7 can be H. One of R1, R2, R3, R4, R5, R6, and R7 can be H. Two of R1, R2, R3, R4, R5, R6, and R7 can be H. Three of R1, R2, R3, R5, R6, and R7 can be H. At least one of R1, R2, R3, R4, R5, R6, and R7 can be H. Up to three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph.

[0105] One, two or three of R1, R2, R3, and R4 are H. One of R1, R2, R3, and R4 is H. Two of R1, R2, R3, and R4 are H. Three of R1, R2, R3, and R4 are H. At least one of R1, R2, R3, and R4 is H.

[0106] At least one of R4, R5, R6, and R7 can be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R4, R5, R6, and R7 can be a side chain of 4-guanidino-2-aminobutanoic acid. At least one of R4, R5, R6, and R7 can be a side chain of arginine. At least one of R4, R5, R6, and R7 can be a side chain of homoarginine. At least one of R4, R5, R6, and R7 can be a side chain of N-methylarginine. At least one of R4, R5, R6, and R7 can be a side chain of N,N-dimethylarginine. At least one of R4, R5, R6, and R7 can be a side chain of 2,3-diaminopropionic acid. At least one of R4, R5, R6, and R7 can be a side chain of 2,4-diaminobutanoic acid, lysine. At least one of R4, R5, R6, and R7 can be the side chain of N-methyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N-ethyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least one of R4, R5, R6, and R7 can be the side chain of citrulline. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethyllysine, β-homoarginine. At least one of R4, R5, R6, and R7 can be the side chain of 3-(1-piperidinyl)alanine.

[0107] At least two of R4, R5, R6, and R7 can be the side chains of 3-guanidino-2-aminopropionic acid. At least two of R4, R5, R6, and R7 can be the side chains of 4-guanidino-2-aminobutanoic acid. At least two of R4, R5, R6, and R7 can be the side chains of arginine. At least two of R4, R5, R6, and R7 can be the side chains of homoarginine. At least two of R4, R5, R6, and R7 can be the side chains of N-methylarginine. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethylarginine. At least two of R4, R5, R6, and R7 can be the side chains of 2,3-diaminopropionic acid. At least two of R4, R5, R6, and R7 can be the side chains of 2,4-diaminobutanoic acid, lysine. At least two of R4, R5, R6, and R7 can be the side chains of N-methyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N-ethyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least two of R4, R5, R6, and R7 can be the side chains of citrulline. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine, β-homoarginine. At least two of R4, R5, R6, and R7 can be the side chain of 3-(1-piperidinyl)alanine.

[0108] At least three of R4, R5, R6, and R7 can be the side chains of 3-guanidino-2-aminopropionic acid. At least three of R4, R5, R6, and R7 can be the side chains of 4-guanidino-2-aminobutanoic acid. At least three of R4, R5, R6, and R7 can be the side chains of arginine. At least three of R4, R5, R6, and R7 can be the side chains of homoarginine. At least three of R4, R5, R6, and R7 can be the side chains of N-methylarginine. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethylarginine. At least three of R4, R5, R6, and R7 can be the side chains of 2,3-diaminopropionic acid. At least three of R4, R5, R6, and R7 can be the side chains of 2,4-diaminobutanoic acid, lysine. At least three of R4, R5, R6, and R7 can be the side chains of N-methyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N-ethyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least three of R4, R5, R6, and R7 can be the side chains of citrulline. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine, β-homoarginine. At least three of R4, R5, R6, and R7 can be the side chain of 3-(1-piperidinyl)alanine.

[0109] AA SC can be the side chain of an asparagine, glutamine, or homoglutamine residue. SC can be the side chain of a glutamine residue. SCFor example, a cCPP may further comprise a linker conjugated to an asparagine, glutamine, or homoglutamine residue. Thus, a cCPP may further comprise a linker conjugated to an asparagine, glutamine, or homoglutamine residue. A cCPP may further comprise a linker attached to a glutamine residue.

[0110] q can be 1, 2 or 3. q can be 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.

[0111] m can be 1 to 3. m can be 1 or 2. m can be 0. m can be 1. m can be 2. m can be 3.

[0112] The cCPP of formula (A) is a compound of formula (I) [ka] or a protonated form thereof, wherein AA SC , R1, R2, R3, R4, R7, m and q are as defined herein.

[0113] The cCPP of formula (A) is represented by formula (Ia) or formula (Ib): [ka] or its protonated form, SC , R1, R2, R3, R4, and m are as defined herein.

[0114] The cCPP of formula (A) is represented by the formula (I-1), (I-2), (I-3) or (I-4): [ka] or a protonated form thereof, wherein AA SC and m is as defined herein.

[0115] The cCPP of formula (A) is represented by the formula (I-5) or (I-6): [ka] or a protonated form thereof, wherein AA SC is as defined herein.

[0116] The cCPP of formula (A) is represented by the formula (I-1): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0117] The cCPP of formula (A) is represented by the formula (I-2): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0118] The cCPP of formula (A) is represented by the formula (I-3): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0119] The cCPP of formula (A) is represented by the formula (I-4): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0120] The cCPP of formula (A) is represented by the formula (I-5): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0121] The cCPP of formula (A) is represented by the formula (I-6): [ka] or a protonated form thereof, wherein AA SC and m is as defined herein.

[0122] The cCPP may include one of the following sequences: FGFGRGR, GfFGrGr, FfΦGRGR, FfFGRGR, or FfΦGrGr. The cCPP may have one of the following sequences: FGFGRGRQ, GfFGrGrQ, FfΦGRGRQ, FfFGRGRQ, or FfΦGrGrQ.

[0123] The present disclosure also provides a compound of formula (II): [ka] With respect to cCPP having the structure During the ceremony, AA SC is an amino acid side chain, R 1a , R 1b , and R 1c are each independently a 6- to 14-membered aryl or a 6- to 14-membered heteroaryl; R 2a , R 2b , R 2c and R 2d are independently amino acid side chains, R 2a , R 2b , R 2c and R 2d At least one of the [ka] or a protonated form thereof, R 2a , R 2b , R 2c and R 2d at least one of is guanidine or a protonated form thereof; each n" is independently an integer 0, 1, 2, 3, 4, or 5; each n' is independently an integer from 0, 1, 2, or 3; If n' is 0, R 2a , R 2b , R 2b or R 2d does not exist.

[0124] R 2a , R 2b , R 2c and R 2d At least two of the [ka] or its protonated form. 2a , R 2b , R 2c and R 2d Two or three of them are [ka] or its protonated form. 2a , R 2b , R 2c and R 2d One of them is [ka] or its protonated form. 2a , R 2b , R 2c and R 2d At least one of the [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d The remainder of R may be guanidine or its protonated form. 2a , R 2b , R 2c and R 2d At least two of the [ka] or its protonated form. 2a , R 2b , R 2c and R 2d The remainder may be guanidine or its protonated form.

[0125] R 2a , R 2b , R 2c and R 2d All of the above [ka] or its protonated form. 2a , R 2b , R 2c and R 2d At least one of the [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d The remainder of R may be a guaninide or a protonated form thereof. 2a , R 2b , R 2c and R 2d The base is [ka] or its protonated form, R 2a , R 2b , R 2cand R 2d The remainder is guanidine or its protonated form.

[0126] R 2a , R 2b , R 2c and R 2d can each independently be 2,3-diaminopropionic acid, 2,4-diaminobutyric acid side chain, ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homo-lysine, serine, homo-serine, threonine, allo-threonine, histidine, 1-methylhistidine, 2-aminobutanedioic acid, aspartic acid, glutamic acid, or homo-glutamic acid.

[0127] AA SC teeth, [ka] where t can be an integer from 0 to 5. SC teeth, [ka] where t can be an integer from 0 to 5. t can be 1 to 5. t is 2 or 3. t can be 2. t can be 3.

[0128] As used herein, AC is AA SC In an embodiment, the linker (L) couples AC to AA SC In an embodiment, the linker (L) is covalently attached to the backbone of AC.

[0129] AA SC may be the side chain of an asparagine, glutamine or homoglutamine residue. SC can be the side chain of a glutamine residue. SC For example, the linker may include a linker conjugated to an asparagine, glutamine, or homoglutamine residue.

[0130] R1a , R 1b , and R 1c Each R can independently be 6- to 14-membered aryl. 1a , R 1b , and R 1c R may each independently be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, or S. 1a , R 1b , and R 1c Each R may be independently selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl, or isoquinolyl. 1a , R 1b , and R 1c Each R may be independently selected from phenyl, naphthyl, or anthracenyl. 1a , R 1b , and R 1c Each R may independently be phenyl or naphthyl. 1a , R 1b , and R 1c may each independently be selected from pyridyl, quinolyl, or isoquinolyl.

[0131] Each n' can independently be 1 or 2. Each n' can be 1. Each n' can be 2. At least one n' can be 0. At least one n' can be 1. At least one n' can be 2. At least one n' can be 3. At least one n' can be 4. At least one n' can be 5.

[0132] Each n" can independently be an integer from 1 to 3. Each n" can independently be 2 or 3. Each n" can be 2. Each n" can be 3. At least one n" can be 0. At least one n" can be 1. At least one n" can be 2. At least one n" can be 3.

[0133] Each n" can be independently 1 or 2, and each n' can be independently 2 or 3. Each n" can be independently 1, and each n' can be independently 2 or 3. Each n" can be independently 1, and each n' can be 2. Each n" is 1, and each n' is 3.

[0134] The cCPP of formula (II) is represented by the formula (II-1): [ka] may have the structure In the formula, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , A.A. SC , n′ and n″ are as defined herein.

[0135] The cCPP of formula (II) has the formula (IIa): [ka] may have the structure In the formula, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , A.A. SC and n' are as defined herein.

[0136] The cCPP of formula (II) has the formula (IIb): [ka] may have the structure In the formula, R 2a , R 2b , A.A. SC and n' is as defined herein.

[0137] cCPP has the formula (IIb): [ka] or its protonated form, During the ceremony, AA SC and n' are as defined herein.

[0138] The cCPP of formula (IIa) has the following structure: [ka] wherein AA SC and n is as defined herein.

[0139] The cCPP of formula (IIa) has the following structure: [ka] wherein AA SC and n is as defined herein.

[0140] The cCPP of formula (IIa) has the following structure: [ka] wherein AA SC and n is as defined herein.

[0141] The cCPP of formula (II) has the structure: [ka] may have:

[0142] The cCPP of formula (II) has the structure: [ka] may have:

[0143] cCPP has the structure of formula (III): [ka] It may have During the ceremony, AA SC is an amino acid side chain, R 1a , R 1b , and R 1c are each independently a 6- to 14-membered aryl or a 6- to 14-membered heteroaryl; R 2a and R 2c are each independently H, [ka] or its protonated form, R 2b and R 2d are each independently guanidine or a protonated form thereof; each n″ is independently an integer from 1 to 3; each n' is independently an integer from 1 to 5; Each p' is independently an integer from 0 to 5.

[0144] As used herein, AC is AA SC The linker can couple AC to AA SC The linker may be covalently attached to the backbone of the AC, the 5' end of the AC or the 3' end of the AC.

[0145] The cCPP of formula (III) is represented by the formula (III-1): [ka] may have the structure During the ceremony, AA SC , R 1a , R 1b , R 1c , R 2a , R 2c , R 2b , R 2d , n', n" and p' are as defined herein.

[0146] The cCPP of formula (III) has the formula (IIIa): [ka] may have the structure During the ceremony, AA SC , R 2a , R 2c , R 2b , R 2d , n', n'', and p' are as defined herein.

[0147] In formulae (III), (III-1), and (IIIa), R a and R c can be H. R a and R c can be H, R b and R d R may each independently be guanidine or a protonated form thereof. a can be H. R b can be H. p' can be 0. R a and R c may be H, and each p' may be 0.

[0148] In formulae (III), (III-1) and (IIIa), R a and R c can be H, R b and R d can each independently be guanidine or a protonated form thereof; n″ can be 2 or 3; and each p′ can be 0.

[0149] p' can be 0. p' can be 1. p' can be 2. p' can be 3. p' can be 4. p' can be 5.

[0150] cCPP has the structure: [ka] may have:

[0151] The cCPP of formula (A) can be selected from: [Table 5]

[0152] The cCPP of formula (A) can be selected from: [Table 6]

[0153] In embodiments, the cCPP is selected from the following: [Table 7]

[0154] In embodiments, the cCPP is not selected from the following: [Table 8]

[0155] cCPP has the formula (D): [ka] or a protonated form thereof, During the ceremony, R1, R2, and R3 can each independently be H or an amino acid residue having a side chain containing an aromatic group; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain, Y is [ka] It is. q is 1, 2, 3 or 4; each m is independently an integer 0, 1, 2, or 3; Each n is independently an integer 0, 1, 2, or 3.

[0156] The cCPP of formula (D) is represented by the formula (DI): [ka] or its protonated form, During the ceremony, R1, R2, and R3 can each independently be H or an amino acid residue having a side chain containing an aromatic group; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain, q is 1, 2, 3 or 4; each m is independently an integer 0, 1, 2, or 3; Y is [ka] It is.

[0157] The cCPP of formula (D) is represented by the formula (D-II): [ka] or its protonated form, During the ceremony, R1, R2, and R3 can each independently be H or an amino acid residue having a side chain containing an aromatic group; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain, q is 1, 2, 3 or 4; each m is independently an integer 0, 1, 2, or 3; each n is independently an integer 0, 1, 2, or 3; Y is [ka] It is.

[0158] The cCPP of formula (D) is represented by the formula (D-III): [ka] or its protonated form, During the ceremony, R1, R2, and R3 can each independently be H or an amino acid residue having a side chain containing an aromatic group; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain, q is 1, 2, 3 or 4; each m is independently an integer 0, 1, 2, or 3; each n is independently an integer 0, 1, 2, or 3; Y is [ka] It is.

[0159] The cCPP of formula (D) is represented by formula (D-IV): [ka] or its protonated form, During the ceremony, R1, R2, and R3 can each independently be H or an amino acid residue having a side chain containing an aromatic group; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AASC is an amino acid side chain, q is 1, 2, 3 or 4; each m is independently an integer 0, 1, 2, or 3; Y is [ka] It is.

[0160] The cCPP of formula (D) is represented by the formula (DV): [ka] or its protonated form, During the ceremony, R1, R2, and R3 can each independently be H or an amino acid residue having a side chain containing an aromatic group; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain, q is 1, 2, 3 or 4; each m is independently an integer 0, 1, 2, or 3; Y is [ka] It is.

[0161] AA SC can be conjugated to a linker.

[0162] Linker The cCPP of the present disclosure can be conjugated to a linker. The linker can link AC to the cCPP. The linker can be attached to the side chain of an amino acid of the cCPP, and AC can be attached to a suitable position on the linker.

[0163] The linker may be any suitable moiety capable of conjugating the cCPP to one or more further moieties, such as an exocyclic peptide (EP) and / or an AC. Prior to attachment to the cCPP and one or more further moieties, the linker has two or more functional groups, each of which can independently form a covalent bond to the cCPP and one or more further moieties. The linker may be covalently attached to the 5' end of the AC or the 3' end of the AC. The linker may be covalently attached to the 5' end of the AC. The linker may be covalently attached to the 3' end of the AC. The linker may be any suitable moiety capable of conjugating the cCPP described herein to an AC.

[0164] The linker may comprise a hydrocarbon linker.

[0165] The linker may comprise a cleavage site, which may be a disulfide or a caspase cleavage site (e.g., Val-Cit-PABC).

[0166] The linker may be (i) one or more D or L amino acids, each of which is optionally substituted; (ii) an optionally substituted alkylene; (iii) an optionally substituted alkenylene; (iv) an optionally substituted alkynylene; (v) an optionally substituted carbocyclyl; (vi) an optionally substituted heterocyclyl; (vii) one or more -(R 1- JR 2 )z″-subunits, wherein R 1 and R 2 each, at each occurrence, is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl; and each J is independently selected from C, NR 3 , -NR 3 C(O)-, S, and O, where R 3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z″ is an integer from 1 to 50; 1-J)z”- or (JR 1 )z″-, wherein each R 1 is, at each occurrence, independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl; each J is, independently, C, NR 3 , -NR 3 C(O)-, S, or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z″ is an integer from 1 to 50; or (ix) the linker may include one or more of (i)-(x).

[0167] The linker may be one or more D or L amino acids and / or (R 1- JR 2 )z″-, wherein R 1 and R 2 each, at each occurrence, is independently alkylene; and each J is independently C, NR 3 , -NR 3 C(O)-, S, and O, where R 4 is independently selected from H and alkyl, and z″ is an integer from 1 to 50, or a combination thereof.

[0168] The linker is -(OCH2CH2) z’ - (e.g., as a spacer), where z' is an integer from 1 to 23, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. "-(OCH2CH2)z'" can also be referred to as polyethylene glycol (PEG).

[0169] The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise -(OCH2CH2) z’-, where z' is an integer from 1 to 23, and a peptide. The peptide may contain 2 to 10 amino acids. The linker may further include a functional group (FG) that can react via click chemistry. FG may be an azide or an alkyne, and a triazole is formed when AC is conjugated to the linker.

[0170] The linker is (i) a β-alanine residue and a lysine residue, (ii) -(JR 1 )z″-, or (iii) combinations thereof. Each R 1 can be independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can be independently C, NR 3 , -NR 3 C(O)-, S, or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z″ can be an integer from 1 to 50. Each R 1 can be alkylene and each J can be O.

[0171] The linker may be (i) a residue of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof, and (ii) -(R 1- J)z”- or (JR 1 )z″-. Each R 1 can be independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can be independently C, NR 3 , -NR 3 C(O)-, S, or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z″ can be an integer from 1 to 50. Each R 1can be alkylene and each J can be O. The linker can include glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0172] The linker can be a trivalent linker. The linker has the structure: [ka] wherein A1, B1 and C1 are independently a hydrocarbon linker (e.g., NRH-(CH2) n -COOH), PEG linker (e.g., NRH-(CHO) n -COOH, where R is H, methyl or ethyl), or one or more amino acid residues, and Z is independently a protecting group. Linkers also include disulfides [NH2-(CHO) n -SS-(CH2O) n -COOH], or a cleavage site such as a caspase cleavage site (Val-Cit-PABC) can be incorporated.

[0173] The carbohydrate may be a residue of glycine or beta-alanine.

[0174] The linker is bivalent and can link a cCPP to an AC. The linker is bivalent and can link a cCPP to an exocyclic peptide (EP).

[0175] The linker may be trivalent and may link the cCPP to the AC and the EP.

[0176] The linker is a divalent or trivalent C1-C 50It can be alkylene, where 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl, or cycloalkenyl. The linker can be a divalent or trivalent C1-C 50 It can be alkylene, where 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or combinations thereof.

[0177] AC may be coupled to glutamic acid of a cyclic peptide, thereby converting glutamic acid to glutamine. A linker (L) may couple AC to glutamine / glutamic acid of a cyclic peptide. In an embodiment, the linker (L) is covalently attached to the backbone of AC.

[0178] The linker has the structure: [ka] wherein each AA is independently an amino acid residue; * AA SC AA SC is a side chain of an amino acid residue of cCPP, where x is an integer from 1 to 10, y is an integer from 1 to 5, and z is an integer from 1 to 10. x can be an integer from 1 to 5. x can be an integer from 1 to 3. x can be 1. y can be an integer from 2 to 4. y can be 4. z can be an integer from 1 to 5. z can be an integer from 1 to 3. z can be 1. Each AA can be independently selected from glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminohexanoic acid.

[0179] The cCPP can be linked to the AC via a linker ("L"). The linker can be conjugated to the AC via a linking group ("M").

[0180] The linker has the structure: [ka] wherein x is an integer from 1 to 10, y is an integer from 1 to 5, z is an integer from 1 to 10, and each AA is independently an amino acid residue; * AA SC AA SC is the side chain of an amino acid residue of a cCPP, and M is a linking group as defined herein.

[0181] The linker has the structure: [ka] It may have In the formula, x' is an integer from 1 to 23, y is an integer from 1 to 5, and z' is an integer from 1 to 23. * AA SC AA SC is the side chain of an amino acid residue of a cCPP, and M is a linking group as defined herein.

[0182] The linker has the structure: [ka] It may have In the formula, x' is an integer from 1 to 23, y is an integer from 1 to 5, and z' is an integer from 1 to 23. * AA SC AA SC are the side chains of amino acid residues of cCPP.

[0183] x can be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.

[0184] x' can be an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 (including all ranges and subranges therebetween). x' can be an integer from 5 to 15. x' can be an integer from 9 to 13. x' can be an integer from 1 to 5. x' can be 1.

[0185] y can be an integer from 1 to 5, for example, 1, 2, 3, 4, or 5 (including all ranges and subranges therebetween), and y can be an integer from 2 to 5. y can be an integer from 3 to 5. y can be 3 or 4. y can be 4 or 5. y can be 3. y can be 4. y can be 5.

[0186] z can be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.

[0187] z' can be an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. z' can be an integer from 5 to 15. z' can be an integer from 9 to 13. z' can be 11.

[0188] As described above, the linker or M (where M is part of the linker) can be covalently attached to the AC at any suitable position on the AC. The linker or M (where M is part of the linker) can be covalently attached to the 3' end of the AC or the 5' end of the AC. The linker or M (where M is part of the linker) can be covalently attached to the backbone of the AC.

[0189] The linker can be attached to the side chain of an aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or a modified side chain of a glutamine or asparagine (e.g., a reduced side chain bearing an amino group).The linker can be attached to the side chain of a lysine on the cCPP.

[0190] The linker has the structure: [ka] It may have During the ceremony, M is a group that conjugates L to AC; AA s is the side chain or terminus of an amino acid on the cCPP, Each AA x are independently amino acid residues, o is an integer from 0 to 10; p is an integer from 0 to 5.

[0191] The linker has the structure: [ka] It may have During the ceremony, M is a group that conjugates L to AC; AA s is the side chain or terminus of an amino acid on the cCPP, Each AA x are independently amino acid residues, o is an integer from 0 to 10; p is an integer from 0 to 5.

[0192] M can include alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted. M can be [ka] wherein R is alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl.

[0193] M is [ka] You can choose from R 10 is alkylene, cycloalkyl, or [ka] where a is 0 to 10.

[0194] M is [ka] R 10 teeth, [ka] where a is from 0 to 10. M can be [ka] It could be.

[0195] M is a heterobifunctional crosslinker, e.g., [ka] which is disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-4.41.20, which is incorporated herein by reference in its entirety.

[0196] M can be -C(O)-.

[0197] AA s may be the side chain or terminus of an amino acid on the cCPP. sNon-limiting examples of AA include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group). s is AA as defined herein SC It could be.

[0198] Each AA x are independently natural or unnatural amino acids. x may be a natural amino acid. x may be a non-natural amino acid. x may be a β-amino acid. The β-amino acid may be β-alanine.

[0199] o can be an integer from 0 to 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. o can be 0, 1, 2, or 3. o can be 0. o can be 1. o can be 2. o can be 3.

[0200] p can be 0 to 5, e.g., 0, 1, 2, 3, 4, or 5. p can be 0. p can be 1. p can be 2. p can be 3. p can be 4. p can be 5.

[0201] The linker has the structure: [ka] It may have In the formula, M, AA s , each -(R 1- JR 2 ) z″-, o, and z″ are defined herein. r can be 0 or 1.

[0202] r can be 0. r can be 1.

[0203] The linker has the structure: [ka] It may have In the formula, M, AA s , o, p, q, r, and z″ may be as defined herein.

[0204] z" can be an integer from 1 to 50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, including all ranges and values ​​therebetween. z" can be an integer from 5 to 20. z" can be an integer from 10 to 15.

[0205] The linker has the structure: [ka] It may have During the ceremony, M, A.A. s and o are as defined herein.

[0206] Other non-limiting examples of suitable linkers include: [ka] [ka] In the formula, M and AA s is as defined herein.

[0207] Provided herein is a compound comprising a cCPP and an AC complementary to a target in a pre-mRNA sequence, further comprising L, a linker conjugated to the AC via a linking group (M), wherein M is [ka] The compound is provided,

[0208] Provided herein is a compound comprising a cCPP and an antisense compound (AC), e.g., an antisense oligonucleotide, that is complementary to a target in a pre-mRNA sequence, further comprising L, a linker conjugated to AC via a linking group (M), wherein M is [ka] Selected from R 1 is alkylene, cycloalkyl, or [ka] where t' is 0 to 10, each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, and R 1 but, [ka] and t' is 2.

[0209] The linker has the structure: [ka] It may have During the ceremony, A.A. s is as defined herein, and m' is 0-10.

[0210] The linker has the formula: [ka] It may be of the following type.

[0211] The linker has the formula: [ka] where "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.

[0212] The linker has the formula: [ka] where "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.

[0213] The linker has the formula: [ka] where "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.

[0214] The linker has the formula: [ka] where "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.

[0215] The linker has the formula: [ka] It may be of the following type.

[0216] The linker can be covalently attached at any suitable position on the AC. The linker is covalently attached to the 3' end of the AC or the 5' end of the AC. The linker can be covalently attached to the backbone of the AC.

[0217] The linker can be attached to the side chain of an aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or a modified side chain of a glutamine or asparagine (e.g., a reduced side chain bearing an amino group).The linker can be attached to the side chain of a lysine on the cCPP.

[0218] cCPP-linker conjugates The cCPP may be conjugated to a linker as defined herein. The linker may be any of the AA SC can be conjugated to

[0219] The linker is -(OCH2CH2) z’ subunits (e.g., as spacers), where z' is an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. "-(OCH2CH2) z’ is also referred to as PEG. The cCPP-linker conjugate may have a structure selected from Table 4. [Table 9]

[0220] The linker is -(OCH2CH2) z’ -subunits, where z' is an integer from 1 to 23, and peptide subunits. The peptide subunits may comprise 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table 5. [Table 10]

[0221] The cCPP-linker conjugate can be Ac-PKKKRKVK(cyclo[FfΦGrGrQ])-PEG12-K(N3)-NH2.

[0222] An EEV is provided that includes a cyclic cell penetrating peptide (cCPP), a linker, and an exocyclic peptide (EP). The EEV has the formula (B): [ka] or a protonated form thereof, During the ceremony, R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; EP is an exocyclic peptide as defined herein; each m is independently an integer from 0 to 3; n is an integer from 0 to 2, x' is an integer from 1 to 20; y is an integer from 1 to 5; q is 1 to 4; z' is an integer from 1 to 23.

[0223] R1, R2, R3, R4, R6, EP, m, q, y, x', z' are as described herein.

[0224] n can be 0. n can be 1. n can be 2.

[0225] EEV has the formula (Ba) or (Bb): [ka] or a protonated form thereof, 1 , R 2 , R 3 , R 4 , m and z' are as defined above in formula (B).

[0226] EEV is calculated by the formula (Bc): [ka] or a protonated form thereof, 1 , R 2 , R 3 , R 4and m are as defined above in formula (B), AA is an amino acid as defined herein, M is as defined herein, n is an integer from 0 to 2, x is an integer from 1 to 10, y is an integer from 1 to 5, and z is an integer from 1 to 10.

[0227] The EEV is represented by the formula (B-1), (B-2), (B-3), or (B-4): [ka] [ka] or a protonated form thereof, where EP is as defined above in formula (B).

[0228] The EEV may comprise the formula (B) and the structure: Ac-PKKKRKV-AEEA-K(cyclo[FGFGRGRQ])-PEG 12 -OH, or Ac-PKKKRKV-AEEA-K(cyclo[GfFGrGrQ])-PEG 12 It may have -OH.

[0229] EEV has the formula: [ka] The cCPP may include

[0230] The EEV may comprise the formula: Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3).

[0231] EEV is [ka] It could be. EEV was synthesized using the recombinant Ac-PKKKRKV-K(cyclo(Ff-Nal-GrGrQ)-PEG 12 -K(N3)-NH s It could be.

[0232] EEV is [ka] It could be.

[0233] EEV is Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-AEEA-K(cyclo(Ff-Nal-GrGrQ)-PEG 12 -OH, or Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-AEEA-K(cyclo(FGFGRGRQ)-PEG 12 It can be -OH.

[0234] EEV is [ka] It could be.

[0235] The EEV can be Ac-PKKKRKV-miniPEG-K(cyclo(Ff-Nal-GrGrQ)-PEG-OH.

[0236] EEV is [ka] It could be.

[0237] EEV is [ka] It could be.

[0238] EEV is [ka] It could be.

[0239] EEV is [ka] It could be.

[0240] EEV is [ka] It could be.

[0241] EEV is [ka] It could be.

[0242] EEV is [ka] It could be.

[0243] EEV is [ka] It could be.

[0244] EEV is [ka] It could be.

[0245] EEV is [ka] It could be.

[0246] EEV is [ka] It could be.

[0247] EEV is Ac-rr-miniPEG2-Dap[cyclo(FfΦ-Cit-r-Cit-rQ)]-PEG12-OH, Ac-frr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH, Ac-rfr-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rbfbr-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rrr-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rbr-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rbrbr-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-hh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-hbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-hbhbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rbhbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-hbrbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-frr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rfr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rbfbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rrr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rbrbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-hh-Dap(。(FfΦ-Cit-r-Cit-rQ))-b-OH. Ac-hbh-Dap(。(FfΦ-Cit-r-Cit-rQ))-b-OH. Ac-hbhbh-Dap(シ(FfΦ-Cit-r-Cit-rQ))-b-OH. Ac-rbhbh-Dap(シ(FfΦ-Cit-r-Cit-rQ))-b-OH. Ac-hbrbh-Dap(シロ(FfΦ-Cit-r-Cit-rQ))-b-OH. Ac-KKKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-KGKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKGK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-KKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-KK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KGK-miniPEG2-Lys(シロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBKBK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-KR-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBR-miniPEG2-Lys(シロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2; Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PGKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKGKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKGRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKKGKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKKRGV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKKRKG-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-KKKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-KKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, and Ac-KRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 may be selected from.

[0248] EEV is Ac-PKKKRKV-Lys(cyclo[FfΦGrGrQ])-PEG 12 -K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦGrGrQ])-miniPEG2-K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRQ])-miniPEG2-K(N3)-NH2, Ac-KR-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKGKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKRKG-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-KKKRK-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FFΦGRGRQ])-miniPEG2-K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[βhFfΦGrGrQ])-miniPEG2-K(N3)-NH2, and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦSrSrQ])-miniPEG2-K(N3)-NH2 may be selected from:

[0249] EEV is Ac-PKKKRKV-miniPEG2-Lys(cyclo(GfFGrGrQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFKRKRQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRGRGQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRrRQ])-PEG12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRRRQ])-PEG 12 -OH, and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRRRRQ])-PEG 12 -OH may be selected from:

[0250] EEV is Ac-KKKRKG-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KKKRK-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KKRKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KRKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KKKKR-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-RKKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, and Ac-KKKRK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH may be selected from:

[0251] EEV is Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH2, and Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH may be selected from:

[0252] The cargo may be an AC and the EEV may be Ac-PKKKRKV-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rr-PEG2-K(シクロ[FGFRRRRQ])-PEG 12 -OH、 Ac-rrr-PEG2-K(シクロ[FfΦGrGrQ])-PEG 12 -OH、 Ac-rrr-PEG2-K(シクロ[FfΦCit-r-Cit-rQ])-PEG 12 -OH、 Ac-rrr-PEG2-K(シクロ[FfFGRGRQ])-PEG 12 -OH、 Ac-rrr-PEG2-K(シクロ[FGFGRGRQ])-PEG 12 -OH、 Ac-rrr-PEG2-K(シクロ[GfFGrGrQ])-PEG 12 -OH、 Ac-rrr-PEG2-K(シクロ[FGFGRRRQ])-PEG 12 -OH、 Ac-rrr-PEG2-K(シクロ[FGFRRRRQ])-PEG 12 -OH、 Ac-rhr-PEG2-K(シクロ[FfΦGrGrQ])-PEG 12 -OH、 Ac-rhr-PEG2-K(シクロ[FfΦCit-r-Cit-rQ])-PEG 12 -OH、 Ac-rhr-PEG2-K(シクロ[FfFGRGRQ])-PEG 12 -OH、 Ac-rhr-PEG2-K(シクロ[FGFGRGRQ])-PEG 12 -OH、 Ac-rhr-PEG2-K(シクロ[GfFGrGrQ])-PEG 12 -OH、 Ac-rhr-PEG2-K(シクロ[FGFGRRRQ])-PEG 12 -OH、 Ac-rhr-PEG2-K(シクロ[FGFRRRRQ])-PEG 12 -OH、 Ac-rbr-PEG2-K(シクロ[FfΦGrGrQ])-PEG 12 -OH、 Ac-rbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-rbrbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-rbrbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rbrbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-rbrbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rbrbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-rbrbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rbrbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-rbhbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-rbhbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rbhbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-rbhbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rbhbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-rbhbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rbhbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, and Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH where b is beta-alanine and the exocyclic sequence may be of D or L stereochemistry.

[0253] In embodiments, the compound that comprises cyclic peptide and AC has improved cytoplasmic uptake efficiency compared to the compound that comprises AC alone.Cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of the compound that comprises cyclic peptide and AC with the cytoplasmic delivery efficiency of AC alone.

[0254] Antisense Compounds In various embodiments, the compounds disclosed herein comprise a CPP (e.g., a cyclic peptide) conjugated to an antisense compound (AC). In embodiments, the AC comprises an antisense oligonucleotide directed to a target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" is intended to include oligonucleotides that are complementary to a targeted polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a selected sequence, for example, a target gene mRNA.

[0255] Antisense oligonucleotides may modulate one or more aspects of protein transcription, translation and expression. In embodiments, antisense oligonucleotides are directed to a target sequence in a target pre-mRNA and modulate one or more aspects of pre-mRNA splicing. As used herein, modulation of splicing refers to altering the processing of a pre-mRNA transcript such that the spliced ​​mRNA molecule contains either a different combination of exons as a result of exon skipping or exon incorporation, a deletion of one or more exons, or a deletion or addition of a sequence not normally found in the spliced ​​mRNA (e.g., an intron sequence). In embodiments, hybridization of an antisense oligonucleotide to a target sequence in a pre-mRNA molecule restores natural splicing to the mutated pre-mRNA sequence. In embodiments, hybridization of an antisense oligonucleotide results in alternative splicing of the target pre-mRNA. In embodiments, hybridization of an antisense oligonucleotide results in exon incorporation or exon skipping of one or more exons. In an embodiment, the exon sequence to be skipped comprises a frameshift mutation, a nonsense mutation or a missense mutation. In an embodiment, the exon sequence to be skipped comprises a nucleic acid deletion, substitution or insertion. In an embodiment, the exon to be skipped itself does not comprise a sequence mutation, but the adjacent exon comprises a mutation that leads to a frameshift mutation or a nonsense mutation. In an embodiment, hybridization of an antisense oligonucleotide to a target sequence in a target pre-mRNA prevents incorporation of the exon sequence in a mature mRNA molecule. In an embodiment, hybridization of an antisense oligonucleotide to a target sequence in a target pre-mRNA results in preferential expression of a wild-type target protein isomer. In an embodiment, hybridization of an antisense oligonucleotide to a target sequence in a target pre-mRNA results in expression of a re-spliced ​​target protein that comprises an active fragment of the wild-type target protein.

[0256] The antisense mechanism functions through the hybridization of the antisense oligonucleotide compound with the target nucleic acid. In an embodiment, the antisense oligonucleotide hybridized to the target sequence suppresses the expression of the target protein. In an embodiment, the hybridization of the antisense oligonucleotide to its target sequence suppresses the expression of one or more wild-type target protein isomers. In an embodiment, the hybridization of the antisense oligonucleotide to its target sequence upregulates the expression of the target protein. In an embodiment, the hybridization of the antisense oligonucleotide to its target sequence increases the expression of one or more wild-type target protein isomers.

[0257] In embodiments, antisense compounds can inhibit gene expression by binding to complementary mRNA. Binding to target mRNA can result in inhibition of gene expression by sterically blocking RNA binding proteins involved in translation, thereby preventing translation of the complementary mRNA strand, or by causing degradation of the target mRNA. Antisense DNA can be used to target specific complementary (coding or non-coding) RNA. When binding occurs, the DNA / RNA hybrid can be degraded by the enzyme RNase H. In embodiments, antisense oligonucleotides contain about 10 to about 50 nucleotides, or about 15 to about 30 nucleotides. In embodiments, antisense oligonucleotides may not be completely complementary to the target nucleotide sequence.

[0258] Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and therefore can be used to specifically inhibit the protein synthesis of target genes.The effectiveness of antisense oligonucleotides for inhibiting protein synthesis has been well proven.For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (US Pat. No. 5,739,119 and US Pat. No. 5,759,829). Furthermore, examples of antisense inhibition have been shown for the nuclear protein cyclin, the multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, and human EGF (Jaskulski et al., Science. 1988 Jun 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U.S. Patent No. 5,801,154; U.S. Patent No. 5,789,573; U.S. Patent No. 5,718,709; and U.S. Patent No. 5,610,288). Additionally, antisense constructs have been described that can be used to inhibit and treat various abnormal cell proliferations, such as cancer (U.S. Patent Nos. 5,747,470, 5,591,317, and 5,783,683).

[0259] Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. Selection of an antisense oligonucleotide sequence specific for a given target sequence is based on analysis of the selected target sequence and determination of secondary structure, Tm, binding energy and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins or other secondary structures that reduce or prevent specific binding to the target mRNA in the host cell. Target regions of mRNA can include regions at or near the AUG translation initiation codon and sequences that are substantially complementary to the 5' region of the mRNA. These secondary structure analyses and target site selection studies can be performed, for example, using OLIGO primer analysis software version 4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al, Nucleic Acids Res. 1997, 25(17):3389-402).

[0260] According to the present disclosure, antisense compounds (ACs) alter one or more aspects of splicing, translation or expression of a target gene, for example, by altering the splicing of a target pre-mRNA in a eukaryote. The ACs according to the present disclosure comprise nucleic acid sequences that are complementary to sequences found in the target pre-mRNA sequence, for example, sequences that include at least a portion of an exon, at least a portion of an intron, or both. The use of these ACs provides a direct genetic approach with the ability to modulate the splicing of specific disease-causing genes. The principle behind antisense technology is that antisense compounds that hybridize to a target nucleic acid modulate gene expression activity, for example, splicing or translation, by one of several antisense mechanisms. The sequence specificity of ACs makes this technology highly attractive as a therapeutic agent for selectively modulating the splicing of pre-mRNAs involved in the pathogenesis of any one of a variety of diseases. Antisense technology is an effective means to alter the expression of one or more specific gene products and therefore may prove useful in several therapeutic, diagnostic and research applications.

[0261] The compounds described herein may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which may be defined, with respect to absolute stereochemistry, as (R) or (S), α or β, or (D) or (L). The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0262] Antisense Compound Hybridization Site Antisense mechanism depends on hybridization of antisense compound to target nucleic acid. In an embodiment, the present disclosure provides an antisense compound that is complementary to a target nucleic acid. In an embodiment, the target nucleic acid sequence is present in a pre-mRNA molecule. In an embodiment, the target nucleic acid sequence is present in an exon of a pre-mRNA molecule. In an embodiment, the target nucleic acid sequence is present in an intron of a pre-mRNA molecule.

[0263] Pre-mRNA molecules are made in the nucleus and processed before or during transport to the cytoplasm for translation. Pre-mRNA processing involves the addition of a 5' methylated cap and a poly(A) tail of approximately 200-250 bases to the 3' end of the transcript. The next step in mRNA processing is splicing of the pre-mRNA, which occurs in the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of the primary transcript (or the DNA that encodes them) that are not included in the coding sequence of the mature mRNA. Exons are regions of the primary transcript that remain in the mature mRNA when it reaches the cytoplasm. Exons are spliced ​​together to form the mature mRNA sequence. Splice junctions are also called splice sites, and the 5' side of the junction is often called the "5' splice site" or "splice donor site" and the 3' side is called the "3' splice site" or "splice acceptor site." In splicing, the 3' end of the upstream exon is joined to the 5' end of the downstream exon. Thus, the unspliced ​​RNA (or pre-mRNA) has an exon / intron junction at the 5' end of the intron and an intron / exon junction at the 3' end of the intron. After the intron is removed, the exons are adjacent to each other in the mature mRNA at what are sometimes called exon / exon junctions or boundaries. Cryptic splice sites are splice sites that are used less frequently but may be used when the normal splice sites are blocked or unavailable. Alternative splicing, defined as different combinations of exons being spliced ​​together, often results in multiple mRNA transcripts from a single gene.

[0264] In embodiments, the AC hybridizes to a sequence within a splice site. In embodiments, the AC hybridizes to a sequence that includes a portion of a splice site. In embodiments, the AC hybridizes to a sequence that includes a portion or all of a splice site. In embodiments, the AC hybridizes to a sequence that includes a portion or all of a splice donor site. In embodiments, the AC hybridizes to a sequence that includes a portion or all of a splice acceptor site. In embodiments, the AC hybridizes to a sequence that includes a portion or all of a cryptic splice site. In embodiments, the AC hybridizes to a sequence that includes an exon / intron junction.

[0265] Pre-mRNA splicing involves two sequential biochemical reactions. Both reactions involve spliceosomal transesterification between RNA nucleotides. In the first reaction, the 2'-OH of a specific branch point nucleotide in the intron, determined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site to form a lariat intermediate. In the second reaction, the 3'-OH of the released 5' exon performs a nucleophilic attack on the last nucleotide of the intron at the 3' splice site, thus joining the exons and releasing the lariat intron. Pre-mRNA splicing is regulated by intronic silencer sequences (ISS) and terminal stem-loop (TSL) sequences. As used herein, the terms "intronic silencer sequence (ISS)" and "terminal stem loop (TSL)" refer to sequence elements within introns and exons, respectively, that control alternative splicing by binding of trans-acting protein factors within the pre-mRNA, thereby resulting in differential utilization of splice sites. Typically, intronic silencer sequences are 8-16 nucleotides in length and are less conserved than the splice sites at exon-intron junctions. Terminal stem loop sequences are typically 12-24 nucleotides in length and form secondary loops within the 12-24 nucleotide sequence due to complementarity and therefore binding.

[0266] In embodiments, the AC hybridizes to a sequence that includes part or all of an intronic silencer sequence. In embodiments, the AC hybridizes to a sequence that includes part or all of a terminal stem loop.

[0267] Up to 50% of human genetic diseases caused by point mutations are caused by abnormal splicing. Such point mutations can either disrupt existing splice sites or create new splice sites, resulting in mRNA transcripts that consist of different combinations of exons or have exon deletions. Point mutations can also result in the activation of cryptic splice sites or the disruption of regulatory cis elements (i.e. splicing enhancers or silencers).

[0268] In embodiments, the AC hybridizes to a sequence that includes some or all of an aberrant splice site resulting from a mutation in the target gene. In embodiments, the AC hybridizes to a sequence that includes some or all of a regulatory element. Also provided are antisense compounds directed to cis-regulatory elements. In embodiments, the regulatory element is in an exon. In embodiments, the regulatory element is in an intron.

[0269] In embodiments, the AC may specifically hybridize with a sequence in the translation initiation codon region, the 5' cap region, the intron / exon junction, the coding sequence, the translation termination codon region, or the 5' or 3' untranslated region. In embodiments, the AC may hybridize with some or all of the pre-mRNA splice sites, exon-exon junctions, or intron-exon junctions. In embodiments, the AC may hybridize with aberrant fusion junctions resulting from rearrangements or deletions. In embodiments, the AC may hybridize with specific exons in alternatively spliced ​​mRNAs.

[0270] In embodiments, the AC hybridizes to a sequence that is 5-50 nucleotides in length, which may also be referred to as the length of the AC. In embodiments, the AC is 5-50 nucleotides in length, e.g., 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 nucleotides in length. In embodiments, the AC is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In embodiments, the AC is at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20, and up to about 21, about 22, about 23, about 24, or about 25, and up to about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40, and up to about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 nucleotides in length. In embodiments, the AC is about 10 nucleotides in length. In embodiments, the AC is about 15 nucleotides in length. In embodiments, the AC is about 16 nucleotides in length. In embodiments, the AC is about 17 nucleotides in length. In embodiments, the AC is about 18 nucleotides in length. In embodiments, the AC is about 19 nucleotides in length. In embodiments, the AC is about 20 nucleotides in length. In embodiments, the AC is about 21 nucleotides in length. In embodiments, the AC is about 22 nucleotides in length. In embodiments, the AC is about 23 nucleotides in length. In embodiments, the AC is about 24 nucleotides in length. In embodiments, the AC is about 25 nucleotides in length. In embodiments, the AC is about 26 nucleotides in length. In embodiments, the AC is about 27 nucleotides in length. In embodiments, the AC is about 28 nucleotides in length. In embodiments, the AC is about 29 nucleotides in length. In embodiments, the AC is about 30 nucleotides in length.

[0271] In embodiments, the AC may have less than 100 percent complementarity to the target nucleic acid sequence. As used herein, the term "percent complementarity" refers to the number of nucleobases of the AC that have nucleobase complementarity with the corresponding nucleobases of an oligomeric compound or nucleic acid, divided by the total length (number of nucleobases) of the AC. Those skilled in the art will appreciate that the inclusion of mismatches is possible without eliminating the activity of the antisense compound. In embodiments, the AC may contain up to about 20% nucleotides that inhibit base pairing of the AC to the target nucleic acid. In embodiments, the AC contains about 15% or less, about 10% or less, 5% or less mismatches, or no mismatches. In embodiments, the AC contains 1, 2, 3, 4, or 5 mismatches. In embodiments, the AC has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementarity to the target nucleic acid. The percent complementarity of an oligonucleotide is calculated by dividing the number of complementary nucleobases by the total number of nucleobases of the oligonucleotide. The percent complementarity of a region of an oligonucleotide is calculated by dividing the number of complementary nucleobases in the region by the total number of nucleobases in the region.

[0272] In embodiments, the incorporation of nucleotide affinity modification allows a greater number of mismatches compared to unmodified compounds.Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between oligonucleotides and target nucleic acid, for example, by determining melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, the techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow those skilled in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes.

[0273] Antisense Mechanism The AC according to the present disclosure may modulate one or more aspects of protein transcription, translation and expression. In an embodiment, the AC hybridized to a target sequence in a target pre-mRNA modulates one or more aspects of pre-mRNA splicing. As used herein, modulation of splicing refers to altering the processing of a pre-mRNA transcript such that the spliced ​​mRNA molecule contains either a different combination of exons as a result of exon skipping or exon incorporation, a deletion of one or more exons, or a deletion or addition of a sequence not normally found in the spliced ​​mRNA (e.g., an intron sequence). In an embodiment, hybridization of the AC to a target sequence in a pre-mRNA molecule restores natural splicing to the mutated pre-mRNA sequence. In an embodiment, hybridization of the AC results in alternative splicing of the target pre-mRNA. In an embodiment, hybridization of the AC results in exon incorporation or exon skipping of one or more exons. In an embodiment, the exon sequence to be skipped comprises a frameshift mutation, a nonsense mutation or a missense mutation. In an embodiment, the exon sequence to be skipped comprises a nucleic acid deletion, substitution or insertion. In an embodiment, the skipped exon itself does not comprise a sequence mutation, but the adjacent exon comprises a mutation that leads to a frameshift mutation or a nonsense mutation. In an embodiment, the deletion of the exon that does not comprise a sequence mutation restores the reading frame of the mature mRNA. In an embodiment, hybridization of the AC to the target sequence in the target pre-mRNA results in preferential expression of the wild-type target protein isoform. In an embodiment, hybridization of the AC to the target sequence in the target pre-mRNA results in expression of a re-spliced ​​target protein that comprises an active fragment of the wild-type target protein.

[0274] The antisense mechanism functions through the hybridization of the antisense compound with the target nucleic acid. In an embodiment, the AC hybridized to the target sequence suppresses the expression of the target protein. In an embodiment, the AC hybridized to the target sequence suppresses the expression of one or more wild-type target protein isomers. In an embodiment, the AC hybridized to the target sequence upregulates the expression of the target protein. In an embodiment, the AC hybridized to the target sequence increases the expression of one or more wild-type target protein isomers.

[0275] The efficacy of the ACs of the present disclosure can be evaluated by assessing the antisense activity resulting from their administration. As used herein, the term "antisense activity" refers to any detectable and / or measurable activity resulting from hybridization of an antisense compound to its target nucleic acid. Such detection and / or measurement may be direct or indirect. In an embodiment, the antisense activity is assessed by detecting and / or measuring the amount of the target protein. In an embodiment, the antisense activity is assessed by detecting and / or measuring the amount of the re-spliced ​​target protein. In an embodiment, the antisense activity is assessed by detecting and / or measuring the amount of the target nucleic acid and / or the cleaved target nucleic acid and / or the alternatively spliced ​​target nucleic acid.

[0276] Antisense Compound Design The design of AC according to the present disclosure depends on the sequence to be targeted. Targeting AC to a specific target nucleic acid molecule can be a multi-step process. This process usually begins with the identification of the target nucleic acid whose expression is to be regulated. As used herein, the terms "target nucleic acid" and "nucleic acid encoding a target gene" also encompass DNA encoding a selected target gene, RNA (such as pre-mRNA and mRNA) transcribed from such DNA, and cDNA derived from such RNA. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.

[0277] Those skilled in the art will be able to design, synthesize and screen antisense compounds of different nucleobase sequences to identify sequences that produce antisense activity.For example, antisense compounds can be designed to change the splicing of target pre-mRNA or inhibit the expression of target protein.Methods for designing, synthesizing and screening antisense compounds for antisense activity against preselected target nucleic acid can be found, for example, in "Antisense Drug Technology, Principles, Strategies, and Applications" edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida, which is incorporated by reference in its entirety for any purpose.

[0278] In embodiments, the antisense compounds comprise modified nucleosides, modified internucleoside linkages and / or conjugate groups.

[0279] In embodiments, the antisense compound is "tricyclo-DNA (tc-DNA)," which refers to a class of constrained DNA analogues in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry for torsion angle γ. Homobasic adenine- and thymine-containing tc-DNA forms highly stable AT base pairs with complementary RNA.

[0280] Nucleosides In an embodiment, antisense compounds are provided that include linked nucleosides.In an embodiment, some or all of the nucleosides are modified nucleosides.In an embodiment, one or more nucleosides include modified nucleobases.In an embodiment, one or more nucleosides include modified sugars.Chemically modified nucleosides are routinely incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity to target RNA.

[0281] In general, a nucleobase is any group that contains one or more atoms or groups of atoms that can hydrogen bond to the base of another nucleic acid. In addition to "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for the compounds described herein. Although the terms modified nucleobase and nucleobase mimics can overlap, generally, modified nucleobases refer to nucleobases that are structurally similar to parent nucleobases, such as 7-deazapurines, 5-methylcytosine, or G-clamps, whereas nucleobase mimics include more complex structures, such as tricyclic phenoxazine nucleobase mimics. Methods for preparing the above modified nucleobases are well known to those skilled in the art.

[0282] In embodiments, the ACs provided herein include one or more nucleosides with modified sugar moieties. In embodiments, the furanosyl sugar ring of a natural nucleoside can be modified in a number of ways, including, but not limited to, the addition of a substituent, bridging two non-geminal ring atoms to form a bicyclic nucleic acid (BNA), and replacing the 4'-position ring oxygen with an atom or group such as -S-, -N(R)-, or C(R1)(R2). Modified sugar moieties are well known and can be used to alter (typically increase) the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of modified sugars includes, but is not limited to, non-bicyclic substituted sugars, particularly non-bicyclic 2'-substituted sugars having 2'-F, 2'-OCH3 or 2'-O(CH2)2-OCH3 substituents, and 4'-thio modified sugars. Sugars can also be substituted with sugar mimetic groups, among others, for example, the furanose ring can be replaced with a morpholine ring. Methods for the preparation of modified sugars are well known to those of skill in the art. Some representative patents and publications that teach the preparation of such modified sugars include U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427 ... Nos. 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,792,747, 5,700,920, and 6,600,032, and WO 2005 / 121371.

[0283] In embodiments, the nucleoside comprises a bicyclic modified sugar (BNA), such as LNA (4'-(CH2)-O-2' bridge), 2'-thio-LNA (4'-(CH2)-S-2' bridge), 2'-amino-LNA (4'-(CH2)-NR-2' bridge), ENA (4'-(CH2)2-O-2' bridge), 4'-(CH2)3-2' bridged BNA, 4'-(CH2CH(CH3))-2' bridged BNA, cEt (4'-(CH(CH3)-O-2' bridge), and cMOE BNA (4'-(CH(CHOCH3)-O-2' bridge). Certain such BNAs have been prepared and are disclosed in the patent and scientific literature (e.g., Srivastava, et al. J. Am. Chem. Soc. 2007, ACS Advanced online). publication,10.1021 / ja071106y,Albaek et al.J.Org.Chem.,2006,71,7731-7740,Fluiter,et al.Chembiochem 2005,6,1104-1109,Singh et al.,Chem.Commun.,1998,4,455-456,Koshkin et al. al., Tetrahedron,1998,54,3607-3630, Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638, Kumar et al. (See, e.g., U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,490, 7,102,111, and 6,268,490; WO 94 / 14226; WO 2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; WO 2007 / 090071). Examples of issued U.S. patents and published applications disclosing BNAs include, e.g., U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,490, 6,268,490, 6,770,748, 6,268 ... 99, 7,034,133, and 6,525,191, and U.S. Patent Publication Nos. 2004-0171570, 2004-0219565, 2004-0014959, 2003-0207841, 2004-0143114, and 20030082807.

[0284] Also provided herein are "locked nucleic acids" (LNAs) in which the 2'-hydroxyl group of a ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage to form a bicyclic sugar moiety (reviewed in Eayadi et al. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3,239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic moiety and ENA™ for an ethylene group at this position (Singh et al., Chem. Commun., 1998, 4, 455-456; ENA™: Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogues exhibit very high duplex thermal stability (Tm = +3 to +10°C) for complementary DNA and RNA, stability against 3'-exonuclease degradation, and good solubility properties. Potent, non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0285] A similarly studied isomer of LNA is alpha-L-LNA, which has been shown to have improved stability against 3'-exonucleases. Alpha-L-LNA has been incorporated into antisense gapmers and chimeras that exhibit strong antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0286] The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, as well as their oligomerization and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0287] Analogs of LNA, phosphorothioate-LNA and 2'-thio-LNA, have also been prepared (Kumar et al. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Synthesis of a novel conformationally restricted high affinity oligonucleotide analog, 2'-amino-LNA, has been described in the art (Singh et al. Chem., 1998, 63, 10035-10039). Additionally, 2'-amino and 2'-methylamino-LNA have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0288] Internucleoside bond Internucleoside linking groups that link nucleosides or otherwise modified monomer units together to form antisense compounds are described herein. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates (including phosphorodiamidates), and phosphorothioates. Representative non-phosphorus-containing internucleoside linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-), siloxanes (-O-Si(H)2-O-), and N,N'-dimethylhydrazines (-CH2-N(CH3)-N(CH3)-). Antisense compounds with non-phosphorus internucleoside linkage groups are called oligonucleosides. Modified internucleoside linkages can be used to alter (typically increase) the nuclease resistance of antisense compounds compared to natural phosphodiester linkages. Internucleoside linkages with chiral atoms can be prepared as racemic, chiral, or mixtures. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0289] In embodiments, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are generally considered to form the internucleoside backbone of the oligonucleotide. The common linkage or backbone of RNA and DNA is the 3'-5' phosphodiester bond.

[0290] Conjugate Group In embodiments, the AC is modified by the covalent attachment of one or more conjugating groups. In general, the conjugating group modifies one or more properties of the AC to which it is attached, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugating groups are routinely used in the chemical arts and are linked to a parent compound, such as an AC, directly or via an optional linking moiety or group. Conjugating groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In embodiments, the conjugating group is polyethylene glycol (PEG), and the PEG is conjugated to either the AC or the cyclic peptide.

[0291] Conjugate groups can be lipid moieties, e.g., cholesterol moieties (Letsinger et al. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0292] Linking groups or bifunctional linking moieties, such as those known in the art, can be included with the compounds provided herein. Linking groups are useful for attaching chemical functional groups, conjugate groups, reporter groups, and other groups to selective sites in a parent compound, such as, for example, AC. In embodiments, the bifunctional linking moiety includes a hydrocarbyl moiety with two functional groups. In embodiments, one of the functional groups is selected to attach to a parent molecule or compound of interest, and the other is selected to attach to essentially any selected group, such as a chemical functional group or conjugate group. Any of the linkers described herein can be used. In embodiments, the linker includes a chain structure or oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In embodiments, the bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), and the like. Some non-limiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Included are alkynyls, a non-limiting list of substituents include hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0293] In an embodiment, the AC may be linked to 10 repeats of an arginine-serine dipeptide. AC linked to 10 repeats of an arginine-serine dipeptide has been applied in vitro to artificially recruit splicing enhancer factors to induce the incorporation of BRCA1 and SMN2 exons that would otherwise be skipped. See Cartegni and Krainer 2003, incorporated herein by reference.

[0294] In embodiments, the AC may be 5-50 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, including all values ​​and ranges contained therein) nucleotides in length. In embodiments, the AC may be 5-10 nucleotides in length. In embodiments, the AC may be 10-15 nucleotides in length. In embodiments, the AC may be 15-20 nucleotides in length. In embodiments, the AC may be 20-25 nucleotides in length. In embodiments, the AC may be 25-30 nucleotides in length. In embodiments, the AC may be 30 to 35 nucleotides in length. In embodiments, the AC may be 35 to 40 nucleotides in length. In embodiments, the AC may be 40 to 45 nucleotides in length. In embodiments, the AC may be 45 to 50 nucleotides in length.

[0295] In embodiments, the AC hybridizes to a nucleic acid sequence of the human DMD gene, which encodes dystrophin. In embodiments, the AC binds to exon 45 of DMD. In embodiments, the AC that binds to exon 45 of DMD is about 18 to about 30 nucleic acids in length, for example, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleic acids in length.

[0296] In embodiments, the antisense compounds hybridize to a nucleic acid sequence within an intron of exon 45 of DMD. In embodiments, the antisense compounds hybridize to a nucleic acid sequence within exon 45 of DMD. In embodiments, the antisense compounds hybridize to a nucleic acid sequence spanning an intron-exon or exon-intron junction of exon 45 of DMD.

[0297] The antisense nomenclature system proposed in Mann et al., (2002) "Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy," J Gen Med. 4:644-654 can be used to represent target regions of gene sequences to which antisense compounds can hybridize. According to this nomenclature system, a negative sign ("-") represents an intronic sequence, and a positive sign ("+") represents an exonic sequence. The letter "A" represents that the antisense compound binds to an acceptor splice site at the beginning of an exon, and the letter "D" represents that the antisense compound binds to a donor splice site at the end of an exon. For example, A(-5+15) represents an antisense oligonucleotide that hybridizes to the last 5 bases of the intron preceding the target exon (e.g., exon 45) and the first 15 bases of the target exon. Similarly, D(+15-5) represents an antisense oligonucleotide that hybridizes to the last 5 exonic bases of a target exon (e.g., exon 45) and the first 15 intronic bases following the target exon, corresponding to the annealing site of the antisense site. An antisense oligonucleotide that hybridizes to the entire nucleic acid sequence within an exon can be represented by A(+5+25), e.g., an antisense oligonucleotide that hybridizes to the nucleic acid sequence from the first 5 nucleotide of an exon to the first 25 nucleotide of the same exon. Unless otherwise indicated, the absence of a "+" or "-" sign generally means that the antisense oligonucleotide binds to a nucleic acid sequence in an exon of a target nucleic acid. Lowercase nucleotides are used to represent intronic sequences, and uppercase nucleotides are used to represent exonic sequences.

[0298] In embodiments, the nucleic acid sequence of exon 45 of DMD is set forth as SEQ ID NO:1 (5' to 3', including adjacent upstream (5') and downstream (3') introns): [ka]

[0299] The upstream (5') intronic sequence (residues -50 to -1) and downstream (3') intronic sequence (residues -1 to -44) are shown in lower case italics, and the exonic sequence (residues +1 to +176) is shown in upper case bold and underlined. In an embodiment, the nucleic acid sequence of dystrophin exon 45 of the human Duchenne muscular dystrophy (DMD) gene comprises 176 nucleobases.

[0300] In embodiments, the AC that binds to exon 45 of DMD is selected from any one of the nucleic acid sequences set forth in Tables 6A-6P, Tables 7A-7O or Tables 8A-8C, their reverse complements, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0301] In embodiments, the AC comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to contiguous nucleotides of SEQ ID NO:1 (e.g., the AC is a 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27mer, 28mer, 29mer, or 30mer), and the first nucleotide of the AC is at position +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, or 30mer, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36 , +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, ​​+49, +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, + 99, +100, +101, +102, +103, +104, +105, +106, +107, +108, +109, +110, +111, +112, +113, +114, +115, +116, +117, +118, +119, +120, +121, +122, +123, +1 24, +125, +126, +127, +128, +129, +130, +131, +132, +133, +134, +135, +136, +137, +138, +139, +140, +141, +142, +143, +144, +145, +146, +147, +148, +1 49, +150, +151, +152, +153, +154, +155, +156, +157, +158, +159, +160, +161 , +162, +163, w+164, +165, +1616, +167, +168, +169, +170, +171, +172, +173,It hybridizes to nucleotides at +174, +175 or +176. In embodiments, the AC comprises nucleotides that are complementary to consecutive nucleotides of the 3' intronic sequence following exon 45 (3' intronic sequence not shown). As used herein, "first nucleotide" refers to the 5' nucleotide of the AC.

[0302] In an embodiment, the AC binds to a sequence of exon 45 of DMD selected from the nucleic acid sequences consisting of the sequences shown in Tables 6A-6P, Tables 7A-7O, or Tables 8A-8C. In an embodiment, the AC that binds to exon 45 of DMD is selected from any one of the nucleic acid sequences in Tables 6A-6P, Tables 7A-7O, and Tables 8A-8C, or reverse complements thereof. In an embodiment, the AC that binds to exon 45 of DMD is selected from any one of the nucleic acid sequences shown in Tables 6A-6P, Tables 7A-7O, and Tables 8A-8C, or reverse complements thereof, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto. In embodiments, the AC that binds to exon 45 of DMD comprises one or more modified nucleic acids, one or more modified internucleotide linkages, or a combination thereof. In embodiments, the AC that binds to exon 45 comprises one or more morpholine rings, one or more phosphorodiamidate linkages, or a combination thereof. In embodiments, the AC that binds to exon 45 of DMD is an antisense phosphorodiamidate morpholino oligomer (PMO) having a sequence selected from any one of the nucleic acid sequences in Tables 6A-6P, their reverse complements, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto. [Table 11] [Table 12]

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

[0303] In embodiments, the AC hybridizes to a nucleic acid spanning an intron-exon or exon-intron junction of exon 45 of DMD. In embodiments, the AC is complementary to a target nucleic acid sequence that includes at least one nucleotide of the upstream (5') intron preceding exon 45 (i.e., starting at position -1). In embodiments, the AC is complementary to a target nucleic acid sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and up to 20 consecutive nucleotides (i.e., starting from position -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2 or -1) of the upstream (5') intron preceding exon 45. In embodiments, the AC is complementary to a target nucleic acid sequence comprising at least a first nucleotide at the 5' end of exon 45 (i.e., position +1). In embodiments, the AC is complementary to a target nucleic acid sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, and up to 25 consecutive nucleotides of exon 45 starting from the first nucleotide at the 5' end of exon 45 (i.e., starting at position +1).

[0304] In embodiments, the AC hybridizes to a nucleic acid sequence spanning the intron-exon junction of exon 45 of DMD, including positions -20 to +25 of SEQ ID NO: 1. The intron-exon junction of exon 45 of DMD, including positions -20 to +25, is represented by SEQ ID NO: 2: [ka] It is expressed as:

[0305] The upstream (5') intronic sequence of SEQ ID NO:2 is shown in italics (residues -20 to -1), and the exonic sequence of SEQ ID NO:2 is shown in bold and underlined (residues +1 to +25).

[0306] In embodiments, AC comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to contiguous nucleotides of SEQ ID NO:2 (e.g., AC is a 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27mer, 28mer, 29mer, or 30mer), and the first nucleotide of AC is D It hybridizes to nucleotides at positions -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24 or +25 of SEQ ID NO:2 in exon 45 of MD or in the 5' adjacent intron of exon 45.

[0307] In an embodiment, the AC binding to exon 45 of DMD is selected from any one of the nucleic acid sequences shown in Tables 7A-7O. In an embodiment, the AC binding to exon 45 of DMD comprises one or more modified nucleic acids, one or more modified internucleotide linkages, or a combination thereof. In an embodiment, the AC binding to exon 45 of DMD comprises one or more morpholine rings, one or more phosphorodiamidate linkages, or a combination thereof. In an embodiment, the AC binding to exon 45 of DMD is an antisense phosphorodiamidate morpholino oligomer (PMO) having a sequence selected from any one of the nucleic acid sequences in Tables 7A-7O, their reverse complements, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto. [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41]

[0308] In an embodiment, the AC binding to exon 45 of DMD is selected from any one of the nucleic acid sequences shown in Tables 8A-8C. In an embodiment, the AC binding to exon 45 of DMD comprises one or more modified nucleic acids, one or more modified internucleotide linkages, or a combination thereof. In an embodiment, the AC binding to exon 45 of DMD comprises one or more morpholine rings, one or more phosphorodiamidate linkages, or a combination thereof. In an embodiment, the AC binding to exon 45 of DMD is an antisense phosphorodiamidate morpholino oligomer (PMO) having a sequence selected from any one of the nucleic acid sequences in Tables 8A-8C, their reverse complements, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto. [Table 42-1] [Table 42-2] [Table 42-3] [Table 43] [Table 44]

[0309] In embodiments, any of AC in Tables 6A-6P or Tables 7A-7O or Tables 8A-8C, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto, is selected from the group consisting of phosphorothioate (PS) nucleotides, phosphorodiamidyl nucleotides, and the like. The AC comprises at least one modified nucleotide or nucleic acid selected from phosphorodiamidate morpholino (PMO) nucleotides, locked nucleic acid (LNA), peptide nucleic acid (PNA), nucleotides containing 2'-O-methyl (2'-OMe) modified backbones, 2'O-methoxy-ethyl (2'-MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides, and 2'-deoxy-2'-fluoro-beta-D-arabino nucleic acid (2'F-ANA). In an embodiment, hybridization of the AC with the target sequence reduces or prevents splicing of exon 45. In an embodiment, the AC comprises at least one phosphorodiamidate morpholino (PMO) nucleotide. In an embodiment, each nucleotide in the AC is a phosphorodiamidate morpholino (PMO) nucleotide.

[0310] In embodiments, the compound has the following structure: [ka] wherein CPPs are cell-penetrating peptides. L is a linker, each B is independently a nucleobase complementary to a base in the target sequence; n is an integer from 1 to 50.

[0311] In embodiments, B and n together correspond to a sequence set forth in Tables 6A-6P, or Tables 7A-7O, or Tables 8A-8C, a reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto.

[0312] Cyclic cell-penetrating peptides (cCPPs) conjugated to AC A cyclic cell penetrating peptide (cCPP) can be conjugated to the AC.

[0313] The AC may be conjugated to the cCPP via a linker. The AC may comprise a therapeutic drug moiety. The therapeutic drug moiety may comprise an oligonucleotide, a peptide, or a small molecule. The oligonucleotide may comprise an antisense oligonucleotide. The AC is conjugated to the linker at the terminal carbonyl group to form the following structure: [ka] wherein EP is an exocyclic peptide, M, AA SC , AC, x', y and z' are as defined above; * AA SC x' can be 1. y can be 4. z' can be 11. -(OCH2CH-2) x’ -and / or (OCH2CH-2) z’ can be independently replaced with one or more amino acids, such as, for example, glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0314] The endosomal escape vehicle (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP) and a linker, conjugated to an AC and represented by the formula (C): [ka] or a protonated form thereof, During the ceremony, R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group; R4 and R6 are independently H or an amino acid side chain; EP is an exocyclic peptide as defined herein; AC is as defined herein; each m is independently an integer from 0 to 3; n is an integer from 0 to 2, x' is an integer from 2 to 20; y is an integer from 1 to 5; q is an integer from 1 to 4, z' is an integer from 2 to 20.

[0315] R1, R2, R3, R4, EP, AC, m, n, x', y, q, and z' are as defined herein.

[0316] The EEV can be conjugated to AC, the EEV-conjugate having the formula (Ca) or (Cb): [ka] or a protonated form thereof, where EP, m and z are as defined above in formula (C).

[0317] The EEV can be conjugated to AC, the EEV-conjugate having the formula (Cc): [ka] or a protonated form thereof, 1 , R 2 , R 3 , R 4and m are as defined above in formula (III), AA can be an amino acid as defined herein, n can be an integer from 0 to 2, x can be an integer from 1 to 10, y can be an integer from 1 to 5, and z can be an integer from 1 to 10.

[0318] The EEV can be conjugated to an AC, and the EEV-oligonucleotide conjugate has the formula (C-1), (C-2), (C-3), or (C-4): [ka] [ka] The structure may include:

[0319] In the above formula, EP is an exocyclic peptide and AC may have a sequence of 15-30 nucleic acids that is complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence. In embodiments, AC may be selected from an oligonucleotide set forth in Tables 6A-6P, Tables 7A-7O, and Tables 8A-8C, a reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto.

[0320] In embodiments, the compounds described herein form multimers. In embodiments, multimerization occurs through non-covalent interactions, for example, hydrophobic interactions, ionic interactions, hydrogen bonds, or dipole-dipole interactions. In embodiments, the compounds form dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, or nonamers. In embodiments, the compounds include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cyclic peptides. In embodiments, the compounds include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ACs. In embodiments, the compounds include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 EPs. In embodiments, the compounds include 1-10 cyclic peptides and 1-10 ACs. In embodiments, the compounds include 1-10 cyclic peptides, 1-10 ACs, or 1-10 EPs. Includes.

[0321] In embodiments, the compounds of the present disclosure include any one of the following structures: The following compounds are exemplary only, and any one of the cyclic peptides, linkers, and ACs in any of the following structures may be replaced with any one of the cyclic peptides, linkers, or ACs described herein. [ka] [ka] [ka]

[0322] Cytoplasmic delivery efficiency Modifications to cyclic cell penetrating peptides (cCPPs) can improve cytoplasmic delivery efficiency. Improved cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of a cCPP having a modified sequence with a control sequence that does not contain a particular substituted amino acid residue (such as, but not limited to, arginine, phenylalanine, and / or glycine) in the modified sequence but is otherwise identical.

[0323] In embodiments, the compound that comprises cyclic peptide and AC has improved cytoplasmic uptake efficiency compared to the compound that comprises AC alone.Cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of the compound that comprises cyclic peptide and AC with the cytoplasmic delivery efficiency of AC alone.

[0324] As used herein, cytoplasmic delivery efficiency refers to the ability of cCPP to cross cell membrane and enter cell cytoplasm.The cytoplasmic delivery efficiency of cCPP does not necessarily depend on receptor or cell type.Cytoplasmic delivery efficiency may refer to absolute cytoplasmic delivery efficiency or relative cytoplasmic delivery efficiency.

[0325] Absolute cytoplasmic delivery efficiency is the ratio of the cytosolic concentration of cCPP (or cCPP-AC conjugate) to the concentration of cCPP (or cCPP-AC conjugate) in growth medium. Relative cytoplasmic delivery efficiency refers to the concentration of cCPP in the cytosol compared to the concentration of control cCPP in the cytosol. Quantification can be achieved by fluorescently labeling cCPP (e.g., with FITC dye) and measuring fluorescence intensity using techniques well known in the art.

[0326] Relative cytoplasmic delivery efficiency is determined by comparing (i) the amount of the cCPP of the present invention internalized by a cell type (e.g., HeLa cells) with (ii) the amount of a control cCPP internalized by the same cell type. To measure relative cytoplasmic delivery efficiency, the cell type may be incubated in the presence of the cCPP for a certain period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), and then the amount of the cCPP internalized by the cell is quantified using methods known in the art, such as fluorescence microscopy. Separately, the same concentration of the control cCPP is incubated in the presence of the cell type for the same period of time, and the amount of the control cCPP internalized by the cell is quantified.

[0327] Relative cytoplasmic delivery efficiency is the IC of cCPPs with modified sequences for intracellular targets 50Measure the IC of cCPP with modified sequence 50 can be determined by comparing to a control sequence (as described herein).

[0328] The relative cytoplasmic delivery efficiency of cCPPs compared to cyclo(FfΦRrRrQ) ranges from about 50% to about 450%, e.g., about 60%, about 70%, about 80%, about 90%, about 100%, 200%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 600%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about 770%, about 780%, about 790%, about 800%, about 810%, about 820%, %, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580% or about 590% (including all values ​​and subranges therebetween). The relative cytoplasmic delivery efficiency of the cCPP may be improved by more than about 600% compared to a cyclic peptide comprising cyclo(FfΦRrRrQ).

[0329] The absolute cytoplasmic delivery efficiency is from about 40% to about 100%, e.g., about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% (including all values ​​and subranges therebetween).

[0330] The cCPPs of the disclosure provide an increased cytoplasmic delivery efficiency of about 1.1 fold to about 30 fold, e.g., about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2.0 fold, about 2.5 fold, about 3.0 fold, about 3.5 fold, about 4.0 fold, about 4.5 fold, about 5.0 fold, about 5.5 fold, about 6.0 fold, about 6.5 fold, about 7.0 fold, about 7.5 fold, about 8.0 fold, about 8.5 fold, about 9.0 fold, about 10 fold, about 10.5 fold, about 11.0 fold, about 11.5 fold, about 12.0 fold, about 12.5 fold, about 13.0 fold, about 13.5 fold, about 14. The improvement may be 0 fold, about 14.5 fold, about 15.0 fold, about 15.5 fold, about 16.0 fold, about 16.5 fold, about 17.0 fold, about 17.5 fold, about 18.0 fold, about 18.5 fold, about 19.0 fold, about 19.5 fold, about 20 fold, about 20.5 fold, about 21.0 fold, about 21.5 fold, about 22.0 fold, about 22.5 fold, about 23.0 fold, about 23.5 fold, about 24.0 fold, about 24.5 fold, about 25.0 fold, about 25.5 fold, about 26.0 fold, about 26.5 fold, about 27.0 fold, about 27.5 fold, about 28.0 fold, about 28.5 fold, about 29.0 fold, or about 29.5 fold (including all values ​​and subranges therebetween).

[0331] Re-spliced ​​target protein A "target protein" is an amino acid sequence resulting from transcription and translation of a target gene. A "re-spliced ​​target protein" as used herein refers to a protein encoded as a result of binding of an AC to a target pre-mRNA transcribed from a target gene. A "wild-type target protein" refers to a naturally occurring correctly translated protein isomer resulting from proper splicing of a target pre-mRNA encoded by a wild-type target gene. The compounds and methods of the present invention may result in a re-spliced ​​target protein containing one or more amino acid substitutions, deletions and / or insertions compared to the wild-type target protein. In embodiments, the re-spliced ​​target protein retains some wild-type target protein activity. In embodiments, the re-spliced ​​target protein produced by administration of a compound of the present invention is homologous to the wild-type target protein. In embodiments, the re-spliced ​​target protein has an amino acid sequence that is at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, and up to 100% identical to the wild-type target protein. In embodiments, the re-spliced ​​target protein is substantially identical to the wild-type target protein. In embodiments, the amino acid sequence of the re-spliced ​​target protein is at least 50% identical to the amino acid sequence of the wild-type target protein. In embodiments, the amino acid sequence of the re-spliced ​​target protein is at least 75% identical to the amino acid sequence of the wild-type target protein. In embodiments, the amino acid sequence of the re-spliced ​​target protein is at least 90% identical to the amino acid sequence of the wild-type target protein. In embodiments, the re-spliced ​​target protein is a truncated form of the wild-type target protein.

[0332] In embodiments, the re-spliced ​​target protein can rescue one or more phenotypes or symptoms of a disease associated with the transcription and translation of the target gene. In embodiments, the re-spliced ​​target protein can rescue one or more phenotypes or symptoms of a disease associated with the expression of the target protein. In embodiments, the re-spliced ​​target protein is an active fragment of the wild-type target protein. In embodiments, the re-spliced ​​target protein functions in a manner substantially similar to the wild-type target protein. In embodiments, the re-spliced ​​target protein allows a cell to function substantially similar to a similar cell expressing the wild-type target protein. In embodiments, the re-spliced ​​target protein does not cure a disease associated with the target gene or target protein, but improves one or more symptoms of the disease. In embodiments, the re-spliced ​​target protein results in an improvement in target protein function of at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 205, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%, and up to about 100%.

[0333] In embodiments, the re-spliced ​​target protein may have an amino acid sequence that is reduced by about one or more amino acids from the size of the wild-type target protein, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175 or about 180 or more amino acids.

[0334] In embodiments, the re-spliced ​​target protein may have one or more improved properties compared to the target protein. In embodiments, the re-spliced ​​target protein may have one or more improved properties compared to the wild-type target protein. In embodiments, the activity or stability of the enzyme may be enhanced by promoting the differential splicing of the target pre-mRNA. In embodiments, the re-spliced ​​target protein may have a sequence that is identical or substantially similar to a wild-type target protein isomer that has improved properties compared to another wild-type target protein isomer.

[0335] In embodiments, one or more properties of the target protein are either absent (eliminated) or reduced in the re-spliced ​​target protein. In embodiments, one or more properties of the wild-type target protein are either absent (eliminated) or reduced in the re-spliced ​​target protein. Non-limiting examples of properties that may be reduced or eliminated include immunogenic, angiogenic, thrombogenic, aggregatory, and ligand binding properties.

[0336] In embodiments, the re-spliced ​​target protein contains one or more amino acid substitutions compared to the wild-type target protein. In embodiments, the substitutions can be conservative or non-conservative. Examples of conservative amino acid substitutions include replacing one amino acid with another amino acid from one of the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). In embodiments, structurally similar amino acids are replaced to reverse the charge of the residue (e.g., glutamic acid is replaced with glutamine, or vice versa, asparagine is replaced with aspartic acid, or vice versa). In embodiments, phenylalanine is replaced with tyrosine, or vice versa. Other non-limiting examples of amino acid substitutions are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0337] In embodiments, the re-spliced ​​target protein may comprise substitutions, deletions and / or insertions at one or more (e.g., several) positions compared to the wild-type target protein, In embodiments, the number of amino acid substitutions, deletions and / or insertions in the amino acid sequence of the re-spliced ​​target protein is 200 or less, 150 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0338] Treatment methodIn embodiments, an AC of the present disclosure is administered to a patient diagnosed with Duchenne muscular dystrophy (DMD) at a dose of about 0.1 mg / kg to about 1000 mg / kg, for example, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, including all values ​​and ranges included therein and therebetween. , about 9mg / kg, about 10mg / kg, about 11mg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, about 20mg / kg, about 21mg / kg, about 22mg / k g, about 23 mg / kg, about 24 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 30 mg / kg, about 31 mg / kg, about 32 mg / kg, about 33 mg / kg, about 34 mg / kg, about 35 mg / kg, about 36 mg / kg kg, approximately 37mg / kg, approximately 38mg / kg, approximately 39mg / kg, approximately 40mg / kg, approximately 41mg / kg, approximately 42mg / kg, approximately 43mg / kg, approximately 44mg / kg, approximately 45mg / kg, approximately 46mg / kg, approximately 47mg / kg, approximately 48mg / kg, approximately 49mg / kg, approximately 50m g / kg, about 51mg / kg, about 52mg / kg, about 53mg / kg, about 54mg / kg, about 55mg / kg, about 56mg / kg, about 57mg / kg, about 58mg / kg, about 59mg / kg, about 60mg / kg, about 61mg / kg, about 62mg / kg, about 63mg / kg, about 64 mg / kg, about 65 mg / kg, about 66 mg / kg, about 67 mg / kg, about 68 mg / kg, about 69 mg / kg, about 70 mg / kg, about 71 mg / kg, about 72 mg / kg, about 73 mg / kg, about 74 mg / kg, about 75 mg / kg, about 76 mg / kg, about 77 mg / kg, about 78mg / kg, about 79mg / kg, about 80mg / kg, about 81mg / kg, about 82mg / kg, about 83mg / kg, about 84mg / kg, about 85mg / kg, about 86mg / kg, about 87mg / kg, about 88mg / kg, about 89mg / kg, about 90mg / kg, about 91mg / kg,About 92mg / kg, about 93mg / kg, about 94mg / kg, about 95mg / kg, about 96mg / kg, about 97mg / kg, about 98mg / kg, about 99mg / kg, about 100mg / kg, about 110mg / kg, about 120mg / kg, about 130mg / kg, about 140mg / kg, about 1 50mg / kg, approximately 160mg / kg, approximately 170mg / kg, approximately 180mg / kg, approximately 190mg / kg, approximately 200mg / kg, approximately 210mg / kg, approximately 220mg / kg, approximately 230mg / kg, approximately 240mg / kg, approximately 250mg / kg, approximately 260mg / kg, approximately 270mg / kg, about 280mg / kg, about 290mg / kg, about 300mg / kg, about 310mg / kg, about 320mg / kg, about 330mg / kg, about 340mg / kg, about 350mg / kg, about 360mg / kg, about 370mg / kg, about 380mg / kg, about 390mg / kg, About 400mg / kg, about 410mg / kg, about 420mg / kg, about 430mg / kg, about 440mg / kg, about 450mg / kg, about 460mg / kg, about 470mg / kg, about 480mg / kg, about 490mg / kg, about 500mg / kg, about 510mg / kg, about 520 mg / kg, approximately 530 mg / kg, approximately 540 mg / kg, approximately 550 mg / kg, approximately 560 mg / kg, approximately 570 mg / kg, approximately 580 mg / kg, approximately 590 mg / kg, approximately 600 mg / kg, approximately 610 mg / kg, approximately 620 mg / kg, approximately 630 mg / kg, approximately 640 mg / k g, about 650mg / kg, about 660mg / kg, about 670mg / kg, about 680mg / kg, about 690mg / kg, about 700mg / kg, about 710mg / kg, about 720mg / kg, about 730mg / kg, about 740mg / kg, about 750mg / kg, about 760mg / kg, about 7 The compound is administered at a dose of about 70 mg / kg, about 780 mg / kg, about 790 mg / kg, about 800 mg / kg, about 810 mg / kg, about 820 mg / kg, about 830 mg / kg, about 840 mg / kg, about 850 mg / kg, about 860 mg / kg, about 870 mg / kg, about 880 mg / kg, about 890 mg / kg, about 900 mg / kg, about 910 mg / kg, about 920 mg / kg, about 930 mg / kg, about 940 mg / kg, about 950 mg / kg, about 960 mg / kg, about 970 mg / kg, about 980 mg / kg, about 990 mg / kg or about 1000 mg / kg.

[0339] The present disclosure provides a method for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, comprising administering a compound disclosed herein. In an embodiment, the target gene is DMD. In an embodiment, the target sequence comprises at least a portion of exon 44 of DMD, at least a portion of the 3' intron adjacent to exon 44 of DMD, at least a portion of the 5' intron adjacent to exon 44 of DMD, or a combination thereof.

[0340] In various embodiments, treatment refers to the partial or complete alleviation, amelioration, alleviation, suppression, delay in onset, reduction in severity and / or incidence of one or more symptoms in a subject.

[0341] In embodiments, there is provided a method of altering expression of a target gene in a subject in need thereof, comprising administering a compound disclosed herein. In embodiments, the treatment results in decreased expression of the target protein. In embodiments, the treatment results in expression of a re-spliced ​​target protein. In embodiments, the treatment results in preferential expression of a wild-type target protein isomer.

[0342] In embodiments, as a result of treatment according to the present disclosure, expression of a target protein in a subject is reduced by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, as compared to the average level of the target protein in the subject prior to treatment or in one or more untreated control individuals having a similar disease. In embodiments, as a result of treatment according to the present disclosure, expression of the re-spliced ​​target protein in a subject is increased by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the target protein in the subject prior to treatment or in one or more untreated control individuals having a similar disease. In embodiments, as a result of treatment according to the present disclosure, expression of a wild-type target protein isomer in a subject is increased or decreased by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, as compared to the average level of the target protein in the subject prior to treatment or in one or more untreated control individuals having a similar disease.

[0343] The terms "improve," "increase," "reduce," "decrease," and similar terms, as used herein, refer to values ​​relative to a control. In embodiments, a suitable control is a baseline measurement, e.g., a measurement in the same individual before starting a treatment described herein, or a measurement in a control individual (or control individuals) in the absence of a treatment described herein. A "control individual" is an individual suffering from a disease who is approximately the same age and / or sex as the individual being treated (to ensure that the stage of the disease is similar in the treated and control individual(s)).

[0344] The individual to be treated (also referred to as a "patient" or "subject") is an individual (fetal, infant, child, adolescent, or adult human) who has a disease or has the potential to develop a disease. The individual may have a disease mediated by aberrant gene expression or aberrant gene splicing. In various embodiments, the individual with the disease may have a wild-type target protein expression or activity level that is about 1% to 99% of the normal protein expression or activity level in an individual not suffering from the disease. In embodiments, such ranges include, but are not limited to, about 80-99%, about 65-80%, about 50-65%, about 30-50%, about 25-30%, about 20-25%, about 15-20%, about 10-15%, about 5-10%, or about 1-5% of the normal thymidine phosphorylase expression or activity level. In embodiments, the individual may have a target protein expression or activity level that is about 1% to about 500% higher than the normal wild-type target protein expression or activity level. In embodiments, the ranges include, but are not limited to, about 1-10%, about 10-50%, about 50-100%, about 100-200%, about 200-300%, about 300-400%, about 400-500%, or about 500-1000% higher target protein expression or activity levels.

[0345] In an embodiment, the individual is one who has recently been diagnosed with the disease. Typically, early treatment (treatment initiated as soon as possible after diagnosis) is important to minimize the effects of the disease and maximize the benefits of treatment.

[0346] In embodiments, the efficacy of the disclosed compounds and ACs against DMD is evaluated in animal models of DMD. Animal models are valuable resources for studying disease pathogenesis and provide a means to test dystrophin-related activity. In embodiments, compounds of the present disclosure are evaluated using mdx mice and golden retriever muscular dystrophy (GRMD) dogs, both of which are dystrophin-negative (see, e.g., Collins & Morgan, Int J Exp Pathol 84:165-172, 2003). In embodiments, compounds of the present disclosure are evaluated using C57BL / 10ScSn-Dmdmdx / J (Bl10 / mdx), or D2.B10-Dmdmdx / J (D2 / mdx) mouse models. In embodiments, compounds of the present disclosure are evaluated using transgenic mice carrying the human DMD gene and lacking the mouse Dmd gene (hDMD / Dmd null mice). This mouse can be generated by crossing male hDMD mice (available from Jackson Laboratory, Bar Harbor, ME) with female DMD null mice. Each of the following references describes these models and is incorporated by reference in its entirety: J Neuromuscul Dis. 2018; 5(4): 407-417., Proc Natl Acad Sci US A. 1984; 81(4): 1189-92., Am J Pathol. 2010; 176(5): 2414-24., J Clin Invest. 2009; 119(12): 3703-12, International Publication No. WO2019014772. These and other animal models can be used to measure the functional activity of various dystrophin proteins.

[0347] In an embodiment, an in vitro model is used to evaluate the effectiveness of the composition of the present disclosure.In an embodiment, the in vitro model is an immortalized muscle cell model.This model is described in the following paper, which is incorporated herein by reference in its entirety: Nguyen et al.J Pers Med.2017 Dec;7(4):13.

[0348] Manufacturing method The compounds described herein can be prepared by various methods known to those skilled in the art of organic synthesis, or variations thereof as will be understood by those skilled in the art.The compounds described herein can be prepared from readily available starting materials.Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0349] Modification of the compounds described herein includes the addition, removal, or movement of various components as described for each compound. Similarly, the chirality of the molecule can be changed if one or more chiral centers are present in the molecule. Furthermore, synthesis of the compounds can include protection and deprotection of various chemical groups. Those skilled in the art can determine the use of protection and deprotection, and the selection of appropriate protecting groups. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0350] Starting materials and reagents used in preparing the compounds and compositions of the disclosure are available from Aldrich Chemical Corporation (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Abercrombie & Fitch (New York, NY), and other companies. These compounds are available from commercial suppliers such as Eppendorf Ingelheim (Ingelheim, Germany), AstraZeneca (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering-Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or may be purchased from Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989) according to procedures known to those skilled in the art.Other materials disclosed herein, such as the pharmaceutical carriers, can be obtained from commercial sources.

[0351] The reactions to produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions, i.e., temperature and pressure, at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of two or more solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) It can be monitored by infrared spectroscopy, spectrophotometry (eg, UV-visible), or mass spectrometry, or by chromatography, for example, high performance liquid chromatography (HPLC) or thin layer chromatography.

[0352] The compounds of the present disclosure can be prepared by solid-phase peptide synthesis in which the amino acid α-N-terminus is protected by an acid or base protecting group. Such a protecting group should have the property of being stable to the conditions of peptide bond formation while being easily removable without disruption of the growing peptide chain or racemization of any of the chiral centers contained therein. Suitable protecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the compounds of the present disclosure. Other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc, and adamantyloxycarbonyl for side chain amino groups such as lysine and arginine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl, and acetyl (Ac) for tyrosine, t-butyl, benzyl, and tetrahydropyranyl for serine, trityl, benzyl, Cbz, p-toluenesulfonyl, and 2,4-dinitrophenyl for histidine, formyl for tryptophan, benzyl and t-butyl for aspartic acid and glutamic acid, and triphenylmethyl (trityl) for cysteine. In solid phase peptide synthesis, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions and are insoluble in the media used.The solid support for the synthesis of α-C-terminal carboxypeptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City, CA). The α-C-terminal amino acid is coupled to the resin by mediated coupling using N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl) in a solvent such as dichloromethane or DMF at a temperature between 10°C and 50°C for about 1 to about 24 hours. When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, before coupling with the α-C-terminal amino acid as described above. One method for coupling to the deprotected 4(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. The coupling of successive protected amino acids can be carried out in an automated polypeptide synthesizer. In one example, the α-N-terminus of the amino acid of the growing peptide chain is protected with Fmoc. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is achieved by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about a 3-fold molar excess and the coupling is preferably carried out in DMF.The coupling agents can be O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either consecutively or in a single operation. Removal and deprotection of the polypeptide can be achieved in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thioanisole, water, ethanedithiol and trifluoroacetic acid. If the α-C-terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification, for example with methanol, followed by aminolysis or direct transamidation. The protected peptide can be purified at this point or taken directly to the next step. Removal of the side chain protecting groups can be achieved using the cleavage cocktail described above. The fully deprotected peptide can be purified by a series of chromatographic steps using any or all of the following types: Ion exchange with weakly basic resins (acetate form), hydrophobic adsorption chromatography with underivatized polystyrene-divinylbenzene (e.g., Amberlite XAD), silica gel adsorption chromatography, ion exchange chromatography with carboxymethylcellulose, partition chromatography with, e.g., Sephadex G-25, LH-20 or countercurrent distribution, high performance liquid chromatography (HPLC), especially reversed-phase HPLC with octyl- or octadecylsilyl-silica bonded phase column packings.

[0353] The polymer, e.g., PEG group, can be attached to AC under any suitable condition used to react a protein with an activated polymer molecule. Any means known in the art can be used, including acylation of a reactive group on the PEG moiety (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimide or hydrazino group) to a reactive group on the AC (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimide or hydrazino group), reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation / ligation methods. Activation groups that can be used to link a water-soluble polymer to one or more proteins include, but are not limited to, sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, aziridine, oxirane, 5-pyridyl, and alpha-halogenated acyl groups (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). The polymer selected for attachment to AC by reductive alkylation should have a single reactive aldehyde so that the degree of polymerization can be controlled. See, e.g., Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002), Roberts et al., Adv. Drug Delivery Rev. 54: 459-476 (2002), and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).

[0354] To directly covalently link an AC to a CPP, the appropriate amino acid residue of the CPP may be reacted with an organic derivatizing agent capable of reacting with selected side chains or the N- or C-terminus of an amino acid. Reactive groups on the peptide or conjugate moiety include, for example, aldehyde, amino, ester, thiol, α-haloacetyl, maleimide or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, or other agents known in the art.

[0355] Methods for synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited to the method for synthesizing AC. In embodiments, compounds having reactive phosphorus groups useful for forming internucleoside linkages, including, for example, phosphodiester and phosphorothioate internucleoside linkages, are provided herein. Methods for preparing and / or purifying precursors or antisense compounds are not limitations of the compositions or methods provided herein. Methods for synthesizing and purifying DNA, RNA and antisense compounds are well known to those skilled in the art.

[0356] Oligomerization of modified and unmodified nucleosides can be routinely carried out following literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and / or RNA (Scaringe, Methods (2001), 23, 206-217; Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36; Gallo et al., Tetrahedron (2001), 57, 5707-5713).

[0357] The antisense compounds provided herein can be easily and routinely made by the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several suppliers, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may also or alternatively be adopted. It is well known to use similar techniques to prepare oligonucleotides, such as phosphorothioates and alkylated derivatives. The present invention is not limited by the method of antisense compound synthesis.

[0358] Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray mass spectrometry. Such synthesis and analysis methods can be carried out in multi-well plates. The method of the present invention is not limited by the method of oligomer purification.

[0359] Method of administration The in vivo application of the compounds of the present disclosure and compositions containing them can be achieved by any suitable method and technique known to those skilled in the art now or in the future.For example, the compounds of the present disclosure can be formulated in a physiologically or pharma-ceutically acceptable form and administered via any suitable route known in the art, such as oral and parenteral administration routes.As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal and intrathecal administration, such as by injection.The administration of the compounds or compositions of the present disclosure can be a single administration, or can be continuous or at different intervals, as can be easily determined by those skilled in the art.

[0360] The compounds disclosed herein and compositions containing them can also be administered using liposome technology, time-release capsules, implantable pumps and biodegradable containers.These delivery methods can advantageously provide a uniform dosage over a long period of time.The compounds can also be administered in their salt derivative form or crystalline form.

[0361] The compounds disclosed herein can be formulated according to known methods for preparing pharma-ceutically acceptable compositions. Formulations are well known and described in detail in several sources readily accessible to those skilled in the art. For example, Remington's Pharmaceutical Science by EW Martin (1995) describes formulations that can be used in connection with the methods of the present disclosure. In general, the compounds disclosed herein can be formulated so that an effective amount of the compound is combined with a suitable carrier to facilitate effective administration of the compound. The compositions used can also be in various forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharma-ceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To enable administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein may advantageously contain a total amount of one or more subject compounds of about 0.1% to 100% by weight, based on the weight of the entire composition, including any carriers or diluents.

[0362] Suitable formulations for administration include aqueous sterile injection solutions that may contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition that only needs to be reconstituted with a sterile liquid carrier (e.g., water for injection) before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, and the like. It is to be understood that in addition to the ingredients specifically mentioned above, the compositions disclosed herein may contain other agents standard in the art having regard to the type of formulation in question.

[0363] The compounds disclosed herein and compositions comprising them can be delivered to cells either through direct contact with the cells or through a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety. Another means for delivering compounds and compositions disclosed herein to cells includes binding the compounds to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Patent No. 6,960,648 and U.S. Patent Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be bound to another composition and allow the composition to translocate across biological membranes. U.S. Patent Application Publication No. 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. Compounds can also be incorporated into polymers, examples of which include poly(DL lactide-co-glycolide) polymer for intracranial tumors, poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (used in GLIADEL), chondroitin, chitin, and chitosan.

[0364] The compounds and compositions disclosed herein, including their pharma- ceutically acceptable salts or prodrugs, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of active agents or their salts can be prepared in water, optionally mixed with non-toxic surfactants. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations may contain preservatives to prevent the growth of microorganisms.

[0365] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, and are optionally encapsulated in liposomes. The final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Optionally, the prevention of microbial activity can be brought about by various other antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0366] Sterile injectable solutions are prepared by incorporating the compounds and / or agents disclosed herein in the required amounts in a suitable solvent with various other ingredients listed above, and then sterilizing by filtration as required. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients present in the previously sterile-filtered solution.

[0367] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known in the art.

[0368] The dosage range for administration of the composition is sufficient to produce the desired effect of acting on the condition or disorder. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. In general, the dosage varies according to the age, condition, sex, and extent of the disease of the patient, and can be determined by those skilled in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. The dosage can vary and can be administered in one or more doses per day for one or several days.

[0369] Also disclosed are pharmaceutical compositions comprising the compounds disclosed herein in combination with pharma- ceutically acceptable carriers. Pharmaceutical compositions comprising an amount of compounds and adapted for oral, topical or parenteral administration constitute preferred embodiments. The dose administered to a patient, particularly a human, must be sufficient to achieve a therapeutic response in the patient over a reasonable time frame without lethal toxicity, and preferably without causing unacceptable levels of side effects or morbidity. Those skilled in the art will understand that the dosage depends on various factors, such as the subject's condition (health), the subject's weight, the type of concomitant treatment, if any, the frequency of treatment, the therapeutic ratio, and the severity and stage of pathological condition.

[0370] Also disclosed are kits comprising a compound disclosed herein in one or more containers. The kits of the present disclosure may optionally comprise a pharma- ceutically acceptable carrier and / or diluent. In one embodiment, the kit comprises one or more other components, auxiliary agents, or adjuvants described herein. In another embodiment, the kit comprises one or more anti-cancer agents, such as agents described herein. In one embodiment, the kit comprises instructions or packaging material describing how to administer the compound or composition of the kit. The containers of the kit may be made of any suitable material, e.g., glass, plastic, metal, etc., and are of any suitable size, shape, or configuration. In one embodiment, the compound and / or agent disclosed herein is provided in the kit in solid form, such as in tablet, pill, or powder form. In another embodiment, the compound and / or agent disclosed herein is provided in the kit in liquid or solution form. In one embodiment, the kit comprises an ampoule or syringe containing the compound and / or agent disclosed herein in liquid or solution form.

[0371] Although several embodiments of the invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0372] Specific Definitions As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an agent" includes mixtures of two or more such agents, reference to "the component" includes mixtures of two or more such components, etc.

[0373] The term "about" when it precedes a numerical value means a range (e.g., plus or minus 10% of the value). For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc., unless the context of this disclosure indicates otherwise or is inconsistent with such an interpretation. For example, in a list of numerical values ​​such as "about 49, about 50, about 55, ...", "about 50" means a range that spans less than half the interval between the preceding value and the following value, e.g., greater than 49.5 to less than 52.5. Furthermore, phrases less than "about" a value or greater than "about" a value should be understood in light of the definition of the term "about" provided herein. Similarly, the term "about" when preceding a series of numerical values ​​or a range of values ​​(e.g., "about 10, 20, 30" or "about 10 to 30") refer to all of the values ​​in the series or to the end points of the range, respectively.

[0374] The terms "miniPEG," "PEG2," and "AEEA" are used interchangeably herein to refer to 2-[2-[2-aminoethoxy]ethoxy]acetic acid.

[0375] As used herein, the term "cyclic cell-penetrating peptide" or "cCPP" refers to a peptide that facilitates the delivery of an AC into a cell.

[0376] As used herein, the term "endosomal escape vehicle" (EEV) refers to a cCPP conjugated to a linker and / or an exocyclic peptide (EP) by a chemical bond (i.e., a covalent bond or a non-covalent interaction). The EEV can be an EEV of formula (B):

[0377] As used herein, the term "EEV conjugate" refers to an endosomal escape vehicle as defined herein conjugated to an AC by chemical bond (i.e., covalent bond or non-covalent interaction). The AC can be delivered into a cell by the EEV. The EEV-conjugate can be an EEV-conjugate of formula (C).

[0378] As used herein, the terms "exocyclic peptide" (EP) and "modulatory peptide" (MP) may be used interchangeably to refer to two or more amino acid residues linked by a peptide bond that may be conjugated to a cyclic cell-penetrating peptide (cCPP) disclosed herein. The EP, when conjugated to a cyclic peptide disclosed herein, may alter the tissue distribution and / or retention of the compound. Typically, the EP contains at least one positively charged amino acid residue, e.g., at least one lysine residue and / or at least one arginine residue. Non-limiting examples of EPs are described herein. The EP may be a peptide identified in the art as a "nuclear localization sequence" (NLS). Non-limiting examples of nuclear localization sequences include the nuclear localization sequence of the large T antigen of the SV40 virus, the minimal functional unit of which is the seven amino acid sequence PKKKRKV, the nucleoplasmin bipartite NLS having the sequence NLSKRPAAIKKAGQAKKKK, the c-myc nuclear localization sequence having the amino acid sequence PAAKRVKLD or RQRRNELKRSF, the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha, the sequences VSRKRPRP and PPKKARED of the sarcoma T protein, the sequence PQPKKKPL of human p53, the sequence PQPKKKPL of mouse c-abl Examples of NLS include the sequence SALIKKKKKMAP of IV, the sequences DRLRR and PKQKKRK of influenza virus NS1, the sequence RKLKKKIKKL of hepatitis virus delta antigen and the sequence REKKKFLKRR of mouse MxI protein, the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase, and the sequence RKCLQAGMNLEARKTKK of steroid hormone receptor (human). Additional examples of NLSs are described in WO 2001 / 038547, which is incorporated herein by reference in its entirety.

[0379] As used herein, "linker" or "L" refers to a moiety that covalently attaches one or more moieties (e.g., an exocyclic peptide (EP) and an AC) to a cyclic cell-penetrating peptide (cCPP). The linker may comprise a natural or unnatural amino acid or polypeptide. The linker may be a synthetic compound that contains two or more suitable functional groups suitable for attaching the cCPP to the AC, thereby forming a compound disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. The cCPP may be covalently attached to the AC via the linker.

[0380] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides of an oligonucleotide can be modified. An oligonucleotide can comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). An oligonucleotide can be composed of natural and / or modified nucleobases, sugars and covalent internucleoside linkages, and can further comprise non-nucleic acid conjugates.

[0381] The terms "peptide", "protein" and "polypeptide" are used interchangeably to refer to natural or synthetic molecules that contain two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of the other amino acid. Two or more amino acid residues may be linked by the carboxyl group of one amino acid to the alpha amino group. Two or more amino acids of a polypeptide may be linked by peptide bonds. A polypeptide may contain peptide backbone modifications in which two or more amino acids are covalently linked by bonds other than peptide bonds. A polypeptide may contain one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide. The term polypeptide includes naturally occurring amino acids and artificially occurring amino acids. The term polypeptide includes peptides that contain, for example, about 2 to about 100 amino acid residues, as well as proteins, such as therapeutic proteins, including, for example, but not limited to, antibodies, enzymes, receptors, soluble proteins, and the like.

[0382] The term "therapeutic polypeptide" refers to a polypeptide having therapeutic, prophylactic or other biological activity. A therapeutic polypeptide can be produced in any suitable manner. For example, a therapeutic polypeptide can be isolated or purified from a naturally occurring environment, can be chemically synthesized, can be recombinantly produced, or a combination thereof.

[0383] The term "small molecule" refers to an organic compound having pharmacological activity and a molecular weight of less than about 2000 daltons, or less than about 1000 daltons, or less than about 500 daltons. Small molecule therapeutic agents are typically produced by chemical synthesis.

[0384] As used herein, the term "adjacent" refers to two amino acids that are covalently connected. For example, [ka] In the context of a representative cyclic cell penetrating peptide (cCPP) such as, AA1 / AA2, AA2 / AA3, AA3 / AA4, and AA5 / AA1 exemplify pairs of adjacent amino acids.

[0385] A residue of a chemical species, as used herein, refers to a derivative of a chemical species present in a particular product. To form a product, at least one atom of the chemical species is replaced by a bond to another moiety such that the product contains a derivative or residue of the chemical species. For example, the cyclic cell-penetrating peptides (cCPPs) described herein have an amino acid (e.g., arginine) incorporated therein through the formation of one or more peptide bonds. An amino acid incorporated into a cCPP may be referred to as a residue, or simply an amino acid. Thus, an arginine or arginine residue may be: [ka] Refers to...

[0386] The term "protonated form thereof" refers to the protonated form of an amino acid. For example, the guanidine group on the side chain of arginine can be protonated to form a guanidinium group. The structure of the protonated form of arginine is: [ka] It is.

[0387] As used herein, the term "chirality" refers to a molecule having two or more stereoisomers that differ in the three-dimensional spatial arrangement of atoms, where one stereoisomer is a non-superimposable mirror image of the other. Amino acids, except for glycine, have a chiral carbon atom adjacent to the carboxyl group. The term "enantiomer" refers to a stereoisomer that is chiral. A chiral molecule can be an amino acid residue that has a "D" and an "L" enantiomer. Molecules that do not have a chiral center, such as glycine, may be referred to as "achiral."

[0388] As used herein, the term "hydrophobic" refers to a moiety that does not dissolve in water or has minimal solubility in water. In general, neutral and / or non-polar moieties, or moieties that are predominantly neutral and / or non-polar, are hydrophobic. Hydrophobicity can be measured using one of the methods disclosed herein below.

[0389] As used herein, "aromatic" refers to an unsaturated cyclic molecule having 4n+2 pi-electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated cyclic molecule not included in the definition of aromatic.

[0390] "Alkyl", "alkyl chain" or "alkyl group" refers to a fully saturated, straight or branched hydrocarbon chain radical having from 1 to 40 carbon atoms and attached to the remainder of the molecule by a single bond. Alkyl containing any number of carbon atoms from 1 to 40 is included. Alkyl containing up to 40 carbon atoms is C1-C 40 Alkyl, alkyl containing up to 10 carbon atoms is C1-C 10 Alkyl, where an alkyl containing up to 6 carbon atoms is C1-C6 alkyl, and an alkyl containing up to 5 carbon atoms is C1-C5 alkyl. C1-C5 alkyl includes C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl, and C1 alkyl (i.e., methyl). C1-C6 alkyl includes all of the moieties listed above for C1-C5 alkyl, but also includes C6 alkyl. C1-C 10 Alkyl includes all of the moieties listed above for C1-C5 alkyl and C1-C6 alkyl, but also includes C7, C8, C9 and C 10 Also includes alkyl. Similarly, C1-C 12 Alkyl includes all of the moieties described above, except that C 11 and C 12 Includes alkyl. C1~C 12Non-limiting examples of alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0391] "Alkylene", "alkylene chain" or "alkylene group" refers to a fully saturated, straight or branched divalent hydrocarbon chain radical having from 1 to 40 carbon atoms. 40 Non-limiting examples of alkylene include butylene ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted.

[0392] "Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups containing any number of carbon atoms from 2 to 40 are included. Alkenyl groups containing up to 40 carbon atoms are C2-C 40 Alkenyl, containing up to 10 carbon atoms, is C2-C 10 An alkenyl group containing up to 6 carbon atoms is C2-C6 alkenyl, and an alkenyl containing up to 5 carbon atoms is C2-C5 alkenyl. C2-C5 alkenyl includes C5 alkenyl, C4 alkenyl, C3 alkenyl, and C2 alkenyl. C2-C6 alkenyl includes all of the moieties listed above for C2-C5 alkenyl, but also includes C6 alkenyl. C2-C 10 Alkenyl includes all of the moieties listed above for C2-C5 alkenyl and C2-C6 alkenyl, but also includes C7, C8, C9 and C 10 Alkenyl is also included. Similarly, C2-C 12Alkenyl includes all of the above moieties, except that C 11 and C 12 Includes alkenyl. C2~C 12 Non-limiting examples of alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-decenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0393] "Alkenylene", "alkenylene chain" or "alkenylene group" refers to a linear or branched divalent hydrocarbon chain radical having from 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. 40 Non-limiting examples of alkenylene include ethene, propene, butene, etc. Unless stated otherwise specifically in the specification, the alkenylene chain can be optional.

[0394] "Alkoxy" or "alkoxy group" refers to the -OR group, where R is alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl, as defined herein. Unless otherwise specifically stated in the specification, an alkoxy group can be optionally substituted.

[0395] "Acyl" or "acyl group" refers to the group -C(O)R, where R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, as defined herein. Unless otherwise specifically stated in the specification, acyl can be optionally substituted.

[0396] An "alkylcarbamoyl" or "alkylcarbamoyl group" is -OC(O)-NR a R b R a and R b are the same or different and are independently alkyl, alkenyl, aryl, heteroaryl, or R a R b may be taken together to form a cycloalkyl or heterocyclyl group, as defined herein. Unless otherwise specifically stated in the specification, an alkylcarbamoyl group may be optionally substituted.

[0397] An "alkylcarboxamidyl" or "alkylcarboxamidyl group" is -C(O)-NR a R b R a and R b are the same or different and are independently an alkyl group, an alkenyl group, an aryl group, a heteroaryl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group, or a heterocyclyl group as defined herein, or R a R b can be taken together to form a cycloalkyl group, as defined herein. Unless stated otherwise specifically in the specification, an alkylcarboxamidyl group can be optionally substituted.

[0398] "Aryl" refers to a hydrocarbon ring system radical containing hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. In the present invention, the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated herein, the term "aryl" is meant to include aryl radicals that are optionally substituted.

[0399] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing a hydrogen atom, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. In the present invention, the heteroaryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems, and the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized. The nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, Examples of phenyl include isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.

[0400] The term "substituted" as used herein refers to any of the above groups (i.e., alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom of any of the groups has been replaced by a non-hydrogen atom, for example, but not limited to, a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl, alkoxy, ester groups; a sulfur atom in groups such as thiol, thioalkyl, sulfone, sulfonyl, sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means any of the above groups in which one or more atoms are replaced by a higher bond (e.g., a double or triple bond) to a heteroatom, such as oxygen in oxo, carbonyl, carboxyl, and ester groups, and nitrogen in groups such as imine, oxime, hydrazone, and nitrile. For example, "substituted" means that one or more atoms are replaced by -NR g R h , -NR g C(=O)R h , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g SO2R h , -OC(=O)NR g R h , -OR g , -SR g -SOR g , -SO2R g , -OSO2R g , -SO2OR g , =NSO2Rg , and -SO2NR g R h "Substituted" also includes any of the above groups in which one or more hydrogen atoms have been replaced with -C(=O)R. g , -C(=O)OR g , -C(=O)NR g R h , -CH2SO2R g , -CH2SO2NR g R h In the above, R g and R h are the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further refers to any of the above groups where one or more atoms are replaced by amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl groups. "Substituted" can also refer to an amino acid in which one or more atoms on the side chain are substituted with an alkyl, alkenyl, alkynyl, acyl, alkylcarboxamidyl, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl. Additionally, each of the aforementioned substituents can also be optionally substituted with one or more of the above-mentioned substituents.

[0401] As used herein, "subject" refers to an individual. Thus, "subject" can include domestic animals (e.g., cats, dogs, etc.), livestock animals (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" can also include mammals (e.g., primates or humans). Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject receiving treatment from a clinician, e.g., a physician.

[0402] The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0403] "Reduce" or other forms of this word, such as "reducing" or "reduction", refers to the reduction of an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, although it is understood that a standard or relative value is not necessarily referenced. For example, "reducing tumor growth" refers to reducing the growth rate of a tumor compared to a standard or control (e.g., an untreated tumor).

[0404] The term "treatment" refers to the medical management of a patient with the intent of curing, ameliorating, stabilizing, or preventing a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically aimed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of an associated disease, pathological condition, or disorder. In addition, the term includes symptomatic treatment, i.e., treatment designed for the relief of symptoms rather than a cure of a disease, pathological condition, or disorder, preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of an associated disease, pathological condition, or disorder, and adjunctive treatment, i.e., treatment used to supplement another specific therapy aimed at ameliorating an associated disease, pathological condition, or disorder.

[0405] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such an amelioration requires only a reduction or alteration, not necessarily elimination.

[0406] The term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0407] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.

[0408] As used herein, the term "pharmaceutical acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose 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 maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents, for example, sugars, sodium chloride, etc. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Suitable inert carriers can include sugars, such as lactose.

[0409] As used herein, the term "sequence identity" refers to the percentage of amino acids that are the same and in the same relative position between two polypeptide sequences. Thus, one polypeptide sequence has a certain percentage of sequence identity compared to another polypeptide sequence. For sequence comparison, typically one sequence serves as a reference sequence to which a test sequence is compared. Those skilled in the art will understand that two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. In embodiments, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), a version of which exists as of the filing date, may be used to determine sequence identity between two amino acid sequences. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The needle-labeled "longest identity" output (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical residues x 100) / (length of alignment - total number of gaps in alignment).

[0410] In an embodiment, sequence identity may be determined using the Smith-Waterman algorithm, a version of which exists as of the filing date.

[0411] As used herein, "sequence homology" refers to the percentage of amino acids that are homologous and in the same relative positions between two polypeptide sequences. Thus, one polypeptide sequence has a certain percentage of sequence homology compared to another polypeptide sequence. As will be appreciated by those skilled in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues with appropriately similar structural and / or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains, and the substitution of one amino acid for another of the same type may often be considered a "homologous" substitution.

[0412] As is well known in the art, amino acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTP, Gapped BLAST, and PSI-BLAST, existing as of the filing date. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above-mentioned programs typically provide an indication of the degree of homology.

[0413] As used herein, the terms "antisense compound" and "AC" are used interchangeably to refer to a polymeric nucleic acid structure (sometimes referred to as an oligonucleotide or polynucleotide) that is at least partially complementary to a target nucleic acid molecule that is hybridized by it (AC). An AC can be a short (in embodiments, less than 50 base pairs) polynucleotide or polynucleotide homolog that contains a sequence that is complementary to a target sequence in a target pre-mRNA strand. An AC can be formed from natural nucleic acids, synthetic nucleic acids, nucleic acid homologs, or any combination thereof. In embodiments, an AC comprises an oligonucleoside. In embodiments, an AC comprises an antisense oligonucleotide. In embodiments, an AC comprises a conjugate group. Non-limiting examples of ACs include, but are not limited to, primers, probes, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, siRNAs, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations thereof. Thus, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpin, and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands that hybridize to form a double-stranded compound, or a single strand with sufficient self-complementarity to allow hybridization and the formation of a complete or partial double-stranded compound. In embodiments, ACs modulate (increase, decrease or change) the expression of target nucleic acids. Various modifications can be made to polymeric nucleic acid structures, such as phosphorodiamidate morpholinos (PMOs). Thus, ACs as used herein encompass any modifications described herein, such as PMOs.

[0414] As used herein, the terms "pre-mRNA" and "primary transcript" refer to a newly synthesized eukaryotic mRNA molecule immediately following DNA transcription. The pre-mRNA must be 5' capped, modified with a 3' polyA tail, and spliced ​​to generate the mature mRNA sequence.

[0415] As used herein, the term "targeting" or "directing" refers to the association of an antisense compound (AC) with a target nucleic acid molecule or a region of a target nucleic acid molecule. In an embodiment, the AC has the ability to hybridize with the target nucleic acid under physiological conditions. In an embodiment, the AC targets a unique portion or site in the target nucleic acid, e.g., a portion of the target nucleic acid having at least one identifiable structure, function or characteristic, e.g., a specific exon or intron, or a selected nucleobase or motif within an exon and / or intron. In an embodiment, the AC targets a region including an intron-exon junction of a gene associated with a disease or disorder. In an embodiment, the AC targets exon 45 of the dystrophin gene. In an embodiment, the AC targets a region including an intronic nucleotide sequence upstream (or 5') of exon 45 of the dystrophin gene. In embodiments, the AC targets a region containing an intronic nucleotide sequence upstream (or 5') of exon 45 of the dystrophin gene.

[0416] As used herein, the terms "target nucleic acid" and "target sequence" refer to a nucleic acid molecule that comprises a nucleic acid sequence that is bound or hybridized by an antisense compound. Target nucleic acids include, but are not limited to, RNA (including, but not limited to, pre-mRNA and mRNA, or portions thereof), cDNA derived from such RNA, and non-translated RNA, such as miRNA. For example, in embodiments, the target nucleic acid may be a cellular gene (or an mRNA transcribed from such a gene) whose expression is associated with a specific disorder or disease state, or a nucleic acid molecule from an infectious agent. In embodiments, the target nucleic acid is a target RNA. In embodiments, the target nucleic acid is a target mRNA. In embodiments, the target nucleic acid is a target pre-mRNA. In embodiments, the target nucleic acid comprises a nucleotide sequence of exon 45 of the dystrophin gene. In embodiments, the target nucleic acid comprises a nucleotide sequence that comprises an intron-exon junction of exon 45 of the dystrophin gene. In embodiments, the target nucleic acid comprises an intronic nucleotide sequence upstream (or 5') of exon 45 of the dystrophin gene.

[0417] As used herein, the term "mRNA" refers to an RNA molecule that codes for a protein, including pre-mRNA and mature mRNA. "Pre-mRNA" refers to a eukaryotic mRNA molecule newly synthesized immediately after DNA transcription. In embodiments, the pre-mRNA is 5' capped, modified with a 3' polyA tail, and / or spliced ​​to generate a mature mRNA sequence. In embodiments, the pre-mRNA contains one or more introns. In embodiments, the pre-mRNA undergoes a process known as splicing, which removes introns and joins exons together. In embodiments, the pre-mRNA contains a polyadenylation site.

[0418] As used herein, the terms "splicing" and "processing" refer to the post-transcriptional modification of pre-mRNA in which introns are removed and exons are spliced ​​together. Splicing occurs in a series of reactions catalyzed by a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs) called the spliceosome. A 3' splice site, a 5' splice site, and a branch site are required within the intron for splicing. The RNA component of the snRNP can interact with the intron and participate in catalysis.

[0419] As used herein, the term "exon" refers to a portion of a pre-mRNA that is typically incorporated into the mature mRNA after splicing.

[0420] As used herein, the term "intron" refers to a portion of a pre-mRNA that is typically excluded from the mature mRNA after splicing.

[0421] As used herein, the term "flanking" refers to an intron located immediately upstream (5') or immediately downstream (3') of the associated exon. For example, a 5' flanking intron of exon 44 refers to an intron that is immediately upstream of (i.e., directly coupled to) exon 44's 5' end. For example, a 3' flanking intron of exon 44 refers to an intron that is immediately downstream of (i.e., directly coupled to) exon 44's 5' end.

[0422] A "target pre-mRNA" is a pre-mRNA that contains a target sequence that is hybridized by an AC.

[0423] A "target mRNA" is an mRNA sequence that is the result of splicing a target pre-mRNA sequence. In embodiments, the target mRNA does not encode a functional protein. In embodiments, the target mRNA retains one or more intron sequences.

[0424] As used herein, the term "gene" refers to a nucleic acid molecule having a nucleic acid sequence that includes a 5' promoter region, and any intron and exon regions, as well as a 3' untranslated region ("UTR"), which are involved in expression of a gene product.

[0425] "Target gene" in this disclosure refers to a gene that encodes a target pre-mRNA.

[0426] "Target protein" refers to the amino acid sequence encoded by the target mRNA. In embodiments, the target protein may not be a functional protein.

[0427] "Wild-type target protein" refers to the naturally occurring functional protein isomer produced by the wild-type, normal or unmutated form of the target gene. Wild-type target protein also refers to a protein derived from a properly spliced ​​target pre-mRNA.

[0428] As used herein, the term "transcript" refers to an RNA molecule transcribed from DNA and includes, but is not limited to, mRNA, mature mRNA, pre-mRNA, and partially processed RNA.

[0429] "Re-spliced ​​target protein" as used herein refers to the protein encoded by the mRNA molecule resulting from splicing of the target pre-mRNA hybridized by AC. The re-spliced ​​target protein may be identical to the wild-type target protein, may be homologous to the wild-type target protein, may be a functional variant of the wild-type target protein, or may be an active fragment of the wild-type target protein.

[0430] As used herein, a "functional fragment" or an "active fragment" refers to a portion of a wild-type eukaryotic target protein that exhibits an activity, e.g., one or more activities of the full-length wild-type target protein, or has another activity. In embodiments, a re-spliced ​​target protein that shares at least one biological activity of the wild-type target protein is considered to be an active fragment of the wild-type target protein. The activity may be any percentage (i.e., higher or lower) of the activity of the full-length wild-type target protein, for example, but not limited to, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 200%, about 300%, about 400%, about 500% or more of the activity compared to the wild-type target protein (including all values ​​and ranges therebetween). Thus, in embodiments, an active fragment may retain at least a portion of one or more biological activities of the wild-type target protein. In embodiments, an active fragment may enhance one or more biological activities of the wild-type target protein.

[0431] As used herein, the term "nucleoside" refers to a glycosylamine that contains a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides with mimetic bases and / or sugar groups. A "natural nucleoside" or an "unmodified nucleoside" is a nucleoside that contains a natural nucleobase and a natural sugar. Natural nucleosides include RNA and DNA nucleosides.

[0432] As used herein, the term "natural sugar" refers to the sugar of a nucleoside that is unmodified from the form naturally occurring in RNA (2'-OH) or DNA (2'-H).

[0433] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group covalently linked to a sugar. Nucleotides may be modified with any of a variety of substituents.

[0434] As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase may include any atom or group of atoms that has the ability to hydrogen bond to the base of another nucleic acid. A natural nucleobase is a nucleobase that is not modified from its naturally occurring form in RNA or DNA.

[0435] As used herein, the term "heterocyclic base moiety" refers to a nucleobase that comprises a heterocycle.

[0436] As used herein, "oligonucleoside" refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0437] As used herein, the term "oligonucleotide" refers to an oligomeric compound that includes multiple linked nucleotides or nucleosides. In certain embodiments, one or more nucleotides of an oligonucleotide are modified. In embodiments, an oligonucleotide includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In embodiments, an oligonucleotide is composed of natural and / or modified nucleobases, sugars, and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.

[0438] As used herein, "internucleoside linkage" refers to a covalent bond between adjacent nucleosides.

[0439] As used herein, "natural internucleotide linkage" refers to a 3' to 5' phosphodiester linkage.

[0440] As used herein, the term "modified internucleoside linkage" refers to any linkage between nucleosides or nucleotides other than a naturally occurring internucleoside linkage.

[0441] As used herein, the term "chimeric antisense compound" or "chimeric AC" refers to an antisense compound having at least one sugar, nucleobase and / or internucleoside linkage that is differently modified compared to other sugars, nucleobases and internucleoside linkages within the same oligomeric compound. The remainder of the sugars, nucleobases and internucleoside linkages can be independently modified or unmodified. In general, chimeric oligomeric compounds are those that have modified nucleosides at separate positions or grouped together in a region that will define a particular motif. Any combination of modifications and / or mimetic groups can constitute the chimeric oligomeric compounds described herein.

[0442] As used herein, the term "mixed backbone antisense oligonucleotide" refers to an antisense oligonucleotide in which at least one internucleoside linkage of the antisense oligonucleotide is different from at least one other internucleoside linkage of the antisense oligonucleotide.

[0443] As used herein, the term "nucleobase complementarity" refers to a nucleobase that has the ability to base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In embodiments, complementary nucleobase refers to the nucleobase of an antisense compound that has the ability to base pair with the nucleobase of a target nucleic acid. For example, if the nucleobase at a specific position of an antisense compound has the ability to hydrogen bond with the nucleobase at a specific position of a target nucleic acid, the hydrogen bond position between the oligonucleotide and the target nucleic acid is considered to be complementary in that nucleobase pair.

[0444] As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds with each other or support hybridization.

[0445] As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize with another oligomeric compound or nucleic acid through nucleobase complementarity. In embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bind to each other and allow stable association between the antisense compound and the target. Those skilled in the art will recognize that it is possible to incorporate mismatches without eliminating the ability of an oligomeric compound to remain associated. Thus, antisense compounds are described herein that may contain up to about 20% mismatched nucleotides (i.e., nucleobases that are not complementary to the corresponding nucleotides of the target). Preferably, the antisense compounds contain no more than about 15%, more preferably no more than about 10%, and most preferably no more than 5% mismatches, or no mismatches. The remaining nucleotides are either complementary or nucleobases that do not interfere with hybridization (e.g., universal bases). One of skill in the art will recognize that the compounds provided herein have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleobase complementarity to the target nucleic acid.

[0446] As used herein, "hybridization" refers to pairing of complementary oligomeric compounds (e.g., antisense compounds and their target nucleic acids). Without being limited to a specific mechanism, the most common pairing mechanism involves hydrogen bonding, which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is a complementary nucleobase to the natural nucleobases thymidine and uracil, which pair by forming hydrogen bonds. The natural base guanine is a complementary nucleobase to the natural bases cytosine and 5-methylcytosine. Hybridization can occur under various circumstances.

[0447] As used herein, the term "specifically hybridize" refers to the ability of an oligomeric compound to hybridize with greater affinity to one nucleic acid site than to another nucleic acid site.In an embodiment, an antisense oligonucleotide specifically hybridizes to more than one target site.In an embodiment, an oligomeric compound specifically hybridizes to its target under stringent hybridization conditions.

[0448] The terms "modulate," "modulating," and "modulation" refer to the perturbation of expression, function, or activity as compared to the level of expression, function, or activity prior to modulation. Modulation can include an increase (stimulation or induction) or a decrease (inhibition or reduction) of expression, function, or activity. In embodiments, modulation can include the perturbation of splice site selection during pre-mRNA processing.

[0449] The terms "inhibit," "inhibiting," or "inhibition" refer to a decrease in activity, expression, function, or other biological parameter, which may, but does not necessarily, include the complete elimination of activity, expression, function, or other biological parameter. Inhibition may include, for example, a reduction in activity, response, condition, or disease by at least about 10% compared to a control. In embodiments, the expression, activity, or function of a gene or protein is reduced by a statistically significant amount.

[0450] As used herein, the term "expression" refers to all of the functions and steps taken to convert a gene's coded information into structures present and functioning within a cell, including, but not limited to, the products of transcription and translation.

[0451] As used herein, the term "2' modified" or "2' substituted" refers to a sugar containing a substituent other than H or OH at the 2' position. 2' modified monomers include, but are not limited to, 2' substituents such as allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ), or O-CH2-C(=O)-N(R m )(R n ) in which Rm and Rn are independently H or substituted or unsubstituted C1-C 10 It is an alkyl.

[0452] As used herein, the term "MOE" refers to a 2'-O-methoxyethyl substituent.

[0453] As used herein, the term "high affinity modified nucleotide" refers to a nucleotide having at least one modified nucleobase, internucleoside linkage, or sugar, such that the modification increases the affinity of the antisense compound that contains the modified nucleotide for a target nucleic acid. High affinity modifications include, but are not limited to, BNA, LNA, and 2'-MOE.

[0454] As used herein, the term "mimetic" refers to a group that is used in place of the sugar, nucleobase, and / or internucleoside linkage in AC. Typically, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization with a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some cases, a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for synthesizing sugar, nucleoside and nucleobase mimetics are well known to those of skill in the art.

[0455] As used herein, the term "bicyclic nucleoside" or "BNA" refers to a nucleoside in which the furanose portion of the nucleoside contains a bridge connecting two atoms on the furanose ring, thereby forming a bicyclic ring system. BNAs include, but are not limited to, α-L-LNA, β-D-LNA, ENA, oxyamino BNA (2'-ON(CH3)-CH2-4'), and aminooxy BNA (2'-N(CH3)-O-CH2-4').

[0456] As used herein, the term "4' to 2' bicyclic nucleoside" refers to a BNA in which the bridge connecting the two atoms of the furanose ring links the 4' and 2' carbon atoms of the furanose ring, thereby forming a bicyclic ring system.

[0457] As used herein, "locked nucleic acid" or "LNA" refers to a nucleotide modified such that the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon of the sugar ring via a methylene group, thereby forming a 2'-C,4'-C-oxymethylene bond. LNA includes, but is not limited to, α-L-LNA and β-D-LNA.

[0458] As used herein, the term "cap structure" or "terminal cap moiety" refers to a chemical modification that is incorporated at either end of an AC.

[0459] As used herein, the term "dosage unit" refers to the form in which a pharmaceutical agent is provided. In an embodiment, the dosage unit is a vial containing lyophilized antisense oligonucleotide. In an embodiment, the dosage unit is a vial containing reconstituted antisense oligonucleotide.

[0460] Although several embodiments of the invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Other embodiments are within the scope of the following claims.

[0461] All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. EXAMPLES

[0462] Example 1. Conjugation of oligonucleotides with cell-penetrating peptides. Conjugation of AC to CPP. As shown in FIG. 1A, AC oligonucleotides with a (NH2-(CH2)5-CH2-) linker at the 5' phosphorothioate terminus are conjugated to a CPP disclosed herein via a carboxylate or N-hydroxysuccinimide ester (NHS ester) functional group on the peptide. As shown in FIG. 1B, AC oligonucleotides are conjugated to a cell penetrating peptide (CPP) by either amide bond formation (left) or click chemistry. The linker / CPP is incorporated on either the 5' or 3' end of the oligonucleotide.

[0463] Synthesis of oligonucleotide-peptide conjugates with PEG spacers. As shown in Figures 2A and 2B, oligonucleotide-peptide conjugates are synthesized without (Figure 2A) and with (Figure 2B) a PEG (polyethylene glycol) linker inserted between the oligonucleotide moiety and the peptide. "R" in the figure represents a palmitoyl group.

[0464] Exemplary antisense compounds bind to or consist of sequences found in Tables 6A-6P, Tables 7A-7O, and Tables 8A-8C. Exemplary CPPs and EEVs are found throughout this disclosure.

[0465] Example 2. Use of cell-penetrating peptides conjugated to oligonucleotides for splicing correction of exon 45 of DMD in an in vitro model. Purpose: This test uses an in vitro model to assess the expression of dystrophin protein using an antisense compound of Tables 6A-6P, Tables 7A-7O or Tables 8A-8C, a reverse complement thereof, or a compound having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto. The effect of a sequence alone or conjugated to a cell-penetrating construct is examined, as well as the effect of a sequence of Table 6A-6P, Table 7A-7O or Table 8A-8C, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto. AC of Tables 6A-6P, Tables 7A-7O or Tables 8A-8C, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto restores the reading frame of the DMD gene.

[0466] This test uses an in vitro model to examine the effect on expression of dystrophin protein of antisense compounds that bind to or comprise the sequences of Tables 6A-6P or Tables 7A-7O or Tables 8A-8C or their reverse complements alone, or ACs that bind to or comprise the sequences of Tables 6A-6P or Tables 7A-7O or Tables 8A-8C or their reverse complements conjugated to a cell penetrating peptide. ACs that bind to or consist of the sequences of Tables 6A-6P or Tables 7A-7O or Tables 8A-8C or their reverse complements restore the reading frame of the DMD gene.

[0467] In vitro models: This study uses primary DMD muscle cells and muscle cells without DMD. This study also uses an immortalized muscle cell model of DMD. Muscle cells derived from DMD patients are transduced with vectors expressing human telomerase reverse transcriptase (hTert) and cyclin-dependent kinase 4 (CDK4) to create muscle stem cell lines with enhanced proliferative potential. These models are described in the following publications: Thorley et al. Skelet Muscle. 2016;6:43. This study also uses the CRL-2061™ muscle cell line, which was derived from a patient with rhabdomyosarcoma.

[0468] Study design: A compound comprising an AC of Table 6A-6P or Table 7A-7O or Table 8A-8C, or its reverse complement, and a cyclic peptide is administered to either immortalized muscle cells, primary DMD muscle cells, or muscle cell lines (e.g., CRL-2061™). Total RNA is extracted from the cells and analyzed by RT-PCR and Western blot to visualize the efficiency of splicing correction and detect dystrophin products. The percentage of exon 45 correction products is determined.

[0469] Example 3. Use of cell-penetrating peptides conjugated to oligonucleotides for splicing correction of exon 45 of DMD in animal models Objective: This study uses a mouse model to examine the effect of a composition comprising an antisense compound of Tables 6A-6P or Tables 7A-7O or Tables 8A-8C, or its reverse complement, alone or conjugated to a cell-penetrating peptide, on the expression of dystrophin protein. AC of Tables 6A-6P or Tables 7A-7O or Table 8C, or its reverse complement, restores the reading frame of the DMD gene.

[0470] Mouse Model: This study uses del52hDMD / mdx mice, described in Veltrop et al. PLoS One. 2018;13(2):e0193289, which is incorporated by reference in its entirety. The del52hDMD / mdx mice carry both the mouse and human DMD genes. The model contains a stop mutation in exon 23 to disrupt expression of mouse dystrophin. The model contains a deletion of exon 52 to disrupt expression of human dystrophin. This study also uses mdx52 mice, which lack exon 52 of mouse dystrophin. This mouse model is described in the following documents, which are incorporated herein by reference: Aoki et al. (2012). PNAS USA. 109(34): 1376-13768, and Araki et al. Biochem Biophys Res Commun. 1997 Sep 18; 238(2): 492-7. Humanized DMD (hDMD) mice are also used. hDMD mice contain a complete human dystrophin gene. This model is described in U.S. Patent No. 9,078,911, which is incorporated herein by reference in its entirety.

[0471] This study evaluates the safety and tolerability of compounds described herein using CD1 mice. Compounds are tested at concentrations ranging from 1 mg / kg mouse body weight to 1 g / kg mouse body weight.

[0472] This study evaluates the efficacy and safety of compounds described herein using non-human primates (NHPs).

[0473] Study design. A composition comprising an AC of Tables 6A-6P or Tables 7A-7O or Tables 8A-8C, or its reverse complement, and a CPP is applied to the above mouse model to evaluate the ability of the compound and AC to skip exon 45 and thus treat DMD. The compound and AC are administered to mice by either intramuscular (IM) or intravenous (IV) injection at the following doses: 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, and 30 mg / kg.

[0474] Total RNA will be extracted from tissue samples and analyzed by RT-PCR and Western blot to visualize the efficiency of splicing correction and detect dystrophin products. The percentage of exon 45 corrected products will be determined.

[0475] Compound synthesis and purification: Compounds were synthesized according to the following procedure. TFA-lysine protected cCPP was reacted with AC from Tables 8A-8C, followed by deprotection to obtain cCPP-AC conjugates. Briefly, cCPP was preactivated by reacting with HATU (2.0 equiv.) and DIPEA (2.0 equiv.) in DMSO (10 mM, 1.8 mL). After 10 min at room temperature, the preactivated solution was combined with a solution of AC in DMSO (10 mM, 1.8 mL) and mixed thoroughly. The reaction was incubated at room temperature for 2 h. The reaction was monitored by LCMS (Q-TOF) using a BEH C18 column (130 Å, 1.7 μm, 2.1 mm×50 mm), Buffer A: water (0.1% FA), Buffer B: acetonitrile (0.1% FA), flow rate: 0.4 mL / min, starting at 2% buffer B and ramping to 98% over 3.4 min. After completion, in situ deprotection of the TFA-protected lysine was initiated by diluting the reaction mixture with 0.2 M KCl (aq) pH 12 (36 mL). The reaction was monitored by LCMS (Q-TOF) using the analytical method described above. The crude mixture was loaded directly onto a C18 reversed phase column (Oligo clarity column, 150 mm×21.2 mm). The crude product was then purified using a gradient of 5-20% over 60 min at a flow rate of 20 mL / min using water and acetonitrile with 0.1% FA as the solvent. Fractions containing the desired product were pooled and the pH of the solution was adjusted to 7 using 0.5 M NaOH. The solution was frozen and lyophilized to obtain a white powder. The cCPP-AC conjugate was reconstituted in 1 M NaCl in water and the formate was exchanged for chloride by repeated washing (centrifugation at 3500 rpm for 20-40 min) through a 3-kD MW cut-off Amicon tube. This process was performed three times with 1 M NaCl and three times with saline (0.9% NaCl, sterile, endotoxin-free). The conductivity of the final filtrate was assessed to confirm the appropriate salt concentration. The solution was further diluted with saline to the desired formulation concentration and sterile filtered in a biosafety cabinet. The concentration of each formulation was remeasured after filtration.

[0476] EEV-PMO-DMD45-5 (see Table 10A) was obtained and the purity and identity of each preparation was assessed by liquid chromatography-mass spectrometry quadrupole time-of-flight mass spectrometry (QTOF-LCMS). EEV-PMO-DMD45-5 was confirmed to be 99% pure by RP-FA and 73.3% pure by CEX. 378 H 609 N 152 O 124 P 21 The calculated MW was 9917.42. The MW determined by QTOF-LCMS was 9917.29. The formulations were further assayed for their endotoxin content, residual free peptide, FA content and pH.

[0477] EEV-PMO-DMD45-7 (see Table 10A) was obtained and the purity and identity of each preparation was assessed by QTOF-LCMS. EEV-PMO-DMD45-7 was 99% pure by RP-FA and 79.7% pure by CEX. 387 H 626 N 156 O 127 P 22 The calculated MW of was 10177.65. The MW determined by QTOF-LCMS was 10177.60.

[0478] Example 4. In vitro screening of AC targeting exon 45 Objective: The objective of this study was to evaluate ACs targeting exon 45.

[0479] Cell culture and treatment: This study uses human rhabdomyosarcoma cells (RMS cells, ATCC CRL-2061). RMS cells were cultured in T75 flasks in RPMI1640 (ATCC) medium supplemented with 10% FBS (VWR) and 1x penicillin-streptomycin (VWR) at 37°C and 5% CO2. 1x10 cells per well were cultured at 1x10 5RMS cells were seeded overnight in 24-well plates in RPMI1640 complete medium. The next day, the culture medium was replaced with 1 mL of fresh medium. 5 μM or 10 μM of AC from Table 8 was added to the cell culture and swirled to mix. Then, 6 μL of Endo-Porter (Gene-tools) per well (1 mL of culture medium) was added to the cell culture and swirled to mix immediately. After 24 hours of incubation, cells were washed twice with PBS (VWR) and then 350 μL of RLT lysis buffer (QIAGEN) was added to each well. Cells were left at room temperature for 5 minutes for cell lysis and pipetted several times for thorough mixing. Lysates were collected in Sample Tube RB (QIAGEN).

[0480] RNA extraction: RNA was extracted using a QIAcube according to the protocol provided by the manufacturer. Total RNA concentration was determined using a NanoDrop 8000 (Thermo Fisher Scientific).

[0481] Nested PCR: Nested PCR was performed using 200 ng of extracted total RNA and the QIAGEN One-Step RT-PCR kit for primary amplification. DMD-specific primers were used to prepare 50 μL of reaction solution according to the manufacturer's protocol. Forward primer: 5'-CAATGCTCCTGACCTCTGTGC-3' Reverse primer: 5'-GCTCTTTTCCAGGTTCAAGTGG-3').

[0482] The RT-PCR program used was as follows:

[0483] Reverse transcription at 50°C for 30 min.

[0484] Initial PCR activation was at 95°C for 15 min.

[0485] 20 cycles: denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min.

[0486] Final extension at 72°C for 5 min.

[0487] Incubate at 4 °C overnight.

[0488] Secondary amplification PCR reactions were prepared using OneTaq® Hot Start 2X Master Mix according to the manufacturer's protocol. Forward primer: 5'-GTCTACAACAAAGCTCAGGTCG-3', Reverse primer: 5'-GCAATGTTATCTGCTTCCTCCAACC-3').

[0489] The RT-PCR program used was as follows:

[0490] 95℃ for 1 minute.

[0491] 25 cycles: denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 1 min.

[0492] Final extension at 72°C for 5 min.

[0493] Incubate at 4 °C overnight.

[0494] Gel electrophoresis analysis for exon skipping efficiency: 4 μL of the above PCR products were analyzed by 2% E-gel (Invitrogen™) and imaged with an E-Gel™ Power Snap electrophoresis system (Thermo Fisher Scientific). The intensities of skipped and full-length bands were analyzed. The polynucleotide levels of bands with exon 45 skipping "A" and bands without exon 45 skipping "B" were measured using ImageJ. Based on these measurements of "A" and "B", the skipping efficiency was determined by the following equation: skipping efficiency (%)=A / (A+B)×100.

[0495] Results: Figure 4 shows the exon 45 skipping efficiency of the ACs in Table 9A. [Table 45]

[0496] The ACs in Table 9B skip exon 45 more effectively than the approved exon 45 antisense oligonucleotide, casimersen. [Table 46]

[0497] Example 5: Sustained and repeated dose effects on D2MDX mice after administration of EEV-PMO-MDX23-1 Methods: We used mouse PMO-EEV of 20, 40 or 80 mpk to induce exon 23 skipping in the D2 / MDX mouse model (EEV-PMO-MDX23-1:EEV=Ac-PKKKRKV-miniPEG-K(cyclo(GfFGrGrQ))-PEG 12 D2 / MDX mice were administered 40mpk once a week for 4 weeks and sacrificed 1 week after the final dose to study repeat dose effects.

[0498] Results: After a single dose of 20, 40 or 80mpk, exon skipping was observed in all four tissues one week after injection (Figure 5). Exon skipping peaked two weeks after injection and was maintained in skeletal muscle (triceps and tibialis anterior) for at least eight weeks (Figure 6). A drop in exon skipping was observed in the diaphragm and heart after four and eight weeks (Figure 6). After four weekly doses at 40mpk, exon skipping was observed in all four tissues (Figure 7) (tissues were harvested one week after the last dose).

[0499] Example 6: Functional assays in D2DMX Methods: Six groups of male D2 / MDX and DBA / 2J (wild-type) mice (n=8 per group) were administered intravenously once every 2 weeks for a total of six doses of: wild-type vehicle (saline), D2.mdx vehicle (saline), PMO only (PMO-MDX23: 5'-GGCCAAACCTCGGCTTACCTGAAAT-3'), or two EEV-PMO constructs targeting exon 23: (EEV-PMO-MDX23-1: EEV=Ac-PKKKRKV-miniPEG-K(cyclo(GfFGrGrQ))-PEG 12 -OH, PMO=5'-GGCCAAACCTCGGCTTACCTGAAAT-3'; and EEV-PMO-MDX23-2 (EEV=Ac-PKKKRKV-Lys(FfΦ-GrGrQ)-PEG12-K(N3)-NH2, PMO=5'-GGCCAAACCTCGGCTTACCTGAAAT-3'-C4COT). C4COT=cyclooct-2-yn-1-O-(CH2)4-OC(O). Dosages are listed above. Creatine kinase levels, grip strength, and wire dangling time were determined using known methods once every 4 weeks for a total of 4 doses.

[0500] Results: Treatment with 80mpk EEV-PMO-MDX23-1 every 2 weeks resulted in longer hanging times compared to the remaining groups 2 weeks after the first injection and continued to show a prolonged and statistically significant improvement compared to the vehicle D2.mdx group at both 4 and 8 weeks after the first injection (Figure 8). After 12 weeks of treatment, animals treated with 80mpk EEV-PMO-MDX23-1 every 2 weeks had wire hanging times that were statistically indistinguishable from wild-type animals (Figure 8). Treatment with EEV-PMO-MDX23-1 at 40mpk every 2 weeks and EEV-PMO-MDX23-2 at 15mpk every 2 weeks with the loading dose showed significantly longer wire dangling times than the vehicle D2.mdx group, beginning 8 weeks after the first treatment and appearing to remain stable through 12 weeks of treatment when signs of phenotypic improvement were first evident (Figure 8). PMO treatment alone appeared to follow the same trend as the vehicle D2.mdx group and although the vehicle control group was lower at 2 weeks, they were still consistent with published data for 4, 8 and 12 weeks, indicating that the study was performed correctly (Figure 8).

[0501] Serum CK was determined at four time points: before administration, and at weeks 4, 8, and 12. Similar to the wild type, the EEV-PMO treatment group showed a significant decrease in serum CK. The PMO only treatment group did not show a significant decrease at any time point after treatment (Figures 9A-B and 10A-B).

[0502] Grip strength was measured before (Figure 11A) and at 12 weeks (Figure 11B). A dose-dependent increase in grip strength was observed in PMO-EEV-treated mice. Vehicle and PMO-treated mice showed no significant improvement.

[0503] Example 7: hDMD and exon 45 skipping Methods: A commercially available exon 45 skipping PMO, casimersen (5'-CAATGCCATCCTGGAGTTCCTG-3'), was conjugated to EEV (Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH, EEV-PMO-DMD45-1) and used as a positive control to test the in vivo system (hDMD).

[0504] Eight- to nine-week-old hDMD mice were intravenously injected with 40, 60, or 80 mpk of EEV-PMO-DMD45-1 positive control. Tissues were harvested one week later and exon skipping was determined by PCR (Labchip quantification).

[0505] Results: Dose-dependent exon 45 skipping was observed in muscle tissues: diaphragm (Figure 12A), heart (Figure 12B), biceps (Figure 12C), and tibialis anterior (Figure 12D).

[0506] Example 8: Library Screening Methods: 60 mpk of positive control (casimersen conjugated to EEV, EEV-PMO-DMD45-1), or candidate PMO conjugated to EEV, were injected intravenously into 8-9 week old hDMD mice. The respective PMO sequences are shown in Table 10A. EEV had the sequence Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG-OH.

[0507] Tissues were harvested one week later and exon skipping was assessed by PCR in the tibialis anterior muscle, diaphragm, and heart.

[0508] Results: Exon 45 skipping at 60 mpk for each exon 45 EEV-PMO was greater than that of the positive control (EEV-PMO-DMD45-1) (Figure 13). To further differentiate the efficacy of the candidate exon 45 EEV-PMOs, exon 45 skipping was tested at 30 mpk. Candidates EEV-PMO-DMD45-10, EEV-PMO-DMD45-11, and EEV-PMO-DMD45-3 showed lower efficacy in tibialis anterior and diaphragm tissues compared to the other EEV-PMOs (Figure 14). Low exon skipping outliers were consistent across all tissues and were all female.

[0509] Example 9: Patient-derived cell data Methods: DMDΔ46-48 iPSC-derived myoblasts, which carry a mutation amenable to exon 45 skipping, were treated with 30 μM EEV-PMO-DMD45-1 (positive control) and 10 EEV-PMO compounds (see Table 10A) for 24 hours and then differentiated for 7 days. Exon skipping was determined by RT-PCR. Dystrophin protein expression was determined by Western blot.

[0510] Results: All 10 EEV-PMOs showed superior exon skipping and dystrophin expression compared to the positive control (Figures 15A-15B).

[0511] DMDΔ46-48 iPSC-derived cardiomyocytes were treated with 30 μM positive control (EEV-PMO-DMD45-1), EEV-PMO-DMD45-5, or EEV-PMO-DMD45-7 for 24 h and analyzed 72 h later. Robust exon 45 skipping and dystrophin protein production was observed for all three constructs (Figures 15C-15D).

[0512] DMDΔ46-48 iPSC-derived cardiomyocytes were treated with 20, 10, 5, or 1 μM of positive control (EEV-PMO-DMD45-1), EEV-PMO-DMD45-5, or EEV-PMO-DMD45-7 for 24 h and analyzed 72 h later. Robust exon 45 skipping was observed for all three constructs (Figure S15E). [Table 47]

[0513] Example 10: Cell viability in human primary proximal tubular epithelial cells (RPTEC) Methods: Ten exon 45 PMO-EEVs (see Table 10B below) were screened for viability in RPTECs (proximal tubular epithelial cells). The EEVs of the ten PMO-EEVs had the sequence Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH. The PMO sequence of each construct and the concentration range tested are shown in Table 10B.

[0514] Two positive controls were used: (1) positive control (EEV-PMO-control: EEV = Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfΦGrGrQ)-AEEA-K(N3), PMO = 5'-TGAAAACGCCGCCATTTCTCAACAG-3'-PEG4COT (PEG4COT = cyclooct-2-yn-1-O-(PEG4)-OC(O))), and (2) melittin (used at 16.6 uM). PBS was used as vehicle (negative control).

[0515] EEV-PMO was resuspended in saline (serial 1:2 dilutions) to give the concentration ranges shown in Table 10B. Data were normalized to melittin controls.

[0516] Results: EEV-PMO-DMD45-2 showed some toxicity only at the two highest concentrations. EEV-PMO-DMD45-3, EEV-PMO-DMD45-4, and EEV-PMO-DMD45-5 had little toxicity at the concentrations tested but showed a trend towards reduced viability at the highest tested concentration (Figure 16). EEV-PMO-DMD45-6, EEV-PMO-DMD45-7, EEV-PMO-DMD45-8, and EEV-PMO-DMD45-9 had little toxicity at the concentrations tested (Figure 17). EEV-PMO-DMD45-10 showed toxicity at the two highest concentrations tested, EEV-PMO-DMD45-11 had little toxicity at the concentrations tested, and the EEV-PMO-control was used as a positive control for toxicity (Figure 18). [Table 48]

[0517] Example 11: Localization The subcellular localization of PMO alone (PMO), PMO conjugated to EEV (EEV-PMO), and PMO conjugated to EEV and a nuclear localization signal (EEV-NLS-PMO) was determined. [Table 49]

[0518] Briefly, THP-1 monocytes were contacted with 3 μM of either PMO, EEV-PMO or EEV-NLS-PMO, incubated for 24 h and examined by LC-MS.

[0519] Figure 22A shows the total cellular uptake for PMO and EEV-PMO and EEV-NLS-PMO contrasted. Both EEV-PMO and EEV-NLS-PMO showed a significant increase in cellular uptake compared to PMO alone. EEV-PMO: approx. 3 times higher than PMO EEV-NLS-PMO: approx. 58 times higher than PMO EEV-NLS-PMO: approx. 19 times faster than EEV-PMO

[0520] Figure 22B contrasts the subcellular localization in THP cells for PMO, EEV-PMO, and EEV-NLS-PMO, as determined using LC-MS / MS. As shown in Figure 22B, EEV-PMO shows improved cell permeability compared to PMO alone. Addition of NLS further improved cell permeability. Figure 22C shows the nuclear uptake of the three constructs.

Claims

1. A compound comprising an endosomal escape vehicle (EEV), wherein the EEV is exocyclic peptides (EPs), cell-penetrating peptides (cCPPs), and Linker Including, The EEV is conjugated to an antisense compound (AC) that is complementary to a target sequence comprising at least a portion of exon 45 of the DMD gene in a pre-mRNA sequence, and the AC comprises a phosphorodiamidate morpholino nucleotide (PMO) comprising 15 to 30 nucleotides; wherein the compound has the following structure: 【Chemical 1】 Including, where * includes the point of attachment to an amino acid side chain (AA SC ) of a cCPP or its protonated form of the formula: 【Chemistry 2】 [During the ceremony, R １ , R ２ and R ３ are independently H or the side chain of an amino acid that contains an aryl or heteroaryl group; R ４ and R6 is independently H or an amino acid side chain; each m independently comprises an integer from 0 to 3; q includes 1; M includes a bonding group. the EP comprises 2 to 10 amino acid residues, wherein one or two amino acid residues of said EP comprise a side chain of a guanidine group or its protonated form; the EP comprises two, three, or four lysine residues, and the EP comprises at least two amino acid residues having hydrophobic side chains; The compound.

2. Two of R 1 , R 2 , and R 3 independently comprise a side chain of phenylalanine, or R 1 , R 2 , and R 3 independently comprise the side chain of phenylalanine; or 2. The compound of claim 1, wherein R1, R2, and R3 independently comprise the side chain of phenylalanine, and wherein R4 is the side chain of arginine.

3. The compound of claim 1, wherein R 4 and R 6 independently comprise H, citrulline, or serine.

4. The compound of claim 1, wherein any one of R 1 , R 2 , and R 3 contains H.

5. The compound of claim 1, wherein R 4 and R 6 independently comprise H.

6. The compound of claim 1, wherein the EP comprises Ac-PKKKRKV.

7. M is 【Chemistry 3】 2. The compound of claim 1, comprising:

8. The compound of claim 1, wherein M comprises —C(O)—, z′ is 11, and x′ is 1.

9. The compound of claim 1, wherein the cyclic peptide is selected from Ff-Nal-GrGrQ, Ff-Nal-GRGRQ, FfFGRGRQ, FGFGRGRQ, GfFGrGrQ, FGFGRRRQ, and FGFRRRRQ.

10. The method of claim 1, wherein the EEV has the formula: Ac-PKKKRKV-PEG 2 -K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG 2 -K(cyclo[FGFGRGRQ])-PEG 2 -K(N 3 )-NH 2 , Ac-PKKKRKV-PEG 2 -K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-PKKKRKV-PEG 2 -K(cyclo[GfFGrGrQ])-PEG 2 -K(N 3 )-NH 2 , Ac-PKKKRKV-PEG 2 -K(cyclo[FfFGRGRQ])-PEG 2 -K(N3)-NH 2 , or Ac-PKKKRKV-PEG 2 -K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH 2. The compound of claim 1 having the formula:

11. The cyclic peptide of claim 1, wherein the cyclic peptide is represented by formula (I-1), (I-2), (I-3), or (I-4): 【Chemistry 4-1】 【Chemistry 4-2】 or its protonated form, wherein AA SC comprises an amino acid side chain; 2. The compound of claim 1, wherein each m is independently an integer 0, 1, 2, or 3.

12. The AC is 5'-CCCAATGCCATCCTGGAGTTCCT-3' 2. The compound of claim 1, comprising a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence comprising:

13. The AC is 5'-CCCAATGCCATCCTGGAGTTCCT-3' at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a base sequence comprising 2. The compound of claim 1, comprising a sequence comprising:

14. The AC is 5'-AATGCCATCCTGGAGTTCCTG-3', 5'-ATGCCATCCTGGAGTTCCTGT-3', 5'-CCCAATGCCATCCTGGAGTTCC-3', 5'-ATGCCATCCTGGAGTTCCTGTA-3', 5'-GCCCAATGCCATCCTGGAGTTCC-3', 5'-CCCAATGCCATCCTGGAGTTCCT-3', 5'-CCCAATGCCATCCTGGAGTTCCTG-3', 5'-TGCCCAATGCCATCCTGGAGTTCCT-3', 5'-CCCAATGCCATCCTGGAGTTCCTGT-3', 5'-CAATGCCATCCTGGAGTTCCTGT-3', or Any reverse complement thereof 2. The compound of claim 1 having a sequence comprising:

15. The cCPP is FGFGRGRQ, wherein Q comprises AA sc ; x' is 1, z' is 11, y is 4; and The compound of claim 1 , wherein M comprises —C(O)—.

16. The cCPP is GfFGrGrQ, wherein Q comprises AA sc ; x' is 1, z' is 11, y is 4; and The compound of claim 1 , wherein M comprises —C(O)—.

17. Formula (C-3) or (C-4): 【Chemistry 5】 or a protonated form thereof, wherein EP comprises an exocyclic peptide and AC comprises an antisense oligonucleotide.

18. The structure: 【Chemistry 6】 or a protonated form thereof, wherein AC comprises said antisense compound having a nucleotide sequence comprising 5'-CCCAATGCCATCCTGGAGTTCCT-3'.

19. The structure: 【Chemistry 7】 or a protonated form thereof, wherein AC comprises said antisense oligonucleotide having a nucleotide sequence comprising 5'-CCCAATGCCATCCTGGAGTTCCT-3'.

20. Formula (C-3): 【Chemistry 8】 or a protonated form thereof, wherein EP comprises the exocyclic peptide and AC comprises said antisense oligonucleotide; AC has a nucleotide sequence comprising 5'-CCCAATGCCATCCTGGAGTTCCT-3', and the EP is Ac-PKKKRKV.

2. The compound of claim 1, comprising:

21. Formula (C-4): 【Chemistry 9】 or a protonated form thereof, wherein EP comprises the exocyclic peptide and AC comprises said antisense oligonucleotide; AC has a nucleotide sequence comprising 5'-CCCAATGCCATCCTGGAGTTCCT-3', and the EP is Ac-PKKKRKV.

2. The compound of claim 1, comprising:

22. A pharmaceutical composition comprising the compound of claim 1.

23. The pharmaceutical composition of claim 22 for the treatment of DMD in a patient in need thereof.

24. The pharmaceutical composition of claim 23, for oral or parenteral administration, wherein parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intratarsal, or intrathecal administration.

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