Compositions and methods for intracellular therapy

Abstract

Provided herein are compounds comprising a cyclic cell-penetrating peptide, a therapeutic moiety, and a regulatory peptide, where the regulatory peptide modulates tissue distribution and / or retention of the compound in vivo. Also provided herein are methods of modulating tissue distribution and / or retention of intracellular therapeutics using the aforementioned compounds.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 186,664, filed May 10, 2021, U.S. Provisional Patent Application No. 63 / 214,085, filed June 23, 2021, U.S. Provisional Patent Application No. 63 / 239,671, filed September 1, 2021, U.S. Provisional Patent Application No. 63 / 290,960, filed December 17, 2021, U.S. Provisional Patent Application No. 63 / 298,565, filed January 11, 2022, U.S. Provisional Patent Application No. 63 / 268,577, filed February 25, 2022, and U.S. Provisional Patent Application No. 63 / 362,295, filed March 31, 2022, the disclosures of each of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Biologics, such as proteins, peptides, and nucleic acids, are promising approaches for the treatment of a wide variety of diseases and disorders. In particular, the therapeutic application of oligonucleotides, such as antisense compounds, is very broad because these compounds can be synthesized with any nucleotide sequence directed against virtually any target gene or genome segment. However, the plasma membrane presents a major challenge in both drug discovery and therapy, especially for therapeutic agents such as biologics. For example, a major challenge in the therapeutic use of oligonucleotide-based biologics is that these formulations have limited ability to access intracellular compartments when administered systemically. Intracellular delivery of oligonucleotide compounds can be facilitated by the use of carrier systems such as polymers, cationic liposomes, or by chemical modification of the construct, for example, by covalent attachment of cholesterol molecules. Nevertheless, intracellular delivery efficiency remains low.

[0003] One potential strategy for disrupting membrane barriers and delivering therapeutic agents, such as biologics, into cells is to conjugate them to cell-penetrating peptides (CPPs). CPPs that enter cells via endocytosis must leave the endocytic vesicle to reach the cytosol. Unfortunately, the endosomal membrane has proven to be a significant barrier to cytoplasmic delivery by these CPPs. In many cases, only a negligible fraction of the peptides leaks into the cell interior (see, e.g., El-Sayed, A et al. AAPS J., 2009, 11, 13-22; Varkouhi, AK et al. J. Controlled Release, 2011, 151, 220-228; Appelbaum, JS et al. Chem. Biol., 2012, 19, 819-830). Cyclic CPPs (cCPPs) with improved properties have been described for use in intracellular delivery of cargo moieties (U.S. Patent Application Publication Nos. 2017 / 0190743 and 2017 / 0355730).

[0004] However, there remains an unmet need for effective compositions and methods for the intracellular delivery of therapeutic agents, particularly in a manner that allows for modulation of the tissue distribution and / or retention of the agent. The compositions and methods disclosed herein address these and other needs. Summary of the Invention

[0005] Described herein are compositions and methods for modulating tissue distribution and / or retention of intracellular therapeutic agents. Compounds containing a cell penetrating peptide (CPP) linked to a therapeutic moiety (TM) have been found to have altered tissue distribution and / or retention when the compound further comprises an exocyclic peptide (EP) as described herein. EPs are typically lysine-containing peptides. EPs have been identified previously known in the art as "nuclear localization signals" (NLSs), such as the nuclear localization sequence of the SV40 virus large T antigen, whose minimal functional unit is the seven-amino acid sequence PKKKRKV. Inclusion of an EP in a CPP-TM compound can alter the levels of TM expression, activity, or function in different tissues, such as different types of muscle tissue or different types of central nervous system tissue (see, e.g., Examples 5 and 7). In one embodiment, the compound is administered intrathecally to modulate the tissue distribution and / or retention of the compound in tissues of the CNS.

[0006] In an embodiment, the following: (a) a cell-penetrating peptide (CPP); (b) Therapeutic part (TM) and; (c) an exocyclic peptide (EP), wherein the tissue distribution or retention of the compound is modulated compared to a compound comprising a CPP and a TM but lacking the EP.

[0007] In one embodiment, the EP is conjugated to a CPP. In one embodiment, the EP is conjugated to a TM. In one embodiment, the CPP is a cyclic cell-penetrating peptide (cCPP). In various embodiments, the therapeutic moiety is a protein, polypeptide, oligonucleotide, or small molecule. In one embodiment, the oligonucleotide is an antisense compound (AC). In one embodiment, the oligonucleotide is other than an antisense compound (AC).

[0008] In embodiments, the therapeutic compound comprises an AC that modulates splicing of DMD exons 2, 8, 11, 17, 19, 23, 29, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 55, and 59. In embodiments, the compound comprises an AC that modulates splicing of DMD exons 2, 8, 11, 23, 43, 44, 45, 50, 51, 53, and 55. In embodiments, the compound comprises an AC that modulates splicing of DMD exons 2, 23, 44, or 51.

[0009] In an embodiment, the compound comprises an AC that modulates the splicing of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7a, and exon 7b of CD33.

[0010] In embodiments, the compound is selected from the group consisting of EEV-PMO-MDX23-1,2,3, EEV-PMO-CD33-1, and the compounds shown in Table C of Example 5 having the structures shown herein.

[0011] In embodiments, a pharmaceutical composition is provided comprising a compound described herein and a pharmaceutically acceptable carrier.

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

[0013] The present disclosure also relates to a method for modulating tissue distribution or retention of a therapeutic agent in a subject in need thereof, the method comprising administering a compound of the present disclosure. In an embodiment, the compound is administered intrathecally to the subject, and the compound modulates tissue distribution and / or retention of the therapeutic agent in tissues of the central nervous system (CNS). In an embodiment, the compound modulates tissue distribution or retention of the therapeutic agent in muscle tissue.

[0014] The present disclosure also relates to a method for treating a disease or disorder in a subject in need thereof, comprising administering a compound of the present disclosure. In an embodiment, the therapeutic agent is an antisense compound (AC), and administration of the compound modulates splicing or expression of a target gene, degrades mRNA, stabilizes mRNA, or sterically blocks mRNA. In an embodiment, administration of the compound modulates splicing of a target pre-mRNA. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows modified nucleotides used in the antisense oligonucleotides described herein. [Figure 2A] FIG. 1 shows the structures of morpholino subunit monomers used in synthesizing phosphorodiamidate-linked morpholino oligomers. FIG. 2 shows the structure of an adenine morpholino monomer. [Figure 2B] FIG. 1 shows the structures of morpholino subunit monomers used in synthesizing phosphorodiamidate-linked morpholino oligomers. FIG. 2 shows the structure of a cytosine morpholino monomer. [Figure 2C] FIG. 1 shows the structures of morpholino subunit monomers used in synthesizing phosphorodiamidate-linked morpholino oligomers. FIG. 2 shows the structure of a guanine morpholino monomer. [Figure 2D] FIG. 1 shows the structures of morpholino subunit monomers used in synthesizing phosphorodiamidate-linked morpholino oligomers. FIG. 2 shows the structure of a thymine morpholino monomer. [Figure 3A] FIG. 1 shows the conjugation chemistry for connecting AC to cyclic cell-penetrating peptides (cCPPs), depicting amide bond formation between a peptide bearing a carboxylic acid group or a TFP-activated ester and a primary amine residue at the 5′ end of AC. [Figure 3B]FIG. 1 shows conjugation chemistry for connecting AC to cyclic cell-penetrating peptides (cCPPs), conjugation of a secondary amine or primary amine-modified AC at 3′ with a peptide-TFP ester via amide bond formation. [Figure 3C] FIG. 1 shows conjugation chemistry for connecting AC to cyclic cell-penetrating peptides (cCPPs). Conjugation of peptide-azide to 5′ cyclooctyne-modified AC via copper-free azide-alkyne cycloaddition. [Figure 3D] FIG. 1 shows conjugation chemistry for connecting ACs to cyclic cell-penetrating peptides (cCPPs). Figure 1 shows another exemplary conjugation between a 3'-modified cyclooctyne AC or a 3'-modified azide AC and a CPP containing a linker-azide moiety or a linker-alkyne / cyclooctyne moiety via copper-free or copper-catalyzed azide-alkyne cycloaddition (click reaction), respectively. [Figure 4] FIG. 1 shows conjugation chemistries for connecting ACs and CPPs with additional linker modalities containing polyethylene glycol (PEG) moieties. [Figure 5A] FIG. 1 shows the structures of three conjugates used in the Examples, and shows the structure of an oligonucleotide conjugate containing a PMO oligonucleotide and a binding group. [Figure 5B] FIG. 1 shows the structures of three conjugates used in the Examples. This figure shows the structure of a cell-penetrating peptide-oligonucleotide conjugate (PMO-NLS-EEV) containing a PMO oligonucleotide, a binding group, a cyclic cell-penetrating peptide, and a nuclear localization signal. [Figure 5C] FIG. 1 shows the structures of three conjugates used in the Examples, including a conjugate containing a linking group, a polyethylene glycol (PEG) linker, and a cyclic cell-penetrating peptide-CPP12-PEG4-dk(LSR). [Figure 6-1]FIG. 1 shows representative structures of cyclic peptide-linker conjugates of the present disclosure. [Figure 6-2] FIG. 1 shows representative structures of cyclic peptide-linker conjugates of the present disclosure. [Figure 7] FIG. 1 shows additional representative structures of cyclic peptide-linker conjugates of the present disclosure. [Figure 8] General schematic diagrams of antigen degradation constructs are provided. Hash boxes indicate optional linker sequences. The orientation of the constructs shown in these figures is not limiting. Various other general conformations of these constructs are contemplated herein. For example, the CPP can be located at any suitable position in the construct (at the N-terminus as shown, at the C-terminus, or at an internal position in the construct). [Figure 9] FIG. 1 shows the direct action of the degradation moiety leading to target antigen degradation. [Figure 10] FIG. 1 shows the indirect action of the degradation moiety leading to target antigen degradation. [Figure 11] FIG. 1 shows the conjugation of an exemplary CPP and an exemplary AC, as described in Example 3. [Figure 12] 12A-D show the levels of exon 23 correction in muscle tissue from MDX mice after 5 days of treatment with the indicated compounds. Results shown are for the diaphragm (FIG. 12A), heart (FIG. 12B), tibialis anterior (FIG. 12C), and triceps (FIG. 12D). [Figure 13] 13A-C show levels of dystrophin expression in muscle tissue of MDX mice after 5 days of treatment with the indicated compounds. Results shown are for the diaphragm (FIG. 13A), heart (FIG. 13B), and tibialis anterior (FIG. 13C). [Figure 14] 14A and 14B are schematic diagrams showing the synthesis of compounds EEV-PMO-CD33-1 (FIG. 14A) and EEV-PMO-CD33-2 (FIG. 14B). [Figure 15]FIG. 1 shows results from experiments performed by intrathecal (IT) injection of PMO-CD33, EEV-PMO-CD33-2, and EEV-PMO-CD33-1 into rats, followed by analysis of expression in the cerebellum, cortex, hippocampus, and olfactory bulb of the rat brain. [Figure 16] Figures A-C show results from experiments performed by intrathecal (IT) injection of PMO-CD33, EEV-PMO-CD33-2, and EEV-PMO-CD33-1 into rats, followed by analysis of expression in the spinal cord, DRG, and CSF of the rat brain. DETAILED DESCRIPTION OF THE INVENTION

[0016] compound Endosomal escape vehicles (EEVs) Provided herein are endosomal escape vehicles (EEVs) that can be used to transport cargo across a cell membrane, for example, to deliver cargo to the cytosol or nucleus of a cell. The cargo may include a macromolecule, such as a peptide or oligonucleotide, or a small molecule. The EEV may include a cell-penetrating peptide (CPP), such as a cyclic cell-penetrating peptide (cCPP) conjugated to an exocyclic peptide (EP). The EP may be interchangeably referred to as a modulatory peptide (MP). The EP may include a nuclear localization signal (NLS) sequence. The EP may be bound to the cargo. The EP may be bound to the cCPP. The EP may be bound to the cargo and the cCPP. The coupling between the EP, cargo, cCPP, or a combination thereof may be non-covalent or covalent. The EP may be bound to the N-terminus of the cCPP via a peptide bond. The EP may be bound to the C-terminus of the cCPP via a peptide bond. The EP may be bound to the cCPP via a side chain of an amino acid in the cCPP. The EP can be attached to the cCPP via a lysine side chain, which can be conjugated to the glutamine side chain in the cCPP. The EP can be conjugated to the 5' or 3' end of the oligonucleotide cargo. 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 via the C-terminus of the EP and cCPP through a side chain on the cCPP and / or the EP. For example, the EP may contain a terminal lysine, which can then be coupled to a glutamine-containing cCPP via an amide bond. If the EP contains a terminal lysine, and the side chain of the lysine can be used to attach the cCPP, the C-terminus or N-terminus can be attached to the linker on the cargo.

[0017] exocyclic peptides The exocyclic peptide (EP) can contain 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 can contain 6 to 9 amino acid residues. The EP can contain 4 to 8 amino acid residues.

[0018] 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 homolog of a natural amino acid in that it has a structure similar to that of a 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 also be a D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, alloisoleucine, 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, the amino acid can be A, G, P, K, R, V, F, H, NaI, or citrulline.

[0019] 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 its protonated form. The EP may contain one or two amino acid residues containing a side chain containing a guanidine group or its protonated form. 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.

[0020] The EP may contain at least two, at least three, or at least four or more lysine residues. The EP may contain 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 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. Protecting groups may be included to enable amide conjugation. The protecting groups may be removed after the EP is conjugated to the cCPP.

[0021] The EP may comprise at least two amino acid residues having hydrophobic side chains. The amino acid residues having hydrophobic side chains may be selected from valine, proline, alanine, leucine, isoleucine, and methionine. The amino acid residues having hydrophobic side chains may be valine or proline.

[0022] 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.

[0023] 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, KRKKG, 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 β-alanine. The amino acids in the EP can have D or L stereochemistry.

[0024] The EP can 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. The EP can include PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is β-alanine. The amino acids in the EP can have D or L stereochemistry.

[0025] The EP can consist 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. The EP can consist of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is β-alanine. The amino acids in the EP can have D or L stereochemistry.

[0026] 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 amino acids 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 amino acids selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK.

[0027] All exocyclic sequences may also contain an N-terminal acetyl group. Thus, for example, an EP may have the structure: Ac-PKKKRKV.

[0028] Cell-penetrating peptides (CPPs) The cell-penetrating peptide (CPP) can contain 6 to 20 amino acid residues. The cell-penetrating peptide can 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 translocate a cargo (e.g., a therapeutic moiety (TM), such as an oligonucleotide, peptide, or small molecule) to penetrate the cell membrane. The cCPP can deliver the cargo to the cytosol of the cell. The cCPP can deliver the cargo to a cellular location where the target (e.g., pre-mRNA) is located. To conjugate the cCPP to a cargo (e.g., a peptide, oligonucleotide, or small molecule), at least one bond or lone pair on the cCPP can be replaced.

[0029] 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 to 10 amino acid residues can be represented by Formulas IA to IE:

[0030] [ka] wherein AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and AA 10 is an amino acid residue.

[0031] The cCPP may contain 6 to 8 amino acids. The cCPP may contain 8 amino acids.

[0032] 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 homolog of a natural amino acid in that it has a structure similar to that of a 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 also 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.

[0033] [Table 1] * One-letter abbreviations: when shown herein in capital letters, they refer to the L-amino acid form; when shown herein in lower case letters, they refer to the D-amino acid form.

[0034] A cCPP can contain 4 to 20 amino acids, where (i) at least one amino acid has a side chain containing a guanidine group or its protonated form, and (ii) at least one amino acid has no side chain, or

[0035] [ka] or a protonated form thereof, and (iii) at least two amino acids have side chains that independently include an aromatic group or a heteroaromatic group.

[0036] At least two of the amino acids may have no side chains, or

[0037] [ka] or a side chain including its protonated form. As used herein, when no side chain is present, an amino acid has two hydrogen atoms on a carbon atom connecting the amine and carboxylic acid (e.g., -CH-).

[0038] The amino acid having no side chain may be glycine or β-alanine.

[0039] The cCPP can comprise 6 to 20 amino acid residues forming the cCPP, wherein (i) at least one amino acid can be a glycine, β-alanine, or 4-aminobutyric acid residue, (ii) at least one amino acid can have a side chain comprising an aryl or heteroaryl group, (iii) at least one amino acid can have a guanidine group,

[0040] [ka] or may have a side chain containing the protonated form thereof.

[0041] The cCPP may comprise 6 to 20 amino acid residues forming the cCPP, wherein (i) at least two amino acid residues may independently be glycine, β-alanine, or 4-aminobutyric acid residues, (ii) at least one amino acid may have a side chain containing an aryl or heteroaryl group, and (iii) at least one amino acid may have a side chain containing a guanidine group,

[0042] [ka] or has a side chain containing its protonated form.

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

[0044] [ka] or may have side chains containing the protonated form thereof.

[0045] Glycine and related amino acid residues The cCPP may contain (i) 1, 2, 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) 2 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (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.

[0046] The cCPP may contain (i) 1, 2, 3, 4, 5, or 6 glycine residues. The cCPP may contain (i) 2 glycine residues. The cCPP may contain (i) 3 glycine residues. The cCPP may contain (i) 4 glycine residues. The cCPP may contain (i) 5 glycine residues. The cCPP may contain (i) 6 glycine residues. The cCPP may contain (i) 3, 4, or 5 glycine residues. The cCPP may contain (i) 3 or 4 glycine residues. The cCPP may contain (i) 2 or 3 glycine residues. The cCPP may contain (i) 1 or 2 glycine residues.

[0047] The cCPP may contain (i) three, four, five, or six glycine, β-alanine, or 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) three glycine, β-alanine, or 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) four glycine, β-alanine, or 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) five glycine, β-alanine, or 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) six glycine, β-alanine, or 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) three, four, or five glycine, β-alanine, or 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) three or four glycine, β-alanine, or 4-aminobutyric acid residues, or a combination thereof.

[0048] The cCPP may contain at least three glycine residues. The cCPP may contain (i) 3, 4, 5, or 6 glycine residues. The cCPP may contain (i) 3 glycine residues. The cCPP may contain (i) 4 glycine residues. The cCPP may contain (i) 5 glycine residues. The cCPP may contain (i) 6 glycine residues. The cCPP may contain (i) 3, 4, or 5 glycine residues. The cCPP may contain (i) 3 or 4 glycine residues.

[0049] 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 4-aminobutyric acid residues may be adjacent. Two glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent.

[0050] 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.

[0051] Amino acid side chains containing aromatic or heteroaromatic groups A cCPP may comprise 2, 3, 4, 5, or 6 amino acid residues that (ii) independently have a side chain that includes an aromatic group or a heteroaromatic group. A cCPP may comprise 2 amino acid residues that (ii) independently have a side chain that includes an aromatic group or a heteroaromatic group. A cCPP may comprise 3 amino acid residues that (ii) independently have a side chain that includes an aromatic group or a heteroaromatic group. A cCPP may comprise 4 amino acid residues that (ii) independently have a side chain that includes an aromatic group or a heteroaromatic group. A cCPP may comprise 5 amino acid residues that (ii) independently have a side chain that includes an aromatic group or a heteroaromatic group. A cCPP may comprise 6 amino acid residues that (ii) independently have a side chain that includes an aromatic group or a heteroaromatic group. A cCPP may comprise 2, 3, or 4 amino acid residues that (ii) independently have a side chain that includes an aromatic group or a heteroaromatic group. A cCPP can include (ii) two or three amino acid residues that independently have side chains that include aromatic or heteroaromatic groups.

[0052] The cCPP may comprise 2, 3, 4, 5, or 6 amino acid residues that (ii) independently have a side chain that includes an aromatic group. The cCPP may comprise 2 amino acid residues that (ii) independently have a side chain that includes an aromatic group. The cCPP may comprise 3 amino acid residues that (ii) independently have a side chain that includes an aromatic group. The cCPP may comprise 4 amino acid residues that (ii) independently have a side chain that includes an aromatic group. The cCPP may comprise 5 amino acid residues that (ii) independently have a side chain that includes an aromatic group. The cCPP may comprise 6 amino acid residues that (ii) independently have a side chain that includes an aromatic group. The cCPP may comprise 2, 3, or 4 amino acid residues that (ii) independently have a side chain that includes an aromatic group. The cCPP may comprise 2 or 3 amino acid residues that (ii) independently have a side chain that includes an aromatic group.

[0053] The aromatic group can be a 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 a 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.

[0054] Amino acid residues having a side chain containing an aromatic or heteroaromatic group can each independently be bis(homonaphthylalanine), 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. Amino acid residues having a side chain containing an aromatic or heteroaromatic group can each independently be:

[0055] [ka] wherein H on the N-terminus and / or H on the C-terminus is replaced by a peptide bond.

[0056] Amino acid residues having a side chain containing an aromatic or heteroaromatic group can 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-butylphenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 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 side chains containing an aromatic group may be residues of phenylalanine. Each amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine.

[0057] 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.

[0058] 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.

[0059] An optional substituent can be, for example, any atom or group that does not significantly (e.g., by more than 50%) reduce the cytoplasmic delivery efficiency of the cCPP compared to an otherwise identical sequence lacking the substituent. An optional substituent can be a hydrophobic or hydrophilic substituent. An optional substituent can be a hydrophobic substituent. The substituent can increase the solvent accessible surface area (as defined herein) of the hydrophobic amino acid. A substituent can be halogen, alkyl, alkenyl, alkynylene, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio. A substituent can be halogen.

[0060] Without wishing to be bound by theory, it is believed that amino acids having aromatic or heteroaromatic groups with higher hydrophobicity values ​​(i.e., amino acids having 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.

[0061] [Table 2]

[0062] The size of the aromatic or heteroaromatic group can be selected to improve the cytoplasmic delivery efficiency of cCPPs. 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 cytoplasmic delivery efficiency compared to an otherwise identical sequence having a smaller hydrophobic amino acid. The size of a 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 a 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 an amino acid can be measured in terms of the SASA of the hydrophobic side chain. A hydrophobic amino acid can have a side chain with a SASA equal to or greater than that of alanine, or equal to or greater than that of glycine. A larger hydrophobic amino acid can have a side chain with a SASA greater than that of alanine, or greater than that of 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 The second hydrophobic amino acid (AA H2 ) 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 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 and at least about 370 Å 2 , at least about 380 Å 2 , at least about 390 Å 2 and at least about 400 Å 2 , at least about 410 Å 2 and at least about 420 Å 2 , at least about 430 Å 2 and at least about 440 Å 2 and at least about 450 Å 2 , at least about 460 Å 2 and at least about 470 Å 2 , at least about 480 Å 2 and at least about 490 Å 2 , about 500Å 2 Greater than 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 Å2 , at least about 630 Å 2 , at least about 640 Å 2 , approximately 650 Å 2 Greater than 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 (I). By way of example, and not limitation, a cCPP having a NaI-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 NaI-Phe-Arg motif, and a phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a NaI-Phe-Arg motif.

[0063] As used herein, "hydrophobic surface area" or "SASA" refers to the solvent accessible surface area of ​​an amino acid side chain (square angstroms: Å). 2 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 herein 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.

[0064] 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.

[0065] [Table 3]

[0066] Amino acid residues having a side chain containing a guanidine group, a guanidine substituent, or their protonated forms As used herein, guanidine has the structure:

[0067] [ka] Refers to...

[0068] As used herein, the protonated form of guanidine has the structure:

[0069] [ka] Refers to...

[0070] 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 mimic the hydrogen bond donating and accepting activity of a guanidinium group.

[0071] 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 comprise at least one amino acid having a side chain containing a guanidine or guanidinium substituent. The cCPP may comprise at least two amino acids having side chains containing guanidine or guanidinium substituents. The cCPP may comprise at least three amino acids having side chains containing guanidine or guanidinium substituents.

[0072] The guanidine or guanidinium group may be an isostere of guanidine or guanidinium. The guanidine or guanidinium substituent may be less basic than guanidine.

[0073] As used herein, a guanidine substituent is:

[0074] [ka] or its protonated form.

[0075] 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 its protonated form, and (ii) at least one amino acid residue has no side chain, or

[0076] [ka] or a protonated form thereof, and (iii) at least two amino acid residues have side chains that independently include an aromatic group or a heteroaromatic group.

[0077] At least two of the amino acid residues may have no side chains, or

[0078] [ka] or a side chain including its protonated form. As used herein, when no side chain is present, an amino acid residue has two hydrogen atoms on the carbon atom connecting the amine and carboxylic acid (e.g., -CH2-).

[0079] cCPP consists of the following parts:

[0080] [ka] or at least one amino acid having a side chain comprising one of the following:

[0081] A cCPP can include at least two amino acids, each amino acid independently comprising the following moiety:

[0082] [ka] or one of its protonated forms. At least two amino acids are

[0083] [ka] or its protonated form. At least one amino acid may have a side chain containing the same moiety selected from

[0084] [ka] or a side chain containing the protonated form thereof. At least two amino acids

[0085] [ka] or a side chain containing its protonated form.

[0086] [ka] or a side chain containing the protonated form thereof.

[0087] [ka] or the side chain may include its protonated form.

[0088] [ka] or may have side chains containing the protonated form thereof.

[0089] [ka] Or the protonated form can be attached to the terminus of an amino acid side chain.

[0090] [ka] can be attached to the terminus of an amino acid side chain.

[0091] The cCPP may comprise two, three, four, five, or six amino acid residues that independently have a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise two amino acid residues that independently have a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise three amino acid residues that independently have a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise four amino acid residues that independently have a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise five amino acid residues that independently have a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise six amino acid residues that independently have a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise two, three, four, or five amino acid residues that independently have a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise two, three, or four amino acid residues that independently have a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise two or three amino acid residues that independently have a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise at least one amino acid residue that has a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise two amino acid residues that have a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise three amino acid residues that have a side chain comprising a guanidine group or a protonated form thereof.

[0092] The amino acid residues may independently have side chains containing non-adjacent guanidine groups, guanidine substituents, or their protonated forms. Two amino acid residues may independently have side chains containing guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Three amino acid residues may independently have side chains containing guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Four amino acid residues may independently have side chains containing 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 alternate stereochemistry.

[0093] The amino acid residues independently having side chains containing a guanidine group, a guanidine substituent, or a protonated form thereof can be L-amino acids. The amino acid residues independently having side chains containing a guanidine group, a guanidine substituent, or a protonated form thereof can be D-amino acids. The amino acid residues independently having side chains containing a guanidine group, a guanidine substituent, or a protonated form thereof can be a mixture of L- and D-amino acids.

[0094] Each amino acid residue having a side chain containing a guanidine group or its protonated form can independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid, or its protonated form. Each amino acid residue having a side chain containing a guanidine group or its protonated form can independently be a residue of arginine or its protonated form.

[0095] Each amino acid having a side chain containing a guanidine substituent or its protonated form may independently be

[0096] [ka] or its protonated form.

[0097] Without being bound by theory, it is hypothesized that the guanidine substituent has reduced basicity compared to arginine and, in some cases, is uncharged (e.g., -N(H)C(O)) at physiological pH, allowing it to maintain bidentate hydrogen-bonding interactions with phospholipids on the plasma membrane, which is believed to facilitate effective membrane binding and subsequent internalization. Removal of the positive charge is also believed to reduce the toxicity of cCPPs.

[0098] Those skilled in the art will understand that the N-terminus and / or C-terminus of the non-natural flavor hydrophobic amino acids form an amide bond upon incorporation into the peptides disclosed herein.

[0099] A cCPP can include a first amino acid having a side chain comprising an aromatic or heteroaromatic group and a second amino acid having a side chain comprising 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 comprising 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 comprising 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" can refer to the amino acid located at the N-terminus of a peptide sequence.

[0100] 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.

[0101] The cCPP can include a first amino acid having a side chain comprising a guanidine group or its protonated form, and a second amino acid having a side chain comprising a guanidine group or its protonated form, 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 its protonated form, 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 its protonated form.

[0102] The cCPP may comprise an asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine residue. The cCPP may comprise an asparagine residue. The cCPP may comprise a glutamine residue.

[0103] cCPPs can each independently comprise 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.

[0104] Without wishing to be bound by theory, it is believed that the chirality of amino acids in a cCPP can affect cytoplasmic uptake efficiency. A cCPP can contain at least one D amino acid. A cCPP can contain 1 to 15 D amino acids. A cCPP can contain 1 to 10 D amino acids. A cCPP can contain 1, 2, 3, or 4 D amino acids. A cCPP can contain 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids with alternating D and L chirality. A cCPP can contain three adjacent amino acids with the same chirality. A cCPP can contain two adjacent amino acids with the same chirality. At least two amino acids can have opposite chirality. At least two amino acids with opposite chirality can be adjacent to each other. At least three amino acids can have alternate stereochemistry relative to each other. At least three amino acids with alternate chirality relative to each other can be adjacent to each other. At least four amino acids have alternate stereochemistry relative to one another. At least four amino acids of alternate chirality relative to one another can be adjacent to one another. At least two amino acids can have the same chirality. At least two amino acids of the same chirality can be adjacent to one another. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids of opposite chirality can be adjacent to at least two amino acids of 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.

[0105] 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 contain 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 contain the following sequences: DXD, DXDX, DXDXD, LXL, LXLX, or LXLXL (where X is an achiral amino acid). The achiral amino acid may be glycine.

[0106] [ka] Or, an amino acid having a side chain comprising its protonated form can be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group.

[0107] [ka] or its protonated form can be adjacent to at least one amino acid having a side chain comprising guanidine or its protonated form. An amino acid having a side chain comprising guanidine or its protonated form can be adjacent to an amino acid having a side chain comprising an aromatic group or a heteroaromatic group.

[0108] [ka] Two amino acids having side chains containing guanidine or its protonated form can be adjacent to each other. Two amino acids having side chains containing guanidine or its protonated form can be adjacent to each other. A cCPP comprises at least two adjacent amino acids having side chains that can contain an aromatic or heteroaromatic group, and

[0109] [ka] or at least two non-adjacent amino acids having side chains containing an aromatic or heteroaromatic group, or a protonated form thereof. A cCPP may have at least two adjacent amino acids having side chains containing an aromatic or heteroaromatic group, and

[0110] [ka] or at least two non-adjacent amino acids having side chains containing the protonated form thereof. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chiralities. Other combinations of amino acids may have any arrangement of D and L amino acids, for example, any of the sequences described in the previous paragraph.

[0111] [ka] Or at least two amino acids having a side chain containing a guanidine group or its protonated form alternate with at least two amino acids having a side chain containing a guanidine group or its protonated form.

[0112] cCPP has the formula (A):

[0113] [ka] (In the formula, 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; The cyclic peptide of formula (A) may comprise the structure FfΦRrRrQ (but not FfΦRrRrQ) or a protonated form thereof.

[0114] cCPP has the formula (I):

[0115] [ka] (In the formula, 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) or a protonated form thereof.

[0116] 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 -C1~3 R1, R2, and R3 can each independently be H or -alkylene-aryl. R1, R2, and R3 can each independently be H or -C 1~3 It can be 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, or isoquinolyl. R1, R2, and R3 are each independently selected from H, -C, 1~3 Alkylene-Ph or -C 1~3 R1, R2, and R3 can each independently be H, -CH2Ph, or -CH2naphthyl. R1, R2, and R3 can each independently be H or -CH2Ph.

[0117] 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, or 3-(9-anthryl)-alanine.

[0118] 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.

[0119] 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.

[0120] R3 can be the side chain of tyrosine. R3 can be the side chain of phenylalanine. R3 can be the side chain of 1-naphthylalanine. R3 can be the side chain of 2-naphthylalanine. R3 can be the side chain of tryptophan. R3 can be the side chain of 3-benzothienylalanine. R3 can be the side chain of 4-phenylphenylalanine. R3 can be the side chain of 3,4-difluorophenylalanine. R3 can be the side chain of 4-trifluoromethylphenylalanine. R3 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 can be the side chain of homophenylalanine. R3 can be the side chain of β-homophenylalanine. R3 can be the side chain of 4-tert-butyl-phenylalanine. R3 can be the side chain of 4-pyridinylalanine. R3 can 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.

[0121] 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 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, or isoquinolyl. R4 can be H, -C 1~3 Alkylene-Ph or -C 1~3R4 can be alkylene-naphthyl. R4 can be H or the side chain of an amino acid in 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 -CH2 naphthyl. R4 can be H or -CH2Ph.

[0122] 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 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, or 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 in 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.

[0123] 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 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, or 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 in 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.

[0124] 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 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, or 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 in 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] At least one of R4, R5, R6, and R7 can be the side chain of 3-guanidino-2-aminopropionic acid. At least one of R4, R5, R6, and R7 can be the side chain of 4-guanidino-2-aminobutanoic acid. At least one of R4, R5, R6, and R7 can be the side chain of arginine. At least one of R4, R5, R6, and R7 can be the side chain of homoarginine. At least one of R4, R5, R6, and R7 can be the side chain of N-methylarginine. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethylarginine. At least one of R4, R5, R6, and R7 can be the side chain of 2,3-diaminopropionic acid. At least one of R4, R5, R6, and R7 can be the 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.

[0130] 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.

[0131] 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.

[0132] AA SC can be the side chain of an asparagine, glutamine, or homoglutamine residue. SC may 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.

[0133] 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.

[0134] 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.

[0135] The cCPP of formula (A) is a compound of formula (I)

[0136] [ka] (In the formula, AA SC , R1, R2, R3, R 4- , R6, m and q are as defined herein) or a protonated form thereof.

[0137] The cCPP of formula (A) is represented by formula (Ia) or formula (Ib):

[0138] [ka] (In the formula, AA SC , R1, R2, R3, R4, and m are as defined herein) or a protonated form thereof.

[0139] The cCPP of formula (A) is represented by formula (I-1), (I-2), (I-3) or (I-4):

[0140] [ka] (In the formula, AA SCand m is as defined herein) or a protonated form thereof.

[0141] The cCPP of formula (A) is represented by formula (I-5) or (I-6):

[0142] [ka] (In the formula, AA SC is as defined herein) or a protonated form thereof.

[0143] The cCPP of formula (A) is represented by formula (I-1):

[0144] [ka] (In the formula, AA SC and m is as defined herein), or a protonated form thereof.

[0145] The cCPP of formula (A) is represented by formula (I-2):

[0146] [ka] (In the formula, AA SC and m is as defined herein), or a protonated form thereof.

[0147] The cCPP of formula (A) is represented by formula (I-3):

[0148] [ka] (In the formula, AA SC and m is as defined herein), or a protonated form thereof.

[0149] The cCPP of formula (A) is represented by formula (I-4):

[0150] [ka] (In the formula, AA SC and m is as defined herein), or a protonated form thereof.

[0151] The cCPP of formula (A) is represented by formula (I-5):

[0152] [ka] (In the formula, AA SC and m is as defined herein), or a protonated form thereof.

[0153] The cCPP of formula (A) is represented by formula (I-6):

[0154] [ka] (In the formula, AA SC and m is as defined herein), or a protonated form thereof.

[0155] The cCPP may comprise one of the following sequences: FGFGRGR, GfFGrGr, FfΦGRGR, FfFGRGR, or FfΦGrGr. The cCPP may have one of the following sequences: FGFΦ, GfFGrGrQ, FfΦGRGRQ, FfFGRGRQ, or FfΦGrGrQ.

[0156] The present disclosure also provides a compound of formula (II):

[0157] [ka] (In the formula, 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; R2a , 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

[0158] [ka] or its protonated form; R 2a , R 2b , R 2c and R 2d at least one of which is guanidine or its protonated form; 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, then R 2a , R 2b , R 2b , or R 2d This relates to a cCPP having the structure (wherein α- and β-terminal fragments do not exist).

[0159] R 2a , R 2b , R 2c , and R 2d At least two of the

[0160] [ka] or its protonated form. 2a , R 2b , R 2c , and R 2d Two or three of them are

[0161] [ka] or its protonated form. 2a , R 2b, R 2c , and R 2d One of them is

[0162] [ka] or its protonated form. 2a , R 2b , R 2c and R 2d At least one of the

[0163] [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

[0164] [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d The remainder may be guanidine or its protonated form.

[0165] R 2a , R 2b , R 2c , and R 2d All of this is

[0166] [ka] or its protonated form. 2a , R 2b , R 2c and R 2d At least one of

[0167] [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d The remainder of R may be a guaninide or its protonated form. 2a , R 2b , R 2c and R 2d The base is

[0168] [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d The remainder is guanidine or its protonated form.

[0169] 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.

[0170] AA SC teeth,

[0171] [ka] where t can be an integer from 0 to 5. SC teeth

[0172] [ka] (wherein t can be an integer from 0 to 5. t can be 1 to 5. t can be 2 or 3. t can be 2. t can be 3).

[0173] R 1a , R 1b , and R 1c Each R can independently be a 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 can be each independently selected pyridyl, quinolyl, or isoquinolyl.

[0174] 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.

[0175] 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.

[0176] Each n" can independently be 1 or 2, but each n' can independently be 2 or 3. Each n" can be 1, but each n' can independently be 2 or 3. Each n" can be 1, but each n' can be 2. Each n" is 1 and each n' is 3.

[0177] The cCPP of formula (II) is represented by the formula (II-1):

[0178] [ka] (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).

[0179] The cCPP of formula (II) has the formula (IIa):

[0180] [ka] (In the formula, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , A.A. SC and n' is as defined herein).

[0181] The cCPP of formula (II) may be represented by the formula (IIb):

[0182] [ka] (In the formula, R 2a , R 2b , A.A. SC and n′ is as defined herein).

[0183] cCPP has the formula (IIb):

[0184] [ka] (In the formula, AA SC and n' are as defined herein), or a protonated form thereof.

[0185] The cCPP of formula (IIa) has the following structure:

[0186] [ka] (In the formula, AA SC and n is as defined herein).

[0187] The cCPP of formula (IIa) has the following structure:

[0188] [ka] (In the formula, AA SC and n is as defined herein).

[0189] The cCPP of formula (IIa) has the following structure:

[0190] [ka] (In the formula, AA SC and n is as defined herein).

[0191] The cCPP of formula (II) has the structure:

[0192] [ka] may have:

[0193] The cCPP of formula (II) has the structure:

[0194] [ka] may have:

[0195] cCPP has the formula (III):

[0196] [ka] (In the formula, 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,

[0197] [ka] or its protonated form; R 2b and R 2d are each independently guanidine or its protonated form; 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.

[0198] The cCPP of formula (III) is represented by the formula (III-1):

[0199] [ka] (In the formula, AA SC , R 1a , R 1b , R 1c , R 2a , R 2c , R 2b , R 2d , n', n'' and p' are as defined herein).

[0200] The cCPP of formula (III) has the formula (IIIa):

[0201] [ka] (In the formula, AA SC , R 2a , R 2c , R 2b , R 2d , n', n'', and p' are as defined herein).

[0202] In formulas (III), (III-1), and (IIIa), R a and R c can be H. R a and R c can be H, but R b and R d R can each independently be guanidine or its protonated form. a can be H. R b can be H. p' can be 0. R a and R c can be H, but each p' can be 0.

[0203] In formulas (III), (III-1) and (IIIa), R a and R c can be H, but R b and R dcan each independently be guanidine or its protonated form, n'' can be 2 or 3, and each p' can be 0.

[0204] p' can be 0. p' can be 1. p' can be 2. p' can be 3. p' can be 4. p' can be 5.

[0205] cCPP has the structure:

[0206] [ka] may have:

[0207] The cCPP of formula (A) can be selected from:

[0208] [Table 4]

[0209] The cCPP of formula (A) can be selected from:

[0210] [Table 5]

[0211] AA SC can be conjugated to a linker.

[0212] In embodiments, the cCPP can be selected from the following:

[0213] [Table 6] Φ = L-naphthylalanine; φ = D-naphthylalanine; Ω = L-norleucine

[0214] In embodiments, the cCPP may be selected from:

[0215] [Table 7] Φ = L-naphthylalanine; φ = D-naphthylalanine; Ω = L-norleucine

[0216] Linker The cCPP of the present disclosure can be conjugated to a linker. The linker can link a cargo to the cCPP. The linker can be attached to the side chain of an amino acid of the cCPP, and the cargo can be attached to an appropriate position on the linker.

[0217] The linker may be any suitable moiety capable of conjugating the cCPP to one or more additional moieties, such as an exocyclic peptide (EP) and / or cargo. Prior to attachment to the cCPP and one or more additional moieties, the linker has two or more functional groups, each capable of independently forming a covalent bond to the cCPP and one or more additional moieties. When the cargo is an oligonucleotide, the linker may be covalently attached to the 5'-end of the cargo or the 3'-end of the cargo. The linker may be covalently attached to the 5'-end of the cargo. The linker may be covalently attached to the 3'-end of the cargo. When the cargo is a peptide, the linker may be covalently attached to the N-terminus or C-terminus of the cargo. The linker may be covalently attached to the backbone of an oligonucleotide or peptide cargo. The linker may be any suitable moiety capable of conjugating the cCPP described herein to a cargo such as an oligonucleotide, peptide, or small molecule.

[0218] The linker may comprise a hydrocarbon linker.

[0219] The linker may comprise a cleavage site, which may be a disulfide or a caspase cleavage site (e.g., Val-Cit-PABC).

[0220] 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, where 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; and each J is independently C, NR 3 , -NR 3 C(O)—, S, or O, wherein 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; alternatively, (ix) the linker may include one or more of (i) through (x).

[0221] The linker may comprise 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.

[0222] The linker is -(OCH2CH2) z’ - (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) (e.g., as a spacer). "-(OCHCH) can also be referred to as polyethylene glycol (PEG).

[0223] 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 contain a functional group (FG) that can react via click chemistry. FG may be an azide or an alkyne, and when the cargo is conjugated to the linker, a triazole is formed.

[0224] The linker consists of (i) β-alanine and lysine residues, (ii) -(JR 1 )z-, or (iii) combinations thereof. 1 can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can independently be C, NR 3 , -NR 3 C(O)—, S, or O, wherein 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.

[0225] 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 independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can independently be C, NR 3 , -NR 3 C(O)—, S, or O, wherein 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, while each J can be O. The linker can include glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0226] The linker can be a trivalent linker. The linker has the structure:

[0227] [ka] wherein A1, B1, and C1 can independently be 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, while Z is independently a protecting group. Linkers also include disulfides [NH-(CHO) n -SS-(CH2O) n -COOH], or a cleavage site such as a caspase cleavage site (Val-Cit-PABC) can be incorporated.

[0228] The carbohydrate may be a residue of glycine or β-alanine.

[0229] The linker is bivalent and can link the cCPP to a cargo. The linker is bivalent and can link the cCPP to an exocyclic peptide (EP).

[0230] The linker may be trivalent, but is capable of linking the cCPP to the cargo and the EP.

[0231] The linker is a divalent or trivalent C1-C 50 and alkylene, wherein 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 may be a divalent or trivalent C1-C 50 It can be alkylene, wherein 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or combinations thereof.

[0232] The linker has the structure:

[0233] [ka] wherein each AA is independently an amino acid residue; * AA SC AA SCis 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.

[0234] The cCPP can be linked to the cargo via a linker ("L"), which can be conjugated to the cargo via a linking group ("M").

[0235] The linker has the structure:

[0236] [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 the cCPP, and M is a linking group as defined herein.

[0237] The linker has the structure:

[0238] [ka] and In the formula, x' is an integer of 1 to 23, y is an integer of 1 to 5, and z' is an integer of 1 to 23. * AA SC AA SC is the side chain of an amino acid residue of the cCPP, and M is a linking group as defined herein.

[0239] The linker has the structure:

[0240] [ka] and In the formula, x' is an integer of 1 to 23, y is an integer of 1 to 5, and z' is an integer of 1 to 23. * AA SC AA SC is the side chain of an amino acid residue of the cCPP, and M is a linking group as defined herein.

[0241] The linker has the structure:

[0242] [ka] and In the formula, x' is an integer of 1 to 23, y is an integer of 1 to 5, and z' is an integer of 1 to 23. * AA SC AA SC is the side chain of an amino acid residue in cCPP.

[0243] 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.

[0244] x' can be an integer from 1 to 23, for example, 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.

[0245] y can be an integer from 1 to 5, for example, 1, 2, 3, 4, or 5 (including all ranges and subranges therebetween), but 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.

[0246] 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.

[0247] 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.

[0248] As described above, the linker or M (where M is part of the linker) can be covalently attached to the cargo at any suitable position on the cargo. The linker or M (where M is part of the linker) can be covalently attached to the 3'-end of the oligonucleotide cargo or the 5'-end of the oligonucleotide cargo. The linker or M (where M is part of the linker) can be covalently attached to the N-terminus or C-terminus of the peptide cargo. The linker or M (where M is part of the linker) can be covalently attached to the backbone of the oligonucleotide or peptide cargo.

[0249] The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group). The linker can be attached to the side chain of lysine on the cCPP.

[0250] The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the peptide cargo, or to a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group). The linker can be attached to the side chain of lysine on the peptide cargo.

[0251] The linker has the structure:

[0252] [ka] and During the ceremony, M is a group that conjugates L to a cargo, e.g., an oligonucleotide; 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.

[0253] The linker has the structure:

[0254] [ka] and During the ceremony, M is a group that conjugates L to a cargo, e.g., an oligonucleotide; 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.

[0255] M can include alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted.

[0256] [ka] wherein R is alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl.

[0257] M is

[0258] [ka] You can choose from R 10 is alkylene, cycloalkyl, or

[0259] [ka] where a is 0 to 10.

[0260] M is

[0261] [ka] However, R 10 teeth

[0262] [ka] where a is from 0 to 10. M can be

[0263] [ka] It could be.

[0264] M is a heterobifunctional crosslinker, e.g.,

[0265] [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.

[0266] M can be —C(O)—.

[0267] AA s can 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.

[0268] Each AA x are independently natural or unnatural amino acids. x can be a natural amino acid. x may be an unnatural amino acid. x may be a β-amino acid. The β-amino acid may be β-alanine.

[0269] 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.

[0270] p can be 0 to 5, for example, 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.

[0271] The linker has the structure:

[0272] [ka] and In the formula, M, AA s , each -(R 1- JR 2 )z″-, o, and z″ are defined herein. r can be 0 or 1.

[0273] r can be 0. r can be 1.

[0274] The linker has the structure:

[0275] [ka] and In the formula, M, AA s , o, p, q, r, and z may each be as defined herein.

[0276] z" can be an integer from 1 to 50, 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, 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.

[0277] The linker has the structure:

[0278] [ka] and During the ceremony, M, A.A. s and o are as defined herein.

[0279] Other non-limiting examples of suitable linkers include:

[0280] [ka]

[0281] [ka] are listed, In the formula, M and AA s is as defined herein.

[0282] Provided herein is a compound comprising a cCPP and an AC complementary to a target in a pre-mRNA sequence, further comprising L, wherein a linker is conjugated to the AC via a linking group (M), wherein M is

[0283] [ka] A compound is provided, wherein:

[0284] Provided herein is a compound comprising a cCPP and a cargo comprising an antisense compound (AC), e.g., an antisense oligonucleotide complementary to a target in a pre-mRNA sequence, further comprising L, wherein the linker is conjugated to the AC via a linking group (M), wherein M is

[0285] [ka] Selected from R 1 is alkylene, cycloalkyl, or

[0286] [ka] wherein t' is 0 to 10, each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, and R 1 teeth

[0287] [ka] and t' is 2.

[0288] The linker has the structure:

[0289] [ka] and During the ceremony, A.A. s is as defined herein, and m' is 0-10.

[0290] The linker has the formula:

[0291] [ka] It can be of the following type.

[0292] The linker has the formula:

[0293] [ka] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0294] The linker has the formula:

[0295] [ka] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0296] The linker has the formula:

[0297] [ka] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0298] The linker has the formula:

[0299] [ka] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0300] The linker has the formula:

[0301] [ka] It can be of the following type.

[0302] The linker can be covalently attached to the cargo at any suitable position on the cargo. The linker is covalently attached to the 3' end of the cargo or the 5' end of an oligonucleotide cargo. The linker can be covalently attached to the backbone of the cargo.

[0303] The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group). The linker can be attached to the side chain of lysine on the cCPP.

[0304] cCPP-linker conjugates The cCPP may be conjugated to a linker as defined herein. The linker may be a linker between the AA SC can be conjugated to

[0305] The linker is -(OCH2CH2) z’ wherein 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. z’ is also referred to as PEG. The cCPP-linker conjugate may have a structure selected from Table 4.

[0306] [Table 8]

[0307] The linker is -(OCH2CH2) z’- (where z' is an integer from 1 to 23) subunits 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.

[0308] [Table 9]

[0309] The cCPP-linker conjugate can have the structure shown in FIG. 1 (eg, Compound 1a, Compound 1b, Compound 2a, or Compound 3a) or the sequence listed in Table 4.

[0310] The cCPP-linker conjugate may have a sequence listed in Table 5.

[0311] The cCPP-linker conjugate can be Ac-PKKKRKV-K(cyclo[FfΦGrGrQ])-PEG-K(N3)-NH2. 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):

[0312] [ka] (In the formula, 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; and z' is an integer from 1 to 23), or a protonated form thereof.

[0313] R1, R2, R3, R4, R7, EP, m, q, y, x', z' are as described herein.

[0314] n can be 0. n can be 1. n can be 2.

[0315] EEV has the formula (Ba) or (Bb):

[0316] [ka]

[0317] [ka] (In the formula, EP, R 1 , R 2 , R 3 , R 4 , m and z' are as defined above in formula (B), or a protonated form thereof.

[0318] EEV is calculated using the formula (Bc):

[0319] [ka] (In the formula, EP, R 1 , R 2 , R 3 , R 4 and 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), or a protonated form thereof.

[0320] EEV is represented by the formula (B-1), (B-2), (B-3), or (B-4):

[0321] [ka]

[0322] [ka]

[0323] [ka] where EP is as defined above in formula (B), or a protonated form.

[0324] The EEV may comprise the formula (B), structure: Ac-PKKKRKVAEEA-K(cyclo[FGFGRGRQ])-PEG 12 -OH or Ac-PK-KKR-KV-AEEA-K(cyclo[GfFGrGrQ])-PEG 12 It may have -OH.

[0325] The EEV may include a cCPP of the following formula:

[0326] [ka]

[0327] The EEV may comprise the formula: Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3).

[0328] The EEV can be:

[0329] [ka]

[0330] EEV is

[0331] [ka] It could be.

[0332] The EEV can be Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-miniPEG-K(cyclo-Ff-Nal-GrGrQ)-PEG-OH.

[0333] EEV is

[0334] [ka] It could be.

[0335] The EEV can be Ac-PKKKRKV-miniPEG-K(cyclo(Ff-Nal-GrGrQ)-PEG-OH).

[0336] The EEV can be:

[0337] [ka]

[0338] The EEV can be:

[0339] [ka]

[0340] The EEV can be:

[0341] [ka]

[0342] The EEV can be:

[0343] [ka]

[0344] EEV is

[0345] [ka] It could be.

[0346] The EEV can be:

[0347] [ka]

[0348] The EEV can be:

[0349] [ka]

[0350] The EEV can be:

[0351] [ka]

[0352] EEV is

[0353] [ka] It could be.

[0354] The EEV can be:

[0355] [ka]

[0356] EEV is

[0357] [ka] It could be.

[0358] The EEV may be selected from the following:

[0359] [Table 10]

[0360] The EEV may be selected from the following:

[0361] [Table 11]

[0362] The EEV may be selected from the following:

[0363] [Table 12]

[0364] The EEV may be selected from the following:

[0365] [Table 13]

[0366] The EEV may be selected from the following: Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2 Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-OH Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH2 Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG2-OH

[0367] The cargo may be a protein and the EEV may be selected from: Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12-OH Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FfF-GRGRQ])-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[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[Ff-Nal-Cit-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(cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rrr-PEG2-K(シクロ[FfF-GRGRQ])-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[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[FfF-GRGRQ])-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[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(シクロ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbr-PEG2-K(シクロ[FfF-GRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(シクロ[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[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(cyclo[FGFGRGRQ])-PEG12-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[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FfF-GRGRQ])-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[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FfF-GRGRQ])-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 β-alanine and the exocyclic sequence can be of D or L stereochemistry.

[0368] In embodiments, provided herein is a TM conjugated to two CPPs. Non-limiting examples of the structure of a TM conjugated to two CPPs are provided below. For illustrative purposes only, the TM in the structure shown is an AC. Other TMs (e.g., therapeutic polypeptides) can also be used. The underlines represent exemplary antisense oligonucleotides. The antisense oligonucleotide sequences shown below are for illustrative purposes only and can be replaced with other antisense oligonucleotide sequences depending on the target of interest. In embodiments, provided herein is a TM conjugated to three CPPs. Non-limiting examples of the structure of a TM conjugated to three CPPs are provided below. For illustrative purposes only, the TM in the structure shown is an AC. Other TMs (e.g., therapeutic polypeptides) can also be used. The underlines represent antisense oligonucleotides. The antisense oligonucleotide sequences shown below are for illustrative purposes only and can be replaced with other antisense oligonucleotide sequences depending on the target of interest.

[0369] cargo A cell-penetrating peptide (CPP), for example, a cyclic cell-penetrating peptide (e.g., cCPP), can be conjugated to a cargo. The cargo can be a therapeutic moiety (TM). The cargo can be conjugated to the terminal carbonyl group of a linker. At least one atom of the cyclic peptide can be substituted by a cargo, or at least one lone pair of electrons can form a bond to the cargo. The cargo can be conjugated to the cCPP via a linker. The cargo can be linked to the AA SC At least one atom of the cCPP can be replaced with a therapeutic moiety, or at least one lone pair of electrons of the cCPP can form a bond to a therapeutic moiety. A hydroxyl group on an amino acid side chain of the cCPP can be replaced with a bond to a cargo. A hydroxyl group on a glutamine side chain of the cCPP can be replaced with a bond to a cargo. The cargo can be conjugated to the cCPP by a linker. The cargo can be linked to an AA SCcan be conjugated to

[0370] The cargo may comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof. The cargo may be a peptide, an oligonucleotide, or a small molecule. The cargo may be a peptide sequence or a non-peptidyl therapeutic agent. The cargo may be an antibody or an antigen-binding fragment thereof, such as, but not limited to, an scFv or a nanobody.

[0371] The cargo may include one or more additional amino acids (e.g., K, UK, TRV), a linker (e.g., the bifunctional linker LC-SMCC), coenzyme A, phosphocoumarin aminopropionic acid (pCAP), 8-amino-3,6-dioxaoctanoic acid (miniPEG), L-2,3-diaminopropionic acid (Dap or J), L-β-naphthylalanine, L-pipecolic acid (Pip), sarcosine, trimesic acid, 7-amino-4-methylcoumarin (Amc), fluorescein isothiocyanate (FITC), L-2-naphthylalanine, norleucine, 2-aminobutyric acid, rhodamine B (Rho), dexamethasone (DEX), or a combination thereof.

[0372] The cargo may include any of those listed in Table 6, or derivatives or combinations thereof.

[0373] [Table 14] * pCAP, phosphocoumarin aminopropionic acid; Ω, norleucine; U, 2-aminobutyric acid; D-pThr is D-phosphothreonine; and Pip is L-piperidine-2-carboxylate.

[0374] Detectable Part The compound may comprise a detectable moiety. The detectable moiety can be attached to a cell-penetrating peptide (CPP) at the amino group, carboxylate group, or side chain of any amino acid in the CPP (e.g., at the amino group, carboxylate group, or side chain of any amino acid in the cCPP). The detectable moiety can be attached to a cyclic cell-penetrating peptide (cCPP) at the side chain of any amino acid in the cCPP. The cargo may comprise a detectable moiety. The cargo may comprise a therapeutic agent and a detectable moiety. The detectable moiety may comprise any detectable label. Examples of suitable detectable labels include, but are not limited to, UV-Vis labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable moieties, chelating centers, heavy atoms, radioisotopes, isotopically detectable spin resonance labels, paramagnetic moieties, chromophores, or any combination thereof. The label may be detectable without the addition of additional reagents.

[0375] The detectable moiety may be a biocompatible detectable moiety, such that the compound may be suitable for use in a variety of biological applications. "Biocompatible" and "biologically compatible," as used herein, generally refer to compounds (together with any of their metabolic or degradation products) that are generally non-toxic to cells and tissues and do not cause any significant adverse effects to cells and tissues when the cells and tissues are incubated (e.g., cultured) in their presence.

[0376] The detectable moiety may comprise a luminophore, such as a fluorescent or near-infrared label. Examples of suitable luminophores include, but are not limited to, metalloporphyrins, benzoporphyrins, azabenzoporphyrins, napthoporphyrins, phthalocyanines, polycyclic aromatic hydrocarbons (such as diimines and pyrenes), azo dyes, xanthene dyes, boron dipyrromethenes, aza-boron dipyrromethenes, cyanine dyes, metal-ligand complexes (such as bipyridines, bipyridyls, phenanthrolines, and coumarins), and ruthenium and iridium acetylacetonates, acridines, oxazine derivatives (such as benzophenoxazines), aza-annulenes, squaraines, 8-hydroxyquinolines, polymethines, luminescence-generating nanoparticles (such as quantum dots and nanocrystals), carbostyrils, terbium complexes, inorganic fluorophores, ionophores (such as crown ether series or derivatized dyes), or combinations thereof. Specific examples of suitable luminophores include Pd(II) octaethylporphyrin, Pt(II)-octaethylporphyrin, Pd(II) tetraphenylporphyrin, Pt(II) tetraphenylporphyrin, Pd(II) meso-tetraphenylporphyrin tetrabenzoporphin, Pt(II) meso-tetraphenylmethrylbenzoporphyrin, Pd(II) octaethylporphyrin ketone, Pt(II) octaethylporphyrin ketone, Pd(II) meso-tetra(pentafluorophenyl)porphyrin, Pt(II) meso-tetra(pentafluorophenyl)porphyrin, Ru(II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru(dpp)3), Ru(II) tris(1,10-phenanthroline) (Ru(phen)3), tris(2,2'-Bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3), erythrosin B, fluorescein, fluorescein isothiocyanate (FITC), eosin, iridium(III) ((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)), (enzothiazole) ((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate), Lumogen dye, Macroflex Fluorescent red, Macrolex fluorescent yellow, Texas red, rhodamine B, rhodamine 6G, sulfur rhodamine, m-cresol, thymol blue, xylenol blue, cresol red, chlorophenol blue, bromocresol green, bromocresol red, bromothymol blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, 4-nitirophenol, alizarin, phenolphthalein, o-cresolphthalein, chlorophenol red, Karmaga fluorescein, bromo-xylenol, phenol red, neutral red, nitrazine, 3,4,5,6-tetrabromophenolphthalein, Congo red, fluorescein, eosin, 2',7'-dichlorofluorescein, 5(6)-carboxy-fluorescein, carboxynaphthofluorescein, 8-hydroxypyrene-1,3,6-trisulfonic acid, seminaphthofluorescein, seminaphthofluorescein, tris(4,7-diphenyl-1,10-phenanthroline), Examples of suitable fluorescein-modifying agents include, but are not limited to, (4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride, (4,7-diphenyl-1,10-phenanthroline)ruthenium(II) tetraphenylborate, platinum(II) octaethylporphyrin, dialkylcarbocyanine, dioctadecylcyclooxacarbocyanine, fluorenylmethyloxycarbonyl chloride, 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), and derivatives or combinations thereof.

[0377] The detection moiety may include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), or derivatives or combinations thereof.

[0378] The detectable moiety can be attached to the cell-penetrating peptide (CPP) at the amino group, carboxylate group, or side chain of any amino acid in the CPP (e.g., at the amino group, carboxylate group, or side chain of any amino acid in a cCPP).

[0379] treatment part The compounds of the present disclosure may include a therapeutic moiety. The cargo may include a therapeutic moiety. The detectable moiety may be linked to the therapeutic moiety, or the detectable moiety may also function as the therapeutic moiety. A therapeutic moiety refers to a group that, when administered to a subject, alleviates one or more symptoms of a disease or disorder. The therapeutic moiety may include a peptide, a protein (e.g., an enzyme, an antibody or fragment thereof), a small molecule, or an oligonucleotide.

[0380] Therapeutic moieties can include a wide variety of drugs, such as antagonists (e.g., enzyme inhibitors) and agonists (e.g., transcription factors that result in increased expression of a desired gene product (as will be understood by those skilled in the art, antagonistic transcription factors can also be used)). Additionally, therapeutic moieties include agents that can induce toxicity and / or induce toxicity to healthy and / or unhealthy cells in the body. Therapeutic moieties may also be capable of inducing and / or priming the immune system against potential pathogens.

[0381] The therapeutic moiety may include, for example, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-inflammatory agent, an immunosuppressant, an anesthetic agent, or any combination thereof.

[0382] The therapeutic moiety may comprise an anti-cancer drug, examples of which include 13-cis-retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, accutane, actinomycin-D, adriamycin, adrsil, agrilin, Ala-Cort, aldesleukin, alemtuzumab, alitretinoin, alkaban-AQ, alkeran, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine, aminoglutethimide, and anagrelide. , Anandrone, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Leucovorin calcium, Campas, Camptosar, Camptothecin-11, Capecitabine, Carlac, Carboplatin, Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cervidine, Cetuximab, Chlorambucil, Cisplatin, Citrovorum factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposome, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposome, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, Depocyto, Dexamethasone, Acetic acid Dexamethasone acid, dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, docetaxel, Doxil, doxorubicin, doxorubicin liposome, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, epirubicin, epoetin alfa, Erbitux, Erwinia L-asparaginase, estramustine, Ethyol, Etopophos, etoposide, etoposide phosphate,Evulexin, Evista, Exemestane, Fairston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, Mechlorethamine, Mechlorethamine hydrochloride, Medralone, Medrol, Megrez, Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Menex, Methotrexate, Methotrexate sodium, Methylprednisolone, Mirocel, Letrozole, Neosar, Nuxidex Prolife with Lastasta, Numega, Nepgen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide Acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orpred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG-Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-Asparaginase, Phenylanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelon, Procarbazine, Procrit, Proleukin, Carmustine Implant 20, Purinethol, raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alpha-2a), Rubex, rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solumedrol, STI-571, streptozocin, tamoxifen, Targetretin, Taxol, Taxotere, Temodar, temozolomide, teniposide, TESPA, thalidomide, Thalomid, TheraCys, thioguanine, thioguanine Tabloid, thiophosphoamide, Thioplex, thiotepa, TICE, Toposar, topotecan,Toremifene, trastuzumab, tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, Bepcid, Besanoid, Viadur, vinblastine, vinblastine sulfate, Vincasar Pfs, vincristine, vinorelbine, vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, zoledronic acid, Zometa, Gliadel wafer, Gleevec, GM-CS F, goserelin, granulocyte colony-stimulating factor, halotestin, herceptin, hexadrol, Hexalen, hexamethylmelamine, HMM, Hycamtin, Hydrea, hydrocortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortone phosphate, hydroxyurea, ibritumomab, ibritumomab tiuxetan, idamycin, idarubicin, Ifex, IFN-α, ifosfamide, IL 2, IL-11, imatinib mesylate, imidazole carboxamide, interferon α, interferon α-2b (PEG conjugate), interleukin 2, interleukin-11, Intron A (interferon α-2b), leucovorin, leukeran, leukine, leuprolide, leulocristine, leustatin, liposomal Ara-C, Liquid Pred, lomustine, L-PAM, L-sarcolysin, methycortene, mitomycin, mitomycin-C, mitoxantrone, M-prednisole, MTC, MTX, mustagen, mustine, mutamycin, Myleran, Iressa, irinotecan, isotretinoin, quidrolase, lanacort, L-asparaginase, and LCR. Therapeutic moieties can also include biopharmaceuticals, such as antibodies.

[0383] The therapeutic moiety may include an antiviral agent such as ganciclovir, azidothymidine (AZT), or lamivudine (3TC).

[0384] The therapeutic portion includes acedapsone sodium, alamethicin, alexidine, amdinocillin, amidinocillin pivoxil, amicicline, amifloxacin, amifloxacin mesylate, amikacin, amikacin sulfate, aminosalicylic acid, aminosalicylate sodium, amoxicillin, amphomycin, ampicillin, ampicillin sodium, apalcillin sodium, apramycin, aspartocin, astromycin sulfate, avilamycin, avoparcin, azithromycin, azlocillin, azlocillin sodium, and bacampicin. Bacitracin hydrochloride, bacitracin, bacitracin methylenedisalicylate, bacitracin zinc, babermycin, benzoylpas calcium, verithromycin, betamycin sulfate, biapenem, vinylamycin, biphenamine hydrochloride, bispyrithione magsulfex, buticacin, butirosin sulfate, capreomycin sulfate, carbadox, carbenicillin disodium, carbenicillin indanyl sodium, carbenicillin phenylsodium, carbenicillin potassium, carmonam sodium, cefaclor, cefadroxil, cephalamin Cefandole, cefamandole nafate, cefamandole sodium, cefaparol, cefatrizine, cefazaflur sodium, cefazolin, cefazolin sodium, cefbuperazone, cefdinir, cefepime, cefepime hydrochloride, cefetecol, cefixime, cefmenoxime hydrochloride, cefmetazole, cefmetazole sodium, cefonicid monosodium, cefonicid sodium, cefoperazone sodium, ceforanide, cefotaxime sodium, cefotetan, cefotetan disodium, cefotiam hydrochloride, cefoxal Cytin, cefoxitin sodium, cefpimizole, cefpimizole sodium, cefpiramide, cefpiramide sodium, cefpirome sulfate, cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin sodium, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroxime pivocetil, cefuroxime sodium, acetrile sodium, cephalexin, cephalexin hydrochloride, cephaloglycin, cephaloridine, cephalothin sodium,Cephapirin sodium, cephradine, cetocycline hydrochloride, cetofenicol, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate complex, chloramphenicol sodium succinate, chlorhexidine phosphanilate, chloroxylenol, chlortetracycline bisulfate, chlortetracycline hydrochloride, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride, ciloremycin, clarithromycin, clinafloxacin hydrochloride, clindamycin, clindamycin hydrochloride , clindamycin palmitate hydrochloride, clindamycin phosphate, clofazimine, cloxacillin benzathine, cloxacillin sodium, cloxiquin, colistin metasodium, colistin sulfate, coumermycin, coumermycin sodium, cyclacillin, cycloserine, dalfopristin, dapsone, daptomycin, demeclocycline, demeclocycline hydrochloride, demecycline, denofungin, diaveridine, dicloxacillin, dicloxacillin sodium, dihydrostreptomycin sulfate, dipyrithione, diris Ethromycin, doxycycline, doxycycline calcium, doxycycline phosphatex, doxycycline hyclate, droxacin sodium, enoxacin, epicillin, epitetracycline hydrochloride, erythromycin, erythromycin acystolate, erythromycin estrate, erythromycin ethylsuccinate, erythromycin single septate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, ethambutol hydrochloride, ethionamide, fleroxacin , floxacillin, fludalanine flumequine, fosfomycin, fosfomycin tromethamine, fumoxicillin, furazolium chloride, furazolium tartrate, sodium fusidate, fusidic acid, gentamicin sulfate, gloximonam, gramicidin, haloprogin, hetacillin, hetacillin potassium, hexedine, ibafloxacin, imipenem, isoconazole, isepamicin, isoniazid, josamycin, kanamycin sulfate, kitasamycin, levofuraltadone, levopropylcillin potassium, lexithromycin, lincomycin,Lincomycin hydrochloride, lomefloxacin, lomefloxacin hydrochloride, lomefloxacin mesylate, loracarbef, mafenide, meclocycline, meclocycline sulfosalicylate, megalomycin phosphate potassium, mequidox, meropenem, methacycline, methacycline hydrochloride, methenamine, methenamine hippurate, methenamine mandelate, methicillin sodium, methioprim, metronidazole hydrochloride, metronidazole phosphate, mezlocillin, mezlocillin sodium, minocycline, minocycline hydrochloride, milkamycin hydrochloride, Monette monensin, monensin sodium, nafcillin sodium, nalidixic acid sodium, nalidixic acid, natamycin, nebramycin, neomycin palmitate, neomycin sulfate, neomycin undecylenate, netilmicin sulfate, neutomycin, nifuraden, nifuraldeson, nifuratel, nifuratron, nifurdazil, niflimide, nifurpirinol, nifurquinazole, nifurthiazole, nitrocycline, nitrofurantoin, nitromide, norfloxacin, novobiocin sodium, ofloxacin, onnetoprim, oxacin oxacillin sodium, oximonam, oximonam sodium, oxolinic acid, oxytetracycline, oxytetracycline calcium, oxytetracycline hydrochloride, paldimycin, parachlorophenol, paulomycin, pefloxacin, pefloxacin mesylate, penamecillin, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V, penicillin V benzathine, penicillin V hydrabamine, penicillin V potassium, pentizidone sodium, phenylaminosalicylic acid Licylic acid, piperacillin sodium, pirbenicillin sodium, pyridicillin sodium, pirlimycin hydrochloride, pivampicillin hydrochloride, pivampicillin pamoate, pivampicillin probenate, polymyxin B sulfate, porfiromycin, propikacin, pyrazinamide, zinc pyrithione, quindecamin acetate, quinupristin, racephenicol, ramoplanin, ranimycin, reromycin, repromicin, rifabutin, rifamethan, rifamexyl, rifamide, rifampin, rifapentine, rifaximin, rolitetracycline,Rolitetracycline nitrate, rosaramycin, rosaramycin butyrate, rosaramycin propionate, rosaramycin sodium phosphate, rosaramycin stearate, rosoxacin, roxarsone, roxithromycin, sancycline, sanfetrinemucine sodium, salmoxicillin, salpicillin, scopafungin, sisomicin, sisomicin sulfate, sparfloxacin, spectinomycin hydrochloride, spiramycin, stalimycin hydrochloride, stefimycin, streptomycin sulfate, streptonicozide, sulfabenz, sulfabenzamide, sulfacetamide, sulfacetamide sodium, sulacytine, sulfadiazine, sulfadiazine sodium, sulfadoxine, sulfalene, sulfamerazine, sulfameter, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfamonomethoxine, sulfamoxole, zinc sulfanilate, sulfanilate Antibacterial agents such as lan, sulfasalazine, sulfasomizole, sulfathiazole, sulfazameth, sulfisoxazole, sulfisoxazoleacetyl, sulfisoxazolediolamine, sulfomixin, sulopenem, sultamicillin, sancillin sodium, talampicillin hydrochloride, teicoplanin, temafloxacin hydrochloride, temocillin, tetracycline, tetracycline hydrochloride, tetracycline phosphate complex, tetroxoprim, thiamphenicol, tifencillin potassium, ticarcillin cresyl sodium, ticarcillin disodium, ticarcillin monosodium, ticlaton, thiodonium chloride, tobramycin, tobramycin sulfate, tosufloxacin, trimethoprim, trimethoprim sulfate, trisulfapyrimidine, troleandomycin, trospemycin sulfate, tyrosulicin, vancomycin, vancomycin hydrochloride, virginiamycin, or zorbamycin.

[0385] The therapeutic moiety may include an anti-inflammatory agent.

[0386] The therapeutic moiety may include dexamethasone (Dex).

[0387] The therapeutic moiety can include a therapeutic protein. For example, some people have deficiencies in certain enzymes (e.g., lysosomal storage diseases). Such enzymes / proteins can be delivered to human cells by linking the enzymes / proteins to cyclic cell-penetrating peptides (cCPPs) disclosed herein. The cCPPs of the present disclosure have been tested with proteins (e.g., GFP, PTP1B, actin, calmodulin, troponin C) and found to be functional.

[0388] The therapeutic moiety can be an anti-infective. The term "anti-infective" refers to an agent that can kill, inhibit, or otherwise slow the growth of an infectious pathogen. The term "infectious agent" refers to a pathogenic microorganism, such as bacteria, viruses, fungi, and intracellular or extracellular parasites. Because infectious diseases are caused by infectious agents, anti-infectives can be used to treat infectious diseases.

[0389] The infectious agent may be a gram-negative bacterium. The gram-negative bacterium may be of a genus selected from Escherichia, Proteus, Salmonella, Klebsiella, Providencia, Enterobacter, Burkholderia, Pseudomonas, Acinetobacter, Aeromonas, Haemophilus, Yersinia, Neisseria, Erwinia, Rhodopseudomonas, and Burkholderia. The infectious agent may be a gram-positive bacterium. The gram-positive bacterium may be of a genus selected from Lactobacillus, Azorhizobium, Streptococcus, Pediococcus, Photobacterium, Bacillus, Enterococcus, Staphylococcus, Clostridium, Butyrivibrio, Sphingomonas, Rhodococcus, and Streptomyces. The infectious agent may be an acid-fast bacterium of the genus Mycobacterium, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, and Mycobacterium leprae. The infectious agent may be of the genus Nocardia. The infectious agent may be selected from any one of the following species: Nocardia asteroides, Nocardia brasiliensis, and Nocardia caviae.

[0390] The infectious agent can be a fungus. The fungus can be from the genus Mucor. The fungus can be from the genus Crytococcus. The fungus can be from the genus Candida. The fungus can be selected from any one of Mucor racemosus, Candida albicans, Crytococcus neoformans, or Aspergillus fumigatus.

[0391] The infectious agent can be a protozoan. The protozoan can be of the genus Plasmodium (e.g., P. falciparum, P. vivax, P. ovale, or P. malariae). Protozoans cause malaria.

[0392] Exemplary organisms include Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, These include Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.

[0393] The infectious agent can be a parasite. The parasite can be Cryptosporidium. The parasite can be an endoparasite. The endoparasite can be a heartworm, a tapeworm, or a flatworm. The parasite can be an epiparasite. The parasite causes a disease selected from acanthomaebiasis, babesiosis, balantidiosis, blastocystosis, coccidiosis, amebic dysentery, giardiasis, isosporiasis, cystosporiasis, leishmaniasis, primary amebic meningoencephalitis, malaria, rhinovirus disease, toxoplasmosis, trichomoniasis, trypanosomiasis, Chagas disease, or scabies.

[0394] The infectious agent may be a virus, non-limiting examples of which include sudden acute respiratory coronavirus 2 (SARS-CoV-2), sudden acute respiratory coronavirus (SARS-CoV), Middle East respiratory virus (MERS), influenza, hepatitis C virus, dengue virus, West Nile virus, Ebola virus, hepatitis B, human immunodeficiency virus (HIV), herpes simplex, herpes zoster, and Lassa virus.

[0395] The anti-infective agent can be an antiviral agent. Non-limiting examples of antiviral agents include nucleoside or nucleotide reverse transcriptase inhibitors (e.g., zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), emtricitabine, abacavir succinate, elvucitabine, adefovir dipivoxil, lobucavir (BMS-180194), lodenosine (FddA), and tenofovir (Te tenofovir disoproxil and tenofovir disoproxil fumarate), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine), protease inhibitors (e.g., ritonavir, tipranavir, saquinavir, nelfinavir, indinavir, amprenavir, fosamprenavir, atazanavir, lopinavir, darunavir (TM C-114), lasanavir, and brecanavir (VX-385)), cell entry inhibitors (e.g., CCR5 antagonists (e.g., maraviroc, vicriviroc, INCB9471, and TAK-652) and CXCR4 antagonists (AMD-11070)), fusion inhibitors (e.g., enfuvirtide), integrase inhibitors (e.g., raltegravir, BMS-707035, and elvitegravir), Tat inhibitors (e.g., didehydrocortistatin A (dCA)), maturation inhibitors (e.g., berivitamin A), immunomodulators (e.g., levamisole), and other antivirals (e.g., hydroxyurea, ribavirin, interleukin-2 (IL-2), interleukin-12 (IL-12), pensafuside, peramivir, zanamivir, oseltamivir phosphate, baloxavir marboxil).

[0396] The anti-infective agent can be an antibiotic. Non-limiting examples of antibiotics include aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin-resistant, netilmicin, streptomycin, and tobramycin), cabecephems (e.g., loracarbef), carbapenems (e.g., ertapenem, imipenem / cilastatin, and meropenem), cephalosporins (e.g., cefadroxil, cefazolin, cephalexin, cefaclor), and the like. cephalexin, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, and cefepime), macrolides (e.g., azithromycin, clarithromycin, dirithromycin, erythromycin, and troleandomycin), monobactam penicillins (e.g., amoxicillin, ampicillin, carbenicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, and ticarcillin), polypeptides (e.g., bacitracin, colistin, and polymyxin B), quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, anti-infective agents include steroidal anti-inflammatory agents, such as fluconazole, norfloxacin, ofloxacin, and trovafloxacin, sulfonamides (e.g., mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, and trimethoprim-sulfamethoxazole), tetracyclines (e.g., demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline), and vancomycin. The anti-infective agent may be a steroidal anti-inflammatory agent.Non-limiting examples of steroidal anti-inflammatory agents include fluocinolone, triamcinolone, triamcinolone acetonide, betamethasone, betamethasone dipropionate, diflucortolone, fluticasone, cortisone, hydrocortisone, mometasone, methylprednisolone, beclomethasone dipropionate, clobetasol, prednisone, prednisolone, methylprednisolone, methylprednisolone, betamethasone, budesonide, and dexamethasone. The anti-infective agent may be a non-steroidal anti-inflammatory agent. Non-limiting examples of nonsteroidal anti-inflammatory agents include cerocoxib, nimesulide, rofecoxib, meclofenamic acid, meclofenamate sodium, flunixin, fluprofen, flurbiprofen, sulindac, meloxicam, piroxicam, etodolac, fenoprofen, fenbuprofen, ketoprofen, suprofen, diclofenac, bromfenac sodium, phenylbutazone, thalidomide, and indomethacin.

[0397] The anti-infective agent may be an anti-fungal agent. Non-limiting examples of anti-fungal agents include amphotericin B, caspofungin, fluconazole, flucytosine, itraconazole, ketoconazole, amlorafine, butenafine, naftifine, terbinafine, elubiol, econazole, enoxol, itraconazole, isoconazole, imidazole, miconazole, sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole, terconazole, butoconazole, thiabendazole, and voriconazole. ol, saperconazole, sertaconazole, fenticonazole, posaconazole, bifonazole, flutrimazole, nystatin, pimaricin, natamycin, tolnaftate, mafenide, dapsone, actofunicon, griseofulvin, potassium iodide, crystal violet, ciclopirox, ciclopirox olamine, haloprogin, undecylenate, silver sulfadiazine, undecylenic acid, undecylenic acid alkanolamide, and carbol-fuchsin.

[0398] The therapeutic moiety can be an analgesic or pain relieving agent. Non-limiting examples of analgesics or pain relievers include aspirin, acetaminophen, ibuprofen, naproxen, procaine, lidocaine, tetracaine, dibucaine, benzocaine, p-butylaminobenzoic acid 2-(diethylamino)ethyl ester HCl, mepivacaine, piperocaine, and dyclonine.

[0399] The therapeutic moiety may be an antibody or antigen-binding fragment. Antibodies and antigen-binding fragments may be derived from any suitable source, such as human, mouse, camelid (e.g., camel, alpaca, llama), rat, ungulate, or non-human primate (e.g., monkey, rhesus).

[0400] Furthermore, it should be understood that the cargoes containing anti-infectives and other therapeutic moieties described herein include their possible salts, the pharmaceutically acceptable salts of which are particularly relevant for therapeutic applications. Salts include acid addition salts and base salts. Examples of acid addition salts are hydrochloride, fumarate, oxalate, etc. Examples of basic salts are salts in which the (remaining) counter ion is an alkali metal such as sodium and potassium, an alkaline earth metal such as calcium salt, potassium salt, and an ammonium ion ( + N(R')4, where R' is independently an optionally substituted C 1~6 -alkyl, optionally substituted C 2~6 -refers to alkenyl, optionally substituted aryl, or optionally substituted heteroaryl).

[0401] The therapeutic moiety can be an oligonucleotide. The oligonucleotide can be an antisense compound (AC). Examples of oligonucleotides include, but are not limited to, antisense oligonucleotides, small interfering RNAs (siRNAs), microRNAs (miRNAs), ribozymes, immunostimulatory nucleic acids, antagomirs, antimirs, microRNA mimics, supermirs, Ul adapters, CRISPR mechanisms, and aptamers. The term "antisense oligonucleotide" or simply "antisense" is meant to include oligonucleotides complementary to a target polynucleotide sequence. Non-limiting examples of antisense oligonucleotides for treating Duchenne muscular dystrophy can be found in U.S. Patent Application Publication No. 2019 / 0365918, U.S. Patent Application Publication No. 2020 / 0040336, U.S. Patent No. 9,499,818, and U.S. Patent No. 9,447,417, each of which is incorporated by reference in its entirety for all purposes.

[0402] The treatment areas include the following diseases: neuromuscular disorders, Pompe disease, beta-thalassemia, dystrophin Kobe, Duchenne muscular dystrophy, Becker muscular dystrophy, diabetes, Alzheimer's disease, cancer, cystic fibrosis, merosin-deficient congenital muscular dystrophy type 1A (MDC1A), proximal spinal muscular atrophy (SMA), Huntington's chorea, Huntington's chorea-like 2 (HDL2), myotonic dystrophy, spinocerebellar degeneration, spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis, and frontotemporal muscular atrophy. The therapeutic moiety can be used to treat any one of dementia, fragile X syndrome, fragile X mental retardation 1 (FMR1), fragile X mental retardation 2 (FMR2), fragile XE mental retardation (FRAXE), Friedreich's ataxia (FRDA), fragile X-associated tremor / ataxia syndrome (FXTAS), myoclonic epilepsy, oculopharyngeal muscular dystrophy (OPMD), syndromic or non-syndromic X-linked mental retardation, myotonic dystrophy, myotonic dystrophy type 1, myotonic dystrophy type 2, epilepsy, Dravet syndrome, or Alzheimer's disease. The therapeutic moiety can be used to treat a cancer selected from glioma, acute myeloid leukemia, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, or melanoma. The therapeutic moieties can be used to treat ocular diseases, non-limiting examples of which include refractive error, macular degeneration, cataracts, diabetic retinopathy, glaucoma, amblyopia, or strabismus.

[0403] The therapeutic moiety may include a targeting moiety. The targeting moiety may include, for example, an amino acid sequence that can target one or more enzyme domains. The targeting moiety may include an inhibitor of an enzyme that can play a role in a disease, such as cancer, cystic fibrosis, diabetes, obesity, or a combination thereof. The targeting moiety targets one or more of the following genes: FMR1, AFF2, FXN, DMPK, SCA8, PPP2R2B, ATN1, DRPLA, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, ATP7B, HTT, SCN1A, BRCA1, LAMA2, CD33, VEGF, ABCA4, CEP290, RHO, USH2A, OPA1, CNGB3, PRPF31, GYS1, or RPGR. The therapeutic moiety can be an antisense compound (AC) as described in U.S. Patent Application Publication No. 2019 / 0365918, which is incorporated by reference in its entirety. For example, the targeting moiety can include any of the sequences listed in Table 7.

[0404] [Table 15] * Fpa, Σ: L-4-fluorophenylalanine; Pip, θ: L-homoproline; Nle, Ω: L-norleucine; Phg, Ψ: L-phenylglycine; F2Pmp, Λ: L-4-(phosphonodifluoromethyl)phenylalanine; Dap, L-2,3-diaminopropionic acid; Nal, Φ': L-β-naphthylalanine; Pp, θ: L-pipecolic acid; Sar, Ξ: sarcosine; Tm, trimesic acid.

[0405] The targeting moiety and the cell-penetrating peptide may overlap, i.e., the residues that form the cell-penetrating peptide may also be part of the sequence that forms the targeting moiety, and vice versa.

[0406] The therapeutic moiety can be attached to the cell-penetrating peptide at an amino group, carboxylate group, or side chain of any amino acid of the cell-penetrating peptide (e.g., at an amino group, carboxylate group, or side chain or any amino acid of a cCPP). The therapeutic moiety can be attached to a detectable moiety.

[0407] Therapeutic moieties may include targeting moieties that can act as inhibitors of Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, etc., or combinations thereof.

[0408] Ras is a protein encoded by the RAS gene in humans. Normal Ras proteins perform essential functions in normal tissue signaling, and mutations in the Ras gene are involved in the development of many cancers. Ras can act as a molecular on / off switch; when turned on, Ras recruits and activates proteins necessary for the propagation of growth factor and other receptor signals. Mutant forms of Ras are involved in a variety of cancers, including lung cancer, colon cancer, pancreatic cancer, and various leukemias.

[0409] Protein-tyrosine phosphatase 1B (PTP1B) is a prototypic member of the PTP superfamily and plays many roles during eukaryotic cell signaling. PTP1B is a negative regulator of the insulin signaling pathway and is considered a promising therapeutic target for the treatment of type II diabetes, among other things. PIP1B is also involved in the development of breast cancer.

[0410] Pin1 is an enzyme that binds to a subset of proteins and plays a role as a post-phosphorylation regulator in regulating protein function. Pin1 activity can regulate the outcome of proline-directed kinase signaling, which in turn can regulate cell proliferation and cell survival. Deregulation of Pin1 may play a role in various diseases. Upregulation of Pin1 may be involved in certain cancers, and downregulation of Pin1 may be involved in Alzheimer's disease. Inhibitors of Pin1 may have relevance for the treatment of cancer and immune disorders.

[0411] Grb2 is an adaptor protein involved in signal transduction and cell communication. The Grb2 protein contains one SH2 domain that can bind to tyrosine phosphorylated sequences. Grb2 is widely expressed and is essential for multiple cellular functions. Inhibition of Grb2 function can impair developmental processes and block the transformation and proliferation of various cell types.

[0412] Recently, it has been reported that the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel protein mutated in cystic fibrosis (CF) patients, is negatively regulated by the CFTR-associated ligand (CAL) via its PDZ domain (CAL-PDZ) (Wolde, M et al. J. Biol. Chem. 2007, 282, 8099). Inhibition of the CFTR / CAL-PDZ interaction has been shown to improve the activity of the most common form of CFTR mutation, ΔPhe508-CFTR (Cheng, SH et al. Cell 1990, 63, 827; Kerem, BS et al. Science 1989, 245, 1073), by reducing its proteasome-mediated degradation (Cushing, PR et al. Angew. Chem. Int. Ed. 2010, 49, 9907). Thus, disclosed herein are methods for treating a subject with cystic fibrosis by administering an effective amount of a compound or composition disclosed herein. The compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can act as an inhibitor of CAL PDZ. The compositions or compositions disclosed herein may also be administered together with a molecule that modifies CFTR function.

[0413] The therapeutic moiety can be attached to the cyclic peptide at an amino group or a carboxylate group, or at the side chain of any of the amino acids of the cyclic peptide (e.g., at an amino group or a carboxylate group on the side chain of an amino acid of the cyclic peptide). In some examples, the therapeutic moiety can be attached to a detectable moiety.

[0414] Also disclosed herein are compositions comprising the compounds described herein.

[0415] Pharmaceutically acceptable salts and prodrugs of the disclosed compounds are also disclosed herein. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases, depending on the specific substituents found on the compound. Under conditions where the disclosed compounds are sufficiently basic or acidic to form stable, non-toxic acid or base salts, it may be appropriate to administer the compounds as salts. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids such as acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-sugar phosphate, maleic acid, tosylic acid, and methanesulfonic acid. Thus, disclosed herein are hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartrate, malonate, ascorbate, α-ketoglutarate, α-glycolate, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of the present compounds can be obtained using standard procedures well known in the art (e.g., by reacting a sufficiently basic compound, such as an amine, with an appropriate acid to provide a physiologically acceptable anion). Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0416] The therapeutic moiety may comprise a therapeutic polypeptide, oligonucleotide, or small molecule. The therapeutic polypeptide may comprise a peptide inhibitor. The therapeutic polypeptide may comprise a binding reagent that specifically binds to a target of interest. The binding reagent may comprise an antibody or antigen-binding fragment thereof that specifically binds to a target of interest. The antigen-binding fragment may comprise a Fab fragment, F(ab') fragment, F(ab')2 fragment, Fv fragment, minibody, diabody, nanobody, single domain antibody (dAb), single chain variable fragment (scFv), or multispecific antibody.

[0417] The oligonucleotide may comprise an antisense compound (AC), which may comprise a nucleotide sequence complementary to a target nucleotide sequence encoding a protein target of interest.

[0418] A therapeutic moiety (TM) can be conjugated to a chemically reactive side chain of an amino acid of a cCPP. Any amino acid side chain on a cCPP that can form a covalent bond or can be modified to do so can be used to link the TM to the cCPP. The amino acid on the cCPP can be a natural or unnatural amino acid. The chemically reactive side chain can include an amine group, a carboxylic acid, an amide, a hydroxyl group, a sulfhydryl group, a guanidinyl group, a phenol group, a thioether group, an imidazolyl group, or an indolyl group. The amino acid of the cCPP to which the TM is conjugated can include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, tryptophan, or an analog thereof. The amino acid on the cCPP used to conjugate the TM can be ornithine, 2,3-diaminopropionic acid, or an analog thereof. The amino acid can be lysine or an analog thereof. The amino acid can be glutamic acid or an analog thereof. The amino acid can be aspartic acid or an analog thereof. The side chain can be replaced with a bond or linker to the TM.

[0419] The TM can include a therapeutic polypeptide, and the cCPP can be conjugated to a chemically reactive side chain of an amino acid on the therapeutic polypeptide. Any amino acid side chain on the TM that can form a covalent bond or can be modified to do so can be used to link the cCPP to the TM. The amino acid on the TM can be a natural or unnatural amino acid. The chemically reactive side chain can include an amine group, a carboxylic acid, an amide, a hydroxyl group, a sulfhydryl group, a guanidinyl group, a phenol group, a thioether group, an imidazolyl group, or an indolyl group. The amino acid on the TM to which the cCPP is conjugated can include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, tryptophan, or an analog thereof. The amino acid on the TM used to conjugate the cCPP can be ornithine, 2,3-diaminopropionic acid, or an analog thereof. The amino acid can be lysine or an analog thereof. The amino acid can be glutamic acid or an analog thereof. The amino acid can be aspartic acid or an analog thereof. The side chain of the TM can be replaced with a bond or linker to the cCPP.

[0420] The TM can be an antisense compound (AC) comprising an oligonucleotide conjugated at the 5' or 3' end to a chemically reactive side chain of an amino acid of a cCPP. The AC can be chemically conjugated to a cCPP via a moiety on the 5' or 3' end of the AC. The chemically reactive side chain of the cCPP can include an amine group, a carboxylic acid, an amide, a hydroxyl group, a sulfhydryl group, a guanidinyl group, a phenol group, a thioether group, an imidazolyl group, or an indolyl group. The amino acid of the cCPP to which the AC is conjugated can include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, or tryptophan. The amino acid of the cCPP to which the AC is conjugated can include lysine or cysteine.

[0421] Non-limiting examples of unconjugated AC structures (i.e., before conjugation to a CPP) are provided below: AC in the structures below refers to an antisense oligonucleotide.

[0422] [ka]

[0423] Non-limiting examples of linear CPPs include polyarginines (e.g., R9 or R 11 These include the antennapedia sequence, HIV-TAT, penetratin, Antp-3A (Antp variant), buforin II, transportan, MAP (model amphipathic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol), and BGTC (Bis-Guanidinium-Tren-Cholesterol).

[0424] Oligonucleotides The compound may comprise a cyclic cell-penetrating peptide (cCPP) conjugated to an antisense compound (AC) as a therapeutic moiety. The AC may comprise an antisense oligonucleotide, siRNA, microRNA, antagomir, aptamer, ribozyme, immunostimulatory oligonucleotide, decoy oligonucleotide, supermir, miRNA mimic, miRNA inhibitor, or a combination thereof.

[0425] antisense oligonucleotides The therapeutic moiety may comprise an antisense oligonucleotide. The term "antisense oligonucleotide" or simply "antisense" refers to an oligonucleotide that is complementary to a target polynucleotide sequence. An antisense oligonucleotide may comprise a single strand of DNA or RNA that is complementary to a selected sequence, for example, a target gene mRNA.

[0426] Antisense oligonucleotides can regulate one or more aspects of protein transcription, translation, and expression and function through the hybridization of antisense oligonucleotides with target nucleic acids.The hybridization of antisense oligonucleotides to their target sequences can suppress the expression of target proteins.The hybridization of antisense oligonucleotides to their target sequences can suppress the expression of one or more target protein isoforms.The hybridization of antisense oligonucleotides to their target sequences can up-regulate the expression of target proteins.The hybridization of antisense oligonucleotides to their target sequences can down-regulate the expression of target proteins.

[0427] Antisense compounds can inhibit gene expression by binding to complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression by binding to it, 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. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. Antisense oligonucleotides can contain about 10 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 25 nucleotides. This term also encompasses antisense oligonucleotides that may not be completely complementary to the desired target gene. Therefore, the compounds disclosed herein can be utilized when non-target-specific activity is found in antisense or when an antisense sequence containing one or more mismatches with the target sequence is desired.

[0428] Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and therefore can be used to specifically inhibit protein synthesis by target genes.The effectiveness of antisense oligonucleotides for inhibiting protein synthesis has been well proven.

[0429] Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence of interest. 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 host cells. Target regions of mRNA include the region at or near the AUG translation initiation codon and sequences substantially complementary to the 5' region of the mRNA. These secondary structure analysis and target site selection considerations can be performed using, for example, 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).

[0430] RNA interference nucleic acid The therapeutic moiety can be an RNA interference (RNAi) molecule or a small interfering RNA molecule. RNA interference methods using RNAi or siRNA molecules can be used to inhibit the expression of a gene or polynucleotide of interest.

[0431] Small interfering RNA (siRNA) is an RNA duplex, typically about 16 to about 30 nucleotides long, that can associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Because RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts, siRNA can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA functions through a natural mechanism that evolved to control gene expression via non-coding RNA. Various RNAi reagents, including siRNAs targeting clinically relevant targets, are currently in pharmaceutical development, as described, for example, in de Fougerolles, A. et al., Nature Reviews 6:443-453 (2007).

[0432] The first RNAi molecules described were RNA:RNA hybrids containing both RNA sense and RNA antisense strands, but it has now been demonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrids can mediate RNAi (Lamberton, JS and Christian, AT, (2003) Molecular Biotechnology 24:111-119). RNAi molecules containing any of these different types of double-stranded molecules can be used. Furthermore, it will be understood that RNAi molecules can be used and introduced into cells in a variety of forms. RNAi molecules can include any and all molecules capable of inducing an RNAi response in a cell, including, but not limited to, double-stranded oligonucleotides comprising two separate strands (i.e., a sense strand and an antisense strand) (e.g., small interfering RNA (siRNA)); double-stranded oligonucleotides comprising two separate strands linked to each other by a non-nucleotidyl linker; oligonucleotides comprising a hairpin loop of complementary sequences that form a double-stranded region (e.g., shRNAi molecules), and expression vectors that express one or more polynucleotides that can form a double-stranded polynucleotide, either alone or in combination with another polynucleotide.

[0433] As used herein, "single-stranded siRNA compound" refers to a siRNA compound that is composed of a single molecule.It can comprise a double-stranded region formed by intrastrand pairing, for example, it can be or comprise a hairpin or panhandle structure.Single-stranded siRNA compound can be antisense with respect to target molecule.

[0434] Single-stranded siRNA compound can be long enough to enter RISC and participate in the RISC-mediated cleavage of target mRNA.Single-stranded siRNA compound is at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, or at most about 50 nucleotides long.Single-stranded siRNA is less than about 200, less than about 100, or less than about 60 nucleotides long.

[0435] Hairpin siRNA compounds can have a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs, or at least about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotide pairs. The duplex region can be about 200 or less, about 100 or less, or about 50 or less nucleotide pairs in length. The duplex region ranges from about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21 nucleotide pairs in length. The hairpin can have a single-stranded overhang or a terminal unpaired region. The overhang can be about 2 to about 3 nucleotides in length. The overhang can be on the sense side of the hairpin or the antisense side of the hairpin.

[0436] A "double-stranded siRNA compound," as used herein, is an siRNA compound that contains two or more (and in some cases two) strands capable of interstrand hybridization to form a region of double-stranded structure.

[0437] The antisense strand of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 60 nucleotides in length, or at least about 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 60 nucleotides in length. It can be about 200 or less, about 100 or less, or about 50 or less nucleotides in length. The range can be about 17 to about 25, about 19 to about 23, and about 19 to about 21 nucleotides in length. As used herein, the term "antisense strand" refers to the strand of a siRNA compound that is sufficiently complementary to a target molecule (e.g., a target RNA).

[0438] The sense strand of a double-stranded siRNA compound can be at least about 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 60 nucleotides in length. It can be no more than about 200, no more than about 100, or no more than about 50 nucleotides in length. Ranges can be from about 17 to about 25, from about 19 to about 23, and from about 19 to about 21 nucleotides in length.

[0439] The double-stranded portion of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 60, or at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 60, nucleotide pairs long, but can be no more than about 200, no more than about 100, or about 50, nucleotide pairs long. Ranges can be from about 15 to about 30, from about 17 to about 23, from about 19 to about 23, and from about 19 to about 21, nucleotide pairs long.

[0440] siRNA compounds may be large enough that they can be cleaved by endogenous molecules (eg, by Dicer) to produce smaller siRNA compounds (eg, siRNA agents).

[0441] The sense strand and the antisense strand can be selected so that the double-stranded siRNA compound comprises a single-stranded or unpaired region at one or both ends of the molecule.Therefore, the double-stranded siRNA compound can contain the sense strand and the antisense strand that are paired to contain an overhang, for example, one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3 nucleotides.The overhang can be the result of one strand being longer than the other strand, or it can be the result of two strands of the same length being offset.In some embodiments, it can have at least one 3' overhang.In some embodiments, both ends of the siRNA molecule can have a 3' overhang.The overhang can be 2 nucleotides.

[0442] The length of the duplexed region can range from about 15 to about 30, or about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides in length, for example, the ssiRNA (siRNA with sticky overhang) compounds described above. ssiRNA compounds can be similar in length and can be constructed from the natural Dicer processing products of long dsiRNAs. Also included are embodiments in which the two strands of the ssiRNA compound are linked (e.g., covalently linked). Included are hairpins or other single-stranded structures that provide a duplex region and a 3' overhang.

[0443] The siRNA compound described herein, for example, double-stranded siRNA compound or single-stranded siRNA compound, can mediate the silencing of target RNA, for example, mRNA, for example, the transcript of the gene encoding protein.For convenience, in this specification, this mRNA is also referred to as the mRNA to be silenced.This gene is also referred to as target gene.Generally, the RNA to be silenced is endogenous gene.

[0444] As used herein, the phrase " mediate RNAi " refers to the ability to silence target RNA in a sequence-specific manner.Without wishing to be bound by theory, it is believed that silencing uses RNAi mechanism or process and guide RNA, for example, ssiRNA compound of about 21 to about 23 nucleotides.

[0445] An siRNA compound that is "sufficiently complementary" to a target RNA (e.g., a target mRNA) can silence the production of a protein encoded by the target mRNA. An siRNA compound that is "sufficiently complementary" to an RNA encoding a protein of interest can silence the production of the protein of interest encoded by the mRNA. An siRNA compound can be "exactly complementary" to a target RNA, for example, when the target RNA and the siRNA compound anneal to form a hybrid consisting of only Watson-Crick base pairs in the exact complementary region. A "sufficiently complementary" target RNA can include an internal region (e.g., at least about 10 nucleotides) that is exactly complementary to the target RNA. In embodiments, an siRNA compound specifically discriminates between single nucleotide differences. In this case, the siRNA compound mediates RNAi only when exact complementarity is found in the region of the single nucleotide difference (e.g., within 7 nucleotides).

[0446] microRNA The therapeutic moiety can be a microRNA molecule. MicroRNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals but are not translated into proteins. Processed miRNAs are single-stranded 17-25 nucleotide (nt) RNA molecules that are incorporated into the RNA-induced silencing complex (RISC) and have been identified as important regulators of development, cell proliferation, apoptosis, and differentiation. They are thought to play a role in regulating gene expression by binding to the 3' untranslated region of specific mRNAs. RISC mediates downregulation of gene expression through translational inhibition, transcriptional cleavage, or both. RISC is also involved in transcriptional silencing in the nuclei of various eukaryotic organisms.

[0447] Antagomir The therapeutic moiety can be an antagomir. Antagomirs are RNA-like oligonucleotides with various modifications for RNase protection and pharmacological properties (e.g., enhanced tissue and cellular uptake). They differ from normal RNA, for example, by complete 2'-0-methylation of the sugars, a phosphorothioate backbone, and, for example, a cholesterol moiety at the 3' end. Antagomirs can be used to efficiently silence endogenous miRNAs by forming duplexes containing the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122, as described in Krutzfeldt et al., Nature, 2005, 438:685-689 (expressly incorporated herein by reference in its entirety). AntagomirRNAs can be synthesized using standard solid-phase oligonucleotide synthesis protocols. See U.S. Patent Application Nos. 11 / 502,158 and 11 / 657,341, the disclosures of each of which are incorporated herein by reference.

[0448] Antagomirs may include ligand-binding monomer subunits and monomers for oligonucleotide synthesis. Monomers are described in U.S. Patent Application No. 10 / 916,185, filed August 10, 2004. Antagomirs may have a ZXY structure as described in International Application No. US2004 / 07070, filed March 8, 2004. Antagomirs may be conjugated with an amphiphilic moiety. Amphiphilic moieties for use with oligonucleotide agents are described in International Application No. US2004 / 07070, filed March 8, 2004.

[0449] Aptamers The therapeutic moiety can be an aptamer. Aptamers are nucleic acid or peptide molecules that bind to a particular molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). DNA or RNA aptamers have been successfully produced that bind to many different entities, from large proteins to small organic molecules. See Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct. Biol. 9:324-9 (1999), and Hermann and Patel, Science 287:820-5 (2000). Aptamers can be RNA- or DNA-based and can include riboswitches. Riboswitches are parts of mRNA molecules that can directly bind to small target molecules, and target binding affects gene activity. Thus, mRNAs containing riboswitches are directly involved in regulating their own activity depending on the presence or absence of their target molecules. Generally, aptamers are engineered through repeated rounds of in vitro selection, i.e., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers can be prepared by any known method, including synthetic, recombinant, and purified methods, and can be used alone or in combination with other aptamers specific to the same target. Furthermore, the term "aptamer" also includes "secondary aptamers," which contain consensus sequences derived from comparing two or more known aptamers to a given target. Aptamers can be "intracellular aptamers" or "intramers," which specifically recognize intracellular targets. See Famulok et al., Chem Biol. 2001, Oct, 8(10):931-939; Yoon and Rossi, Adv Drug Deliv Rev. 2018, Sep, 134:22-35 (each incorporated herein by reference).

[0450] Ribozymes The therapeutic moiety can be a ribozyme. Ribozymes are RNA molecular complexes with specific catalytic domains that have endonuclease activity (Kim and Cech, Proc Natl Acad Sci US A. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20). For example, many ribozymes accelerate phosphoester transfer reactions with high specificity, often cleaving only one of several phosphates in an oligonucleotide substrate (Cech et al., Cell. 1981 Dec;27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 Dec 5;216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14;357(6374):173-6). This specificity results from the requirement that the substrate bind to the ribozyme's internal guide sequence ("IGS") through specific base-pairing interactions prior to chemical reaction.

[0451] At least six basic varieties of naturally occurring enzymatic RNAs are currently known. Each is capable of catalyzing the hydrolysis of RNA phosphodiester bonds in trans (and thus cleaving other RNA molecules) under physiological conditions. Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs via the target-binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA, then binds to it through complementary base pairing, and once bound to the correct site, acts enzymatically to cleave the target RNA. This strategic cleavage of the target RNA destroys its ability to direct synthesis of the encoded protein. After an enzymatic nucleic acid binds and cleaves its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0452] Enzymatic nucleic acid molecules can be formed, for example, in hammerhead, hairpin, hepatitis delta virus, group I intron, or RNase P RNA (associated with an RNA guide sequence), or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described by Rossi et al., Nucleic Acids Res. 1992 Sep 11;20(17):4559-65. Examples of hairpin motifs are described by Hampel et al. (EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan 25;18(2):299-304, and U.S. Patent No. 5,631,359. Examples of hepatitis virus motifs are described in Perrotta and Been, Biochemistry. 1992 Dec 1;31(47):11843-52; examples of RNase P motifs are described in Guerrier-Takada et al, Cell. 1983 Dec;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifs are described in Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991 Oct 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar 23;32(11):2795-9); and examples of group I introns are described in U.S. Pat. No. 4,987,071. An enzymatic nucleic acid molecule can have a specific substrate binding site complementary to one or more regions of the target gene DNA or RNA, and have nucleotide sequences within or surrounding that substrate binding site that confer RNA cleavage activity to the molecule. Thus, ribozyme constructs need not be limited to the particular motifs referenced herein.

[0453] Methods for producing ribozymes that target polynucleotide sequences are known in the art. Ribozymes can be designed, synthesized, and tested in vitro and in vivo as described in WO 93 / 23569 and WO 94 / 02595, each of which is specifically incorporated herein by reference.

[0454] Ribozyme activity can be increased by altering the length of the ribozyme binding arms or by chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see, e.g., WO 92 / 07065, WO 93 / 15187, WO 91 / 03162, EP 92110298.4, U.S. Pat. No. 5,334,711, and WO 94 / 13688, which describe various chemical modifications that can be made to the sugar portion of enzymatic RNA molecules, modifications that enhance their effectiveness in cells, and removal of stem Π bases to shorten RNA synthesis time and reduce chemical requirements).

[0455] Immunostimulatory oligonucleotides The therapeutic moiety can be an immunostimulatory oligonucleotide. Immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) can induce an immune response when administered to a patient, which can be a mammal or other patient. ISSs contain, for example, specific palindromes that result in hairpin secondary structures (see Yamamoto S., et al. (1992) J. Immunol. 148:4072-4076), or CpG motifs, as well as other known ISS features (e.g., multi-G domains, see WO 96 / 11266).

[0456] The immune response may be an innate or adaptive immune response. The immune system is divided into a more innate immune system and the adaptive immune system of vertebrates, the latter being further divided into humoral and cellular components. The immune response may be mucosal.

[0457] Immunostimulatory nucleic acids are considered non-sequence-specific if they are not required to specifically bind to and reduce the expression of a target polynucleotide in order to elicit an immune response. Thus, particular immunostimulatory nucleic acids may contain sequences that correspond to regions of a naturally occurring gene or mRNA, yet still be considered non-sequence-specific immunostimulatory nucleic acids.

[0458] The immunostimulatory nucleic acid or oligonucleotide may contain at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. The immunostimulatory nucleic acid may contain at least one CpG dinucleotide with a methylated cytosine. The nucleic acid may contain a single CpG dinucleotide, where the cytosine in the CpG dinucleotide is methylated. The nucleic acid may contain the sequence 5' TAACGTTGAGGG'CAT 3'. The nucleic acid may contain at least two CpG dinucleotides, where at least one cytosine in the CpG dinucleotide is methylated. Each cytosine in the CpG dinucleotide present in the sequence can be methylated. The nucleic acid may contain multiple CpG dinucleotides, where at least one of the CpG dinucleotides contains a methylated cytosine.

[0459] Further specific nucleic acid sequences of oligonucleotides (ODN) suitable for use in the present compositions and methods are described in Raney et al., Journal of Pharmacology and Experimental Therapeutics, 298:1185-1192 (2001). The ODN used in the present compositions and methods can have a phosphodiester ("PO") backbone or a phosphorothioate ("PS") backbone, and / or at least one methylated cytosine residue in CpG motif.

[0460] Decoy oligonucleotides The therapeutic moiety can be a decoy oligonucleotide. Because transcription factors recognize their relatively short binding sequences, short oligonucleotides containing the consensus binding sequence of a specific transcription factor can be used as a tool to manipulate gene expression in living cells, even in the absence of surrounding genomic DNA. This strategy involves intracellular delivery of such a "decoy oligonucleotide," which is then recognized and bound by the target factor. Once the DNA binding site of the transcription factor is occupied by the decoy, the transcription factor is unable to subsequently bind to the promoter region of the target gene. The decoy can be used as a therapeutic agent to either inhibit the expression of genes activated by the transcription factor or upregulate genes repressed by the binding of the transcription factor. An example of the use of decoy oligonucleotides can be found in Mann et al., J. Clin. Invest, 2000, 106:1071-1075, which is expressly incorporated herein by reference in its entirety.

[0461] Supermir The therapeutic moiety may be a supermir. A supermir refers to a single-stranded, double-stranded, or partially double-stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or both, or modifications thereof, having a nucleotide sequence substantially identical to an miRNA and antisense to its target. The term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages and containing at least one non-naturally occurring moiety that functions similarly. Such modified or substituted oligonucleotides have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases. A supermir may not contain a sense strand. A supermir may not self-hybridize to a significant extent. While a supermir may have secondary structure, it is substantially single-stranded under physiological conditions. A substantially single-stranded supermir is single-stranded to the extent that less than about 50% of the supermir (e.g., less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5%) is double-stranded with itself. A supermir can include a hairpin segment (e.g., a sequence) that can self-hybridize, for example, at the "3'" end, to form a duplex region (e.g., a duplex region of at least about 1, about 2, about 3, or about 4 nucleotides, or of less than about 8, about 7, about 6, or about 5 nucleotides, or of about 5 nucleotides). The duplexed regions can be linked by a linker, e.g., a nucleotide linker, e.g., about 3, about 4, about 5, or about 6 dTs, e.g., modified dTs. Supermirs can be duplexed with shorter oligos, e.g., about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length, e.g., at one or both of the 3' and 5' ends, or at one end and a non-end or in the middle of the supermir.

[0462] miRNA mimics The therapeutic moiety may be an miRNA mimic. miRNA mimics represent a class of molecules that can be used to mimic the gene silencing capabilities of one or more miRNAs. Thus, the term "microRNA mimic" refers to a synthetic non-coding RNA (i.e., a miRNA not obtained by purification from an endogenous miRNA source) that can enter the RNAi pathway and regulate gene expression. miRNA mimics can be designed as mature molecules (e.g., single-stranded) or mimic precursors (e.g., pre-miRNAs). miRNA mimics may include nucleic acids (modified or modified nucleic acids) such as, but not limited to, oligonucleotides containing RNA, modified RNA, DNA, modified DNA, locked nucleic acids, or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA), or any combination of the above (including DNA-RNA hybrids). Additionally, miRNA mimics may include conjugates that can affect delivery, intracellular compartmentalization, stability, specificity, functionality, strand usage, and / or efficacy. In one design, miRNA mimics are double-stranded molecules (e.g., having a double-stranded region of about 16 to about 31 nucleotides in length) that contain one or more sequences that share identity with the mature strand of a given miRNA. Modifications can include 2' modifications (including 2'-0 methyl and 2'F modifications) on one or both strands of the molecule, as well as internucleotide modifications (e.g., phosphorothioate modifications) that enhance nucleic acid stability and / or specificity. In addition, miRNA mimics can include overhangs. The overhangs can comprise about 1 to about 6 nucleotides on either the 3' or 5' end of either strand and can be modified to enhance stability or functionality. miRNA mimics can include a double-stranded region of about 16 to about 31 nucleotides and can include one or more of the following chemical modification patterns: That is, the sense strand contains 2'-0-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide) and all of the Cs and Us, while the antisense strand modifications may include 2'F modifications of all of the Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with the two-nucleotide 3' overhang.

[0463] miRNA inhibitors The therapeutic moiety can be an miRNA inhibitor. The terms "anti-mir," "microRNA inhibitor," "miR inhibitor," or "miRNA inhibitor" are synonymous and refer to an oligonucleotide or modified oligonucleotide that interferes with the activity of a specific miRNA. Generally, the inhibitor is a natural or modified nucleic acid, such as an oligonucleotide such as RNA, modified RNA, DNA, modified DNA, locked nucleic acid (LNA), or any combination of the above.

[0464] Modifications include 2' modifications (including 2'-0 alkyl and 2'F modifications) and internucleotide modifications (e.g., phosphorothioate modifications), which can affect delivery, stability, specificity, intracellular compartmentalization, or efficacy. Additionally, miRNA inhibitors can include conjugates, which can affect delivery, intracellular compartmentalization, stability, and / or efficacy. Inhibitors can adopt a variety of configurations, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplexes), and hairpin designs. Generally, microRNA inhibitors include one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand(s) of the targeted miRNA. Furthermore, miRNA inhibitors can also include additional sequences located 5' and 3' relative to the sequence that is the reverse complement of the mature miRNA. The additional sequences may be the reverse complement of the sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequences may be any sequence (having a mixture of A, G, C, or U). One or both of the additional sequences may be any sequence capable of forming a hairpin. The reverse complement of the miRNA may flank the hairpin structure on the 5' and 3' sides. When the microRNA inhibitor is double-stranded, it may contain mismatches between nucleotides on opposite strands. Furthermore, the microRNA inhibitor may be linked to a conjugate moiety to facilitate the uptake of the inhibitor into cells. For example, the microRNA inhibitor may be linked to cholesteryl 5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypentylcarbamate), which allows passive uptake of the microRNA inhibitor into cells. MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13:723-730 (2007), and in WO 2007 / 095387 and WO 2008 / 036825, each of which is incorporated herein by reference in its entirety.One of skill in the art can select a sequence from a database for a desired miRNA and design an inhibitor useful in the methods disclosed herein.

[0465] Antisense Compounds (AC) Therapeutic moieties include antisense compounds (ACs) that can alter one or more aspects of the translation or expression of a target gene. The principle behind antisense technology is that antisense compounds that hybridize to a target nucleic acid regulate gene expression activity, such as translation, through one of several antisense mechanisms. Antisense technology is an effective means for altering the expression of one or more specific gene products and may therefore prove useful in several therapeutic, diagnostic, and research applications.

[0466] 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.

[0467] Antisense Compound Hybridization Site The antisense mechanism relies on hybridization of the antisense compound to a target nucleic acid.

[0468] An AC can hybridize to a sequence of about 5 to about 50 nucleic acids in length, which can also be considered the length of the AC. An AC can be about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 nucleic acids in length. An AC can be 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, about 20, about 21, about 22, about 23, about 24, about 25, 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, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 nucleic acids in length. An AC can be about 10 nucleic acids in length. An AC can be about 15 nucleic acids in length. An AC can be about 20 nucleic acids in length. An AC can be about 25 nucleic acids in length. An AC can be about 30 nucleic acids in length.

[0469] The AC may have less than about 100% 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 understand that mismatches can be included without eliminating the activity of the antisense compound. The AC may contain up to about 20% nucleotides that inhibit base pairing of the AC to the target nucleic acid. The AC may contain about 15% or less, about 10% or less, 5% or less mismatches, or no mismatches. The AC may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 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.

[0470] The incorporation of nucleotide affinity modifications can allow 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 an oligonucleotide and a target nucleic acid, for example, by determining the melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allows those skilled in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.

[0471] Antisense mechanism An AC according to the present disclosure may regulate one or more aspects of protein transcription, translation, and expression.

[0472] ACs can regulate transcription, translation, or protein expression through steric blocking. The following review describes the mechanism and applications of steric blocking: Roberts et al., Nature Reviews Drug Discovery (2020) 19:673-694, which is incorporated herein by reference in its entirety.

[0473] The antisense mechanism functions through hybridization between the antisense compound and the target nucleic acid. The AC can hybridize to its target sequence and downregulate the expression of the target protein. The AC can hybridize to its target sequence and downregulate the expression of one or more target protein isomers. The AC can hybridize to its target sequence and upregulate the expression of the target protein. The AC can hybridize to its target sequence and increase the expression of one or more target protein isomers.

[0474] The effectiveness of ACs 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 the hybridization of an antisense compound to its target nucleic acid. Such detection and / or measurement can be direct or indirect. In embodiments, antisense activity is assessed by detecting and / or measuring the amount of target protein. Antisense activity can be assessed by detecting and / or measuring the amount of target nucleic acid.

[0475] 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.

[0476] Those skilled in the art can design, synthesize and screen antisense compounds of different nucleic acid base sequences to identify sequences that produce antisense activity.For example, antisense compounds can be designed to inhibit the expression of target proteins.Methods for designing, synthesizing and screening antisense compounds for antisense activity against preselected target nucleic acids 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.

[0477] Antisense compounds comprising from about 8 to about 30 linked nucleosides are provided. The antisense compounds may contain modified nucleosides, modified internucleoside linkages and / or conjugate groups.

[0478] The antisense compound may be "tricyclo-DNA (tc-DNA)," which refers to a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and enhance the backbone geometry at a torsion angle γ. Homobasic adenine- and thymine-containing tc-DNA forms highly stable AT base pairs with complementary RNA.

[0479] Nucleosides Antisense compounds may contain linked nucleosides. Some or all of the nucleosides may be modified nucleosides. One or more nucleosides may contain modified nucleobases. One or more nucleosides may contain modified sugars. Chemically modified nucleosides are routinely incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for target RNA. Non-limiting examples of nucleosides are provided in Khvorova et al. Nature Biotechnology (2017) 35:238-248, which is incorporated herein by reference in its entirety.

[0480] Generally, a nucleobase is any group that contains one or more atoms or groups of atoms that can hydrogen bond with the base of another nucleic acid.In addition to "unmodified" or "natural" nucleobases such as purine nucleobases adenine (A) and guanine (G), and 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 their parent nucleobases, such as 7-deazapurine, 5-methylcytosine, or G-clamp, while nucleobase mimics include more complex structures, such as tricyclic phenoxazine nucleobase mimics.Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.

[0481] An AC may contain one or more nucleosides with modified sugar moieties. The furanosyl sugar ring of natural nucleosides can be modified in several ways, including, but not limited to, the addition of substituents, bridging two non-geminal ring atoms to form bicyclic nucleic acids (BNAs), 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 with 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. Methods for preparing modified sugars are well known to those skilled 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.

[0482] Nucleosides can include bicyclic modified sugars (BNAs), such as LNA (4'-(CH)-O-2' bridge), 2'-thio-LNA (4'-(CH)-S-2' bridge), 2'-amino-LNA (4'-(CH)-NR-2' bridge), ENA (4'-(CH)-O-2' bridge), 4'-(CH)-2' bridged BNA, 4'-(CHCH(CH))-2' bridged BNA, cEt (4'-(CH(CH)-O-2' bridge), and cMOE BNA (4'-(CH(CHOCH)-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, 6,794,491 ... Nos. 99, 7,034,133, and 6,525,191, and U.S. Patent Application Publication Nos. 2004-0171570, 2004-0219565, 2004-0014959, 2003-0207841, 2004-0143114, and 2003-0082807.

[0483] Also provided herein are "locked nucleic acids" (LNAs) in which the 2'-hydroxyl group of the 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 Elayadi et al., Curr. 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, but the term LNA is used for the bicyclic moiety, and the term ENA™ is used 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 analogs 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).

[0484] A similarly studied isomer of LNA is α-L-LNA, which has been shown to have excellent stability against 3'-exonucleases and has been incorporated into antisense gapmers and chimeras that exhibit potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0485] The synthesis and preparation of 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.

[0486] Analogs of LNA, such as phosphorothioate-LNA and 2'-thio-LNA, have also been prepared (Kumar et al., Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the 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). Furthermore, 2'-amino and 2'-methylamino-LNA have been prepared, and the thermal stability of their duplexes with complementary RNA and DNA strands has previously been reported.

[0487] 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, and phosphorothioates. Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Antisense compounds with non-phosphorus internucleoside linkages 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.

[0488] 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.

[0489] Conjugated groups Cargos can be modified by the covalent attachment of one or more conjugate groups. Generally, conjugate groups modify one or more properties of the cargo, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in chemistry and are linked to parent compounds directly or via an optional linking moiety or group. Conjugate 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. The conjugate group can include polyethylene glycol (PEG). PEG can be conjugated to either the cargo or the cCPP. The cargo can include a peptide, oligonucleotide, or small molecule.

[0490] Conjugate groups can be lipid moieties, e.g., cholesterol moieties (Letsinger et al., Proc. 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); thiocholesterol (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, such as 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); polyamines 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).

[0491] Linking groups or bifunctional linking moieties, such as those known in the art, are suitable for use 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 an AC. Generally, bifunctional linking moieties comprise a hydrocarbyl moiety having two functional groups. 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. The linker can comprise a chain structure or oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. Bifunctional linking moieties can 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-C10 alkyl, substituted or unsubstituted C2-C6 alkyl, and substituted or unsubstituted C1-C6 alkyl. 10 Alkenyl or substituted or unsubstituted C2-C 10 Included are alkynyls, and a non-limiting list of substituents include hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0492] The AC can be about 5 to about 50 nucleotides in length. The AC can be about 5 to about 10 nucleotides in length. The AC can be about 10 to about 15 nucleotides in length. The AC can be about 15 to about 20 nucleotides in length. The AC can be about 20 to about 25 nucleotides in length. The AC can be about 25 to about 30 nucleotides in length. The AC can be about 30 to about 35 nucleotides in length. The AC can be about 35 to about 40 nucleotides in length. The AC can be about 40 to about 45 nucleotides in length. The AC can be about 45 to about 50 nucleotides in length.

[0493] Clustered regularly interspaced short palindromic repeats (CRISPR) gene editing machinery The compound can comprise one or more cCPPs (or cCPPs) conjugated to CRISPR gene editing mechanism.As used herein, "CRISPR gene editing mechanism" refers to a protein, nucleic acid, or combination thereof that can be used to edit genome.Non-limiting examples of gene editing mechanism include gRNA, nuclease, nuclease inhibitor, and combinations and complexes thereof. The following patent documents describe CRISPR gene editing mechanisms: U.S. Patent No. 8,697,359, U.S. Patent No. 8,771,945, U.S. Patent No. 8,795,965, U.S. Patent No. 8,865,406, U.S. Patent No. 8,871,445, U.S. Patent No. 8,889,356, U.S. Patent No. 8,895,308, U.S. Patent No. 8,906,616, U.S. Patent No. 8,932,814, U.S. Patent No. 8,945,839, U.S. Patent No. 8,993,233, U.S. Patent No. 8,999,641, U.S. Patent Application No. 14 / 704,551, and U.S. Patent Application No. 13 / 842,859. Each of the foregoing patent documents is incorporated herein by reference in its entirety.

[0494] The linker can conjugate the cCPP to the CRISPR gene editing machinery. Any linker described in this disclosure or known to one of skill in the art can be utilized.

[0495] gRNA The compound can include a cCPP conjugated to a gRNA, which targets a genomic locus in a prokaryotic or eukaryotic cell.

[0496] The gRNA may be a single-molecule guide RNA (sgRNA). The sgRNA comprises a spacer sequence and a scaffold sequence. The spacer sequence is a short nucleic acid sequence used to target a nuclease (e.g., Cas9 nuclease) to a specific nucleotide region of interest (e.g., a genomic DNA sequence to be cleaved). The spacer may be about 17 to 24 base pairs in length, e.g., about 20 base pairs in length. The spacer may be about 15, about 16, about 17, 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 base pairs in length. The spacer may be 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, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 base pairs in length. The spacer can be about 15, about 16, about 17, 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 base pairs in length. The spacer sequence can have a GC content of about 40% to about 80%.

[0497] The spacer can target the site immediately before the 5' protospacer adjacent motif (PAM). The PAM sequence can be selected based on the desired nuclease. For example, the PAM sequence can be any one of the PAM sequences shown in the following table, where N refers to any nucleic acid, R refers to A or G, Y refers to C or T, W refers to A or T, and V refers to A, C, or G.

[0498] [Table 16]

[0499] The spacer may target a sequence of a mammalian gene, such as a human gene. The spacer may target a mutant gene. The spacer may target a coding sequence.

[0500] The scaffold sequence is a sequence within the sgRNA that is involved in nuclease (e.g., Cas9) binding. The scaffold sequence does not include a spacer / targeting sequence. In embodiments, the scaffold can be about 1 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides in length. The scaffold may be about 1, about 2, about 3, about 4, 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, about 20, about 21, about 22, about 23, about 24, about 25, 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, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68 8, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101 , about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, or about 125 nucleotides in length. The scaffold can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, or at least 125 nucleotides in length.

[0501] The gRNA may be a dual-molecule guide RNA, e.g., a crRNA and a tracrRNA. The gRNA may further comprise a polyA tail.

[0502] The compound comprises a cCPP conjugated to a nucleic acid comprising a gRNA. The nucleic acid may comprise about 1, about 2, about 3, about 4, 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 gRNAs. The gRNAs may recognize the same target. The gRNAs may recognize different targets. The nucleic acid comprising the gRNA comprises a sequence encoding a promoter, and the promoter drives expression of the gRNA.

[0503] nuclease The compound may comprise a cyclic cell-penetrating peptide (cCPP) conjugated to a nuclease. The nuclease may be a type II, type VA, type VB, type VC, type VU, or type VI-B nuclease. The nuclease may be a transcription activator-like effector nuclease (TALEN), meganuclease, or zinc finger nuclease. The nuclease may be a Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. The nuclease may be a Cas9 nuclease or a Cpf1 nuclease.

[0504] The nuclease can be a modified or mutant form of a Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. The nuclease can be a modified or mutant form of a TAL nuclease, meganuclease, or zinc finger nuclease. "Modified" or "mutant" nucleases can be, for example, truncated, fused to another protein (such as another nuclease), catalytically inactivated, etc. The nuclease can have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to a naturally occurring Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, or Cas14 nuclease, or a TALEN, meganuclease, or zinc finger nuclease. The nuclease can be a Cas9 nuclease derived from S. pyogenes (SpCas9). The nuclease can have at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a Cas9 nuclease derived from S. pyogenes (SpCas9). The nuclease may be Cas9 (SaCas9) from Staphylococcus aureus. The nuclease may have at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to Cas9 (SaCas9) from Staphylococcus aureus. The Cpfl may be a Cpfl enzyme from Acidaminococcus (species BV3L6, UniProt accession number U2UMQ6). The nuclease may have at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the Cpfl enzyme from Acidaminococcus (species BV3L6, UniProt accession number U2UMQ6).

[0505] The Cpf1 can be a Cpf1 enzyme from the family Lachnospiraceae (species ND2006, UniProt accession number A0A182DWE3). The nuclease can have at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the Cpf1 enzyme from the family Lachnospiraceae. The nuclease-encoding sequence can be codon-optimized for expression in mammalian cells. The nuclease-encoding sequence can be codon-optimized for expression in human or mouse cells.

[0506] The compound may comprise a cCPP conjugated to a nuclease. The nuclease may be a soluble protein.

[0507] The compound can include a cCPP conjugated to a nucleic acid encoding a nuclease. The nucleic acid encoding the nuclease can include a sequence encoding a promoter, which drives expression of the nuclease.

[0508] gRNA and nuclease combinations The compound may include one or more cCPPs conjugated to a gRNA and a nuclease. One or more cCPPs may be conjugated to a nucleic acid encoding a gRNA and / or a nuclease. The nucleic acid encoding the nuclease and gRNA may include a sequence encoding a promoter, which drives expression of the nuclease and gRNA. The nucleic acid encoding the nuclease and gRNA may include two promoters, with the first promoter controlling expression of the nuclease and the second promoter controlling expression of the gRNA. The nucleic acid encoding the gRNA and nuclease may encode from about 1 to about 20 gRNAs, or from about 1, about 2, about 3, about 4, 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, or about 19 up to about 20 gRNAs. The gRNAs can recognize different targets. The gRNAs can recognize the same target.

[0509] The compound may comprise a circular cell-penetrating peptide (or cCPP) conjugated to a ribonucleoprotein (RNP) that comprises a gRNA and a nuclease.

[0510] A composition can be delivered to a cell that includes (a) a cCPP conjugated to a gRNA and (b) a nuclease. A composition can be delivered to a cell that includes (a) a cCPP conjugated to a nuclease and (b) a gRNA.

[0511] A composition can be delivered to a cell that includes (a) a first cCPP conjugated to a gRNA and (b) a second cCPP conjugated to a nuclease. The first cCPP and the second cCPP can be the same. The first cCPP and the second cCPP can be different.

[0512] Genetic element of interest The compound may comprise a cyclic cell-penetrating peptide (cCPP) conjugated to a genetic element of interest. The genetic element of interest can replace a genomic DNA sequence cleaved by a nuclease. Non-limiting examples of genetic elements of interest include genes, single nucleotide polymorphisms, promoters, or terminators.

[0513] Nuclease inhibitors The compound may include a cyclic cell-penetrating peptide (cCPP) conjugated to a nuclease inhibitor (e.g., a Cas9 inhibitor). A limitation of gene editing is potential off-target editing. Delivery of a nuclease inhibitor can limit off-target editing. The nuclease inhibitor may be a polypeptide, polynucleotide, or small molecule. Nuclease inhibitors are described in U.S. Patent Application Publication No. 2020 / 087354, WO 2018 / 085288, U.S. Patent Application Publication No. 2018 / 0382741, WO 2019 / 089761, WO 2020 / 068304, WO 2020 / 041384, and WO 2019 / 076651, which are incorporated herein by reference in their entireties.

[0514] Therapeutic Polypeptides antibody The therapeutic moiety may comprise an antibody or antigen-binding fragment. The antibodies and antigen-binding fragments may be derived from any suitable source, such as human, mouse, camelid (e.g., camel, alpaca, llama), rat, ungulate, or non-human primate (e.g., monkey, rhesus).

[0515] The term "antibody" refers to an immunoglobulin (Ig) molecule capable of binding to a specific target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies and antigen-binding fragments thereof. A native immunoglobulin molecule generally comprises two heavy chain polypeptides and two light chain polypeptides. Each heavy chain polypeptide associates with a light chain polypeptide through an interchain disulfide bond between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., proteins composed of two heterologous polypeptide chains). The two heterodimeric proteins then associate through an additional interchain disulfide bond between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.

[0516] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one complementarity-determining region (CDR) of an immunoglobulin heavy chain and / or light chain that binds to at least one epitope of an antigen of interest. An antigen-binding fragment may contain one, two, or three CDRs of a variable heavy chain (VH) sequence derived from an antibody that specifically binds to a target molecule. An antigen-binding fragment may contain one, two, or three CDRs of a variable light chain (VL) sequence derived from an antibody that specifically binds to a target molecule. An antigen-binding fragment may contain one, two, three, four, five, or all six CDRs of the variable heavy chain (VH) and variable light chain (VL) sequences derived from an antibody that specifically binds to a target molecule. Antigen-binding fragments include proteins comprising a portion of a full-length antibody, generally the antigen-binding or variable region thereof, e.g., Fab, F(ab')2, Fab', Fv fragments, minibodies, diabodies, single domain antibodies (dAbs), single-chain variable fragments (scFv), nanobodies, multispecific antibodies formed from antibody fragments, and any other modified configuration of an immunoglobulin molecule that may contain an antigen-binding site or fragment of the required specificity.

[0517] The term "F(ab)" refers to two of the protein fragments resulting from proteolytic cleavage of an IgG molecule with the enzyme papain. Each F(ab) may comprise a covalent heterodimer consisting of a VH chain and a VL chain and contains an intact antigen-binding site. Each F(ab) may be a monovalent antigen-binding fragment. The term "Fab'" refers to a fragment derived from F(ab')2 and may contain a small portion of Fc. Each Fab' fragment may be a monovalent antigen-binding fragment.

[0518] The term "F(ab')2" refers to the protein fragment of IgG generated by proteolytic cleavage with the enzyme pepsin. Each F(ab')2 fragment can contain two F(ab') fragments and can therefore be a bivalent antigen-binding fragment.

[0519] "Fv fragment" refers to a noncovalent VH:VL heterodimer containing an antigen-binding site that retains much of the antigen recognition and binding capabilities of a native antibody molecule, but lacks the CH1 and CL domains contained within the Fab. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0520] Bispecific antibodies (BsAbs) are antibodies that can simultaneously bind to two distinct, unique antigens (or different epitopes of the same antigen). Therapeutic moieties can include bispecific antibodies that can simultaneously bind to two different targets of interest. BsAbs can redirect cytotoxic immune effector cells for enhanced tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and other cytotoxic mechanisms mediated by effector cells.

[0521] Recombinant antibody engineering has enabled the generation of recombinant bispecific antibody fragments comprising the variable heavy (VH) and variable light (VL) domains of a parent monoclonal antibody (mab). Non-limiting examples include scFv (single-chain variable fragment), BsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (single domain antibody), and BssdAb (bispecific single domain antibody).

[0522] BsAbs with an Fc region are useful for carrying out Fc-mediated effector functions such as ADCC and CDC. They have the half-life of normal IgG. On the other hand, BsAbs without an Fc region (bispecific fragments) rely solely on their antigen-binding ability to achieve therapeutic activity. Due to their smaller size, these fragments have better solid tumor penetration. BsAb fragments do not require glycosylation and can be produced in bacterial cells. The size, valency, flexibility, and half-life of BsAbs are tailored to the application.

[0523] Using recombinant DNA technology, bispecific IgG antibodies can be constructed from two different heavy and light chains expressed in the same cell line. Random assembly of different chains results in non-functional molecules and the formation of undesired HC homodimers. To address this issue, a second binding moiety (e.g., a single-chain variable fragment) can be fused to the N- or C-terminus of the H or L chain to obtain a tetravalent BsAb containing two binding sites for each antigen. Additional methods for addressing LC-HC mispairing and HC homodimerization are described below.

[0524] Knob-into-hole BsAb IgG. Heavy chain heterodimerization is forced by introducing different mutations into the two CH3 domains, resulting in asymmetric antibodies. Specifically, a "knob" mutation in one HC and a "hole" mutation in the other HC promote heterodimerization.

[0525] Ig-scFv fusions. The direct addition of new antigen-binding moieties to full-length IgG results in tetravalent fusion proteins. Examples include IgG C-terminal scFv fusions and IgG N-terminal scFv fusions.

[0526] Diabody-Fc fusions, which involve replacing the Fab fragment of an IgG with a bispecific diabody (a derivative of an scFv).

[0527] Dual variable domain-IgG (DVD-IgG): The VL and VH domains of an IgG with one specificity were fused to the N-terminus of the VL and VH, respectively, of an IgG of a different specificity via a linker sequence to form a DVD-IgG.

[0528] The term "diabody" refers to a bispecific antibody in which VH and VL domains are expressed in a single polypeptide chain, using a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121-23 (1994)). Diabodies can be engineered to bind to two different antigens and are bispecific antigen-binding constructs.

[0529] The term "nanobody" or "single-domain antibody" refers to an antigen-binding fragment of a single monomeric variable antibody domain, including one variable domain (VH) of a heavy-chain antibody. They have several advantages over traditional monoclonal antibodies (mAbs), such as smaller size (15 kD), stability in a reducing intracellular environment, and ease of production in bacterial systems (Schumacher et al., (2018) Nanobodies: Chemical Functionalization Strategies and Intracellular Applications. Angew. Chem. Int. Ed. 57, 2314; Siontorou, (2013) Nanobodies as novel agents for disease diagnosis and therapy. International Journal of Nanomedicine, 8, 4215-27). These characteristics make nanobodies amenable to genetic and chemical modification (Schumacher et al., (2018) Nanobodies: Chemical Functionalization Strategies and Intracellular Applications. Angew. Chem. Int. Ed. 57, 2314) and facilitate their application as research tools and therapeutic agents (Bannas et al., (2017) Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Frontiers in Immunology, 8, 1603).Over the past decade, nanobodies have been used for protein immobilization (Rothbauer et al., (2008) A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins. Mol. Cell. Proteomics, 7, 282-289), imaging (Traenkle et al., (2015) Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells. Mol. Cell. Proteomics, 14, 707-723), and protein-protein interaction detection (Herse et al., (2013) Visualization and targeted disruption of protein interactions in living cells. Nat. Commun., 4, 2660; Massa et al., (2014) Site-Specific Labeling of Cysteine-Tagged Camelid Single-Domain Antibody-Fragments for Use in Molecular Imaging. Bioconjugate Chem, 25, 979-988) and as macromolecular inhibitors (Truttmann et al., (2015) HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation. J. Biol. Chem. 290, 9087-9100).

[0530] The therapeutic moiety can be an antigen-binding fragment that binds to a target of interest. The antigen-binding fragment that binds to a target of interest can comprise one, two, or three CDRs of the variable heavy chain (VH) sequence from an antibody that specifically binds to the target of interest. The antigen-binding fragment that binds to a target of interest can comprise one, two, or three CDRs of the variable light chain (VL) sequence from an antibody that specifically binds to the target of interest. The antigen-binding fragment that binds to a target of interest can comprise one, two, three, four, five, or all six CDRs of the variable heavy chain (VH) and / or variable light chain (VL) sequence from an antibody that specifically binds to the target of interest. The target-binding antigen-binding fragment can be a portion of a full-length antibody, such as a Fab, F(ab')2, Fab', Fv fragment, minibody, diabody, single domain antibody (dAb), single chain variable fragment (scFv), nanobody, multispecific antibody formed from antibody fragments, or any other modified structure of an immunoglobulin molecule that contains an antigen-binding site or fragment of the required specificity.

[0531] The therapeutic moiety may comprise a bispecific antibody. A bispecific antibody (BsAb) is an antibody that can simultaneously bind to two separate, unique antigens (or different epitopes of the same antigen).

[0532] The therapeutic moiety may comprise a "diabody."

[0533] The therapeutic moiety may comprise a nanobody or a single domain antibody (sometimes referred to herein as an sdAb or VHH).

[0534] The therapeutic moiety may comprise a "minibody." A minibody (Mb) comprises a CH3 domain fused or linked to an antigen-binding fragment (e.g., a CH3 domain fused or linked to an scFv, domain antibody, etc.). The term "Mb" may refer to a single CH3 domain. A CH3 domain may refer to a minibody. (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al., Science 242, 423-426, 1988; Huston et al., PNAS USA 85, 5879-5883, 1988; International Application No. US92 / 09965; International Publication No. WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0535] Therapeutic moieties may include "monobodies." The term "monobody" refers to synthetic binding proteins constructed using fibronectin type III domains (FN3) as a molecular scaffold.

[0536] The therapeutic moiety can be an antibody mimetic. Antibody mimetics are compounds that, like antibodies, can specifically bind to an antigen, but are not structurally related to antibodies. They are typically artificial peptides or proteins with a molar mass of about 3 to 20 kD (compared to the molar mass of an antibody, which is about 150 kDa). Examples of antibody mimetics include affibody molecules (built on a Protein A domain scaffold, Nygren (June 2008). FEBS J. 275(11):2668-76), affilins (built on gamma-B crystalline or ubiquitin scaffolds, see Ebersbach H et al. (September 2007). J. Mol. Biol. 372(1):172-85), affimers (built on a Crystatin scaffold, see Johnson A et al., (Aug 7, 2012). Anal. Chem. 84(15):6553-60), affitins (built on Sac7d from the S. acidocaldarius scaffold, Krehenbrink M et al., (November 2012). 2008). J. Mol. Biol. 383(5):1058-68), alphabodies (built on a triple helical coiled-coil scaffold, see Desmet, J et al., (5 Feb 2014). Nature Communications. 5:5237), anticalins (built on a lipocalin scaffold, see Skerra A (June 2008). FEBS J. 275(11):2677-83), avimers (built on various membrane receptor scaffolds, see Silverman J et al. (December 2005). Nat. Biotechnol. 23(12):1556-61), DARPins (built on an ankyrin repeat motif scaffold, see Stumpp et al., (August 2008). Drug Discov.Today.13(15-16):695-701), Fynomer (built on the scaffold of the SH3 domain of Fyn, Grabulovski et al., (2007). J Biol Chem.282(5):3196-3204), Kunitz domain peptides (built on the scaffold of Kunitz domains of various protease inhibitors, see Nixon et al (March 2006). Curr Opin Drug Discov Dev. 9(2):261-8), and monobodies (built on the scaffold of the type III domain of fibronectin, see Koide et al (2007). Methods Mol. Biol. 352:95-109).

[0537] The therapeutic moiety may comprise a "designed ankyrin repeat" or "DARPin". DARPins are derived from the naturally occurring ankyrin protein, which is composed of at least three repeat motifs, usually four or five repeats.

[0538] The therapeutic moiety may comprise a "dual variable domain-IgG" or "DVD-IgG", which is generated from two parent monoclonal antibodies by fusing the VL and VH domains of an IgG of one specificity to the N-terminus of the VL and VH, respectively, of an IgG of a different specificity via a linker sequence.

[0539] The therapeutic moiety may comprise an F(ab) fragment.

[0540] The therapeutic moiety may comprise an F(ab')2 fragment.

[0541] The therapeutic moiety may comprise an Fv fragment.

[0542] The antigen-binding fragment may include a "single-chain variable fragment" or "scFv." scFv refers to a fusion protein of the variable regions of immunoglobulin heavy (VH) and light (VL) chains linked by a short linker peptide of 10 to approximately 25 amino acids. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. The linker can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Numerous methods have been described for identifying chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains derived from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, for example, U.S. Pat. Nos. 5,091,513 and 5,132,405 to Huston et al. and U.S. Pat. No. 4,946,778 to Ladner et al.

[0543] Antigen-binding constructs can contain two or more antigen-binding moieties. They can bind to two distinct, unique antigens or different epitopes of the same antigen. Knob-into-hole BsAb IgG. Heavy chain heterodimerization can be forced by introducing different mutations into the two CH3 domains, resulting in asymmetric antibodies. Specifically, a "knob" mutation is introduced into one HC and a "hole" mutation is introduced into the other HC to promote heterodimerization.

[0544] Peptide inhibitors The therapeutic moiety may comprise a peptide. The peptide may act as an agonist, increasing the activity of a target protein. The peptide may act as an antagonist, decreasing the activity of a target protein. The peptide may be configured to inhibit protein-protein interactions (PPIs). Protein-protein interactions (PPIs) are important in many biochemical processes, such as the transcription of nucleic acids and various post-translational modifications of translated proteins. PPIs can be experimentally determined by biophysical techniques, such as X-ray crystallography, NMR spectroscopy, surface plasma resonance (SPR), biolayer interferometry (BLI), isothermal titration calorimetry (ITC), radioligand binding, spectrophotometric assays, and fluorescence spectroscopy. Peptides that inhibit protein-protein interactions are sometimes referred to as peptide inhibitors.

[0545] The therapeutic moiety may include a peptide inhibitor. The peptide inhibitor may contain about 5 to about 100 amino acids, about 5 to about 50 amino acids, about 15 to about 30 amino acids, or about 20 to about 40 amino acids. The peptide inhibitor may contain one or more chemical modifications, for example, to reduce proteolysis and / or improve in vivo half-life. The peptide inhibitor may contain one or more synthetic amino acids and / or backbone modifications. The peptide inhibitor may have an α-helical structure.

[0546] Peptide inhibitors can target the dimerization domain of a homodimeric or heterodimeric target protein of interest.

[0547] small molecule The therapeutic moiety may comprise a small molecule. The therapeutic moiety may comprise a small molecule kinase inhibitor. The therapeutic moiety may comprise a small molecule that inhibits a kinase that phosphorylates a target of interest. Inhibiting phosphorylation of a target of interest can block nuclear translocation of the target of interest. The therapeutic moiety may comprise a small molecule inhibitor of MyD88.

[0548] composition Compositions comprising the compounds described herein are provided.

[0549] Pharmaceutically acceptable salts and / or prodrugs of the disclosed compounds are provided. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases, depending on the specific substituents found on the compound. Under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable, non-toxic acid or base salts, it may be appropriate to administer the compounds as salts. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic, sulfuric, and organic acids such as acetic, propionic, benzoic, succinic, fumaric, mandelic, oxalic, citric, tartaric, malonic, ascorbic, α-ketoglutaric, α-sugar phosphate, maleic, tosylic, and methanesulfonic acids. Thus, disclosed herein are hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartrate, malonate, ascorbate, α-ketoglutarate, α-glycolate, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of the present compounds can be obtained using standard procedures well known in the art (e.g., by reacting a sufficiently basic compound, such as an amine, with an appropriate acid to provide a physiologically acceptable anion). Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0550] Mechanisms of oligonucleotide therapeutics and target molecules Many types of oligonucleotides can regulate gene transcription, translation, and / or protein function in cells. Non-limiting examples of such oligonucleotides include, for example, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense, antagomirs, antimirs, microRNA mimics, supermirs, Ul adapters, and aptamers. Further examples include DNA targeting, triplex-forming oligonucleotides, strand invasion oligonucleotides, and synthetic guide strands for CRISPR / Cas. These nucleic acids act through various mechanisms. See Smith and Zain, Annu Rev Pharmacol Toxicol. 2019, 59:605-630, incorporated herein by reference.

[0551] Splice-switching antisense oligonucleotides are short synthetic antisense modified nucleic acids that base pair with pre-mRNA and disrupt the normal splicing repertoire of a transcript by blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is necessary for the proper expression of the majority of protein-coding genes, and thus targeting this process provides a means of manipulating protein production from genes. Splicing modulation is particularly valuable in the case of diseases caused by mutations that inhibit normal splicing, or in cases where interfering with the normal splicing process of gene transcripts can be therapeutic. Such antisense oligonucleotides provide an effective and specific method for targeting and altering splicing in a therapeutic manner. See Havens and Hastings, Nucleic Acids Res. 2016 Aug 19;44(14):6549-6563, incorporated herein by reference.

[0552] In the case of siRNA or miRNA, these nucleic acids can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi). After introduction of siRNA or miRNA into the cytoplasm, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of the siRNA or miRNA is displaced from the RISC complex and provides a template within RISC that can recognize and bind to mRNAs with sequences complementary to those of the bound siRNA or miRNA. Upon binding to the complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strand. RNAi can enable the downregulation of specific proteins by targeting the specific destruction of the corresponding mRNA that encodes protein synthesis.

[0553] Because siRNA and miRNA constructs can be synthesized using any nucleotide sequence directed against a target protein, the therapeutic applications of RNAi are extremely broad. To date, siRNA constructs have demonstrated the ability to specifically downregulate target proteins in both in vitro and in vivo models, as well as in clinical studies.

[0554] Antisense oligonucleotides and ribozymes can also inhibit the translation of mRNA into protein. In the case of antisense constructs, these single-stranded deoxyribonucleotides have sequences complementary to the sequence of the target protein mRNA and can bind to the mRNA through Watson-Crick base pairing. This binding prevents translation of the target mRNA and / or causes RNase H degradation of the mRNA transcript. As a result, antisense oligonucleotides have great potential for specificity of action (i.e., downregulation of specific disease-related proteins). To date, these compounds have shown promise in several in vitro and in vivo models, such as models of inflammatory disease, cancer, and HIV (reviewed in Agrawal, Trends in Biotech. 14:376-387 (1996)). Antisense can also affect cellular activity by specifically hybridizing with chromosomal DNA.

[0555] Immunostimulatory nucleic acids include deoxyribonucleic acids and ribonucleic acids. In the case of deoxyribonucleic acids, specific sequences or motifs have been shown to induce immune stimulation in mammals. These sequences or motifs include CpG motifs, pyrimidine-rich sequences, and palindromic sequences. CpG motifs in deoxyribonucleic acids are thought to be specifically recognized by the endosomal receptor, toll-like receptor 9 (TLR-9), which then triggers both innate and acquired immune stimulation pathways. Specific immunostimulatory ribonucleic acid sequences have also been reported. These RNA sequences are thought to induce immune activation by binding to toll-like receptors 6 and 7 (TLR-6 and TLR-7). Furthermore, double-stranded RNA has also been reported to be immunostimulatory and is thought to activate through binding to TLR-3.

[0556] Non-limiting examples of mechanisms and targets of antisense oligonucleotides (ASOs) for modulating gene transcription, translation and / or protein function are shown in Tables 9A and 9B.

[0557] [Table 17]

[0558] [Table 18]

[0559] Clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins comprise the CRISPR-Cas system. CRISPR-Cas is a gene editing mechanism. The RNA-guided (e.g., gRNA) Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner. The Cas9 endonuclease can be replaced with any nuclease disclosed herein. The gRNA targets the nuclease (e.g., Cas9 nuclease) to a specific nucleotide region of interest (e.g., the genomic DNA sequence to be cleaved) and cleaves the genomic DNA. The genomic DNA can then be replaced with the desired genetic element.

[0560] Methods for modulating tissue distribution and / or retention Provided herein are compounds and methods for modulating the tissue distribution and / or retention of a therapeutic agent in a subject. Tissue distribution, for example, refers to increasing the concentration of a therapeutic agent in a specific region of a tissue, e.g., increasing the concentration in a brain region such as the cerebellum, cortex, hippocampus, or olfactory bulb compared to an unconjugated therapeutic agent. A compound that modulates the tissue degradation of a therapeutic agent can include a cyclic cell-penetrating peptide (cCPP) and an exocyclic peptide (EP). A method for modulating tissue distribution can include administering to a subject a compound comprising a cyclic cell-penetrating peptide (cCPP) and an exocyclic peptide (EP). Modulation of the tissue distribution or retention of a compound can be assessed by measuring the amount, expression, function, or activity of the compound in different tissues in vivo. The tissues can be different tissues of the same biological system, for example, different types of muscle tissue or different tissues within the central nervous system. The tissue can be muscle tissue, and there is modulation of the distribution or retention of the compound in cardiac tissue compared to at least one other type of muscle tissue (e.g., skeletal muscle, including but not limited to, diaphragm, tibialis anterior, and triceps, or smooth muscle). The tissue can be a CNS tissue, provided there is modulation of the distribution or retention of the compound in at least one CNS tissue relative to at least one other type of CNS tissue.

[0561] Any of the EPs described herein are suitable for inclusion in the compounds used in the present methods. The EP can be PKKKRKV. The EP can be 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, and PKKKRKG. EP is KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, K Can be KKKK, KKKRK, KBKBK, KKKRKV, PGKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV and PKKKRKG.

[0562] The EP may comprise PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is β-alanine. The amino acids in the EP may have D or L stereochemistry. The EP may comprise PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is β-alanine. The amino acids in the EP may have D or L stereochemistry.

[0563] 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.

[0564] The amount, expression, function or activity of the compound may be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue compared to a second tissue.

[0565] The amount, expression, function or activity of the compound may be decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue compared to a second tissue.

[0566] The amount or expression of the compound can be assessed in different tissue types by methods known in the art (including, but not limited to, the methodologies described in the Examples). Tissues can be prepared by standard methods. The amount or expression of the compound in different tissues can be measured by techniques well established in the art, such as LC-MS / MS, Western blot analysis, or ELISA. The function or activity of the compound in different tissues can be measured by established techniques for assessing the relevant function or activity, such as using RT-PCR to evaluate the activity of oligonucleotide-based therapeutic moieties. For example, for antisense compounds (ACs) used as therapeutic moieties (TMs) that induce exon skipping in target mRNAs of interest, RT-PCR can be used to quantify the level of exon skipping in different tissues.

[0567] The tissue distribution and / or retention of a therapeutic agent in tissues of the central nervous system (CNS) can be modulated with a compound comprising a cyclic cell-penetrating peptide (cCPP) and an exocyclic peptide (EP). The compound can be administered intrathecally to a subject, and the compound can modulate the tissue distribution and / or retention of the therapeutic agent in tissues of the central nervous system (CNS). Non-limiting examples of CNS tissues include the cerebellum, cortex, hippocampus, olfactory bulb, spinal cord, dorsal root ganglion (DRG), and cerebrospinal fluid (CSF). The compound comprising a cCPP and an EP can be administered intrathecally, and the level of expression, activity, or function of the therapeutic agent can be higher in at least one CNS tissue compared to another CNS tissue. The compound comprising a cCPP and an EP can be administered intrathecally, and the level of expression, activity, or function of the therapeutic agent can be lower in at least one CNS tissue compared to another CNS tissue. The therapeutic agent can include a CD33-targeted therapeutic agent (e.g., a CD33-targeted antisense compound), and the compound is administered intrathecally. The present compounds, including cCPP and EP, can be administered intrathecally at a dose of at least 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 50 mg / kg.

[0568] A method for modulating tissue distribution or retention of a therapeutic agent in the central nervous system (CNS) of a subject includes: (a) a cyclic cell-penetrating peptide (cCPP); (b) a therapeutic moiety (TM) comprising the therapeutic agent; and (c) an exocyclic peptide (EP) comprising at least one positively charged amino acid residue, wherein the amount, expression, function, or activity of the therapeutic agent is modulated by at least 10% in at least one tissue of the subject's CNS compared to a second tissue of the subject's CNS.

[0569] The amount, expression, function, or activity of a Therapeutic Agent may be modulated by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue of the CNS of a subject compared to a second tissue of the CNS of the subject.

[0570] Any of the therapeutic agents described herein for CNS-related diseases or disorders are suitable for inclusion in the compounds used in the methods. Therapeutic agents can include CD33-targeted therapeutic agents, such as any of the CD33-targeted antisense compounds described herein.

[0571] Any of the CPPs described herein are suitable for inclusion in the compounds used in the present methods. The CPP may be a cyclic CPP (cCPP).

[0572] The compounds can be used to treat a subject having a central nervous system disease or disorder or a neuroinflammatory disease or disorder, hi embodiments, the subject has Alzheimer's disease or Parkinson's disease.

[0573] The tissue distribution and / or retention of the therapeutic agent in different types of muscle tissue can be modulated. Non-limiting examples of muscle tissue include the diaphragm, cardiac muscle, tibialis anterior, triceps, other skeletal muscle, and smooth muscle. A compound comprising cCPP, EP, and a therapeutic agent can be administered, and the level of expression, activity, or function of the therapeutic agent can be higher in at least one muscle tissue compared to another muscle tissue. A compound comprising cCPP, EP, and a therapeutic agent can be administered, and the level of expression, activity, or function of the therapeutic agent can be lower in at least one muscle tissue compared to another muscle tissue. The therapeutic agent can be a dystophin-targeting therapeutic agent (e.g., a DMD-targeting antisense compound). The compound can be administered at a dosage of at least 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 50 mg / kg.

[0574] 1. A method for modulating tissue distribution or retention of a therapeutic agent in the musculature of a subject, comprising: (a) a cyclic cell-penetrating peptide (cCPP); (b) a therapeutic moiety (TM) comprising the therapeutic agent; and (c) an exocyclic peptide (EP) comprising at least one positively charged amino acid residue, wherein the amount, expression, function, or activity of the therapeutic agent is modulated by at least 10% in at least one tissue of the subject's muscular system compared to a second tissue of the subject's muscular system.

[0575] The amount, expression, function, or activity of a Therapeutic Agent may be modulated by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue of the subject's muscular system compared to a second tissue of the subject's muscular system.

[0576] Any of the therapeutic agents described herein for diseases or disorders related to the muscular system are suitable for inclusion in the compounds used in the methods. The therapeutic agent may be a DMD-targeted therapeutic agent, such as a DMD-targeted antisense compound.

[0577] Any of the CPPs described herein are suitable for inclusion in the compounds used in the methods. In embodiments, the CPP is a cyclic CPP (cCPP).

[0578] In embodiments, the subject has a neuromuscular or musculoskeletal disorder. In embodiments, the subject has Duchenne muscular dystrophy.

[0579] Diseases Associated with Aberrant Splicing and Exemplary Target Genes The human genome contains over 40,000 genes, approximately half of which correspond to protein-coding genes. However, the number of human protein species is predicted to be orders of magnitude higher due to single amino acid polymorphisms, post-translational modifications, and, importantly, alternative splicing. RNA splicing, which generally occurs in the nucleus, is the process by which precursor messenger RNA (pre-mRNA) is converted into mature messenger RNA (mRNA) by removing non-coding regions (introns) and joining together the remaining coding regions (exons). The resulting mRNA can then be transported from the nucleus and translated into proteins. Alternative or differential splicing is a regulated process in gene expression that results in a single gene encoding multiple proteins. In this process, specific exons of a gene can be included or excluded from the final processed mRNA produced from that gene. Although alternative splicing is a normal phenomenon in eukaryotes and contributes to the biodiversity of proteins encoded by the genome, aberrant variations in splicing are significantly implicated in disease. The majority of human genetic diseases are caused by splicing variants. Aberrant splicing variants contribute to the development of cancer, and splicing factor genes are frequently mutated in different types of cancer.

[0580] Approximately 10% of the approximately 80,000 mutations reported in the Human Gene Mutation Database (HGMD) affect splice sites. In HGMD, there are 3,390 disease-causing mutations that occur at the +1 donor splice site. These mutations affect 2,754 exons in 901 genes. The prevalence is even higher for neuromuscular disorders (NMDs) due to the unusually large size and multi-exon structure of genes encoding muscle structural proteins, further highlighting the importance of these mutations in NMDs.

[0581] Previously, correction of point mutations, e.g., splice site mutations, has been attempted via the homology-directed repair (HDR) pathway, but this is highly inefficient in postmitotic tissues such as skeletal muscle, hindering its therapeutic utility in NMD. Furthermore, standard gene therapy approaches for reintroducing corrected coding regions into the genome are hindered by the large size of genes encoding, for example, muscle structural proteins. Furthermore, many existing therapies rely on inefficient delivery of therapeutic compounds into diseased cells, resulting in impractical in vivo treatment and higher toxicity.

[0582] The target gene of the present disclosure can be any eukaryotic gene containing one or more introns and one or more exons. The target gene can be a mammalian gene. The mammal can be a human, mouse, cow, rat, pig, horse, chicken, sheep, etc. The target gene can be a human gene.

[0583] A target gene can be a gene containing a mutation that results in aberrant splicing. A target gene can be a gene containing one or more mutations. A target gene can be a gene containing one or more mutations such that transcription and translation of the target gene does not result in a functional protein. A target gene can be a gene containing one or more mutations such that transcription and translation of the target gene results in a target protein that is less active or non-functional than the wild-type target protein.

[0584] The target gene may be a gene underlying a genetic disorder. The target gene may have abnormal gene expression in the central nervous system. The target gene may be a gene involved in the pathogenesis of a neuromuscular disorder (NMD). The target gene may be a gene involved in the pathogenesis of a musculoskeletal disorder (NMD). The neuromuscular disease may be Pompe disease, and the target gene may be GYS1.

[0585] Antisense compounds can be used to target genes containing mutations that result in aberrant splicing underlying genetic diseases in order to redirect splicing to obtain the desired splice product (Kole, Acta Biochimica Polonica, 1997, 44, 231-238).

[0586] The CRISPR gene editing machinery can be used to target abnormal genes for removal or to regulate gene transcription and translation.

[0587] The disease may include β-thalassemia (Dominski and Kole, Proc. Natl. Acad. Sci. USA, 1993, 90, 8673-8677; Sierakowska et al., Nucleosides & Nucleotides, 1997, 16, 1173-1182; Sierakowska et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 12840-44; Lacerra et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 9591-9596).

[0588] The disease may involve dystrophin Kobe (Takeshima et al., J. Clin. Invest., 1995, 95, 515-520).

[0589] The disease may include Duchenne muscular dystrophy (Dunckley et al., Nucleosides & Nucleotides, 1997, 16, 1665-1668; Dunckley et al., Human Mol. Genetics, 1998, 5, 1083-90). The target gene may be the DMD gene, which encodes dystrophin. The protein consists of an N-terminal domain that binds to actin filaments, a central rod domain, and a C-terminal cysteine-rich domain that binds to the dystrophin-glycoprotein complex (Hoffman et al., 1987; Koenig et al., 1988; Yoshida and Ozawa, 1990). Mutations in the DMD gene that interrupt the reading frame result in complete loss of dystrophin function, which causes severe Duchenne muscular dystrophy (DMD) [MIM ​​310200]. On the other hand, milder Becker muscular dystrophy (BMD [MIM300376]) results from a non-frameshift mutation in the same gene, resulting in an internally deleted but partially functional dystrophin that retains its N- and C-termini (Koenig et al. 1989, Di Blasi et al. 1996). More than two-thirds of patients with DMD and BMD have deletions of two or more exons (den Dunnen et al. 1989). Notably, patients with very mild BMD who lack up to 67% of the central rod domain have been described (England et al. 1990, Winnard et al. 1993, Mirabella et al. 1998). This suggests that partially functional dystrophin can be generated despite large deletions, provided the deletions result in an in-frame transcript. These observations led to the idea of ​​using AC to alter splicing so that the open reading frame was restored and the severe DMD phenotype was converted to a milder BMD phenotype.Several studies have demonstrated therapeutic AC-induced single-exon skipping in cells derived from the mdx mouse model (Dunckley et al. 1998; Wilton et al. 1999; Mann et al. 2001, 2002; Lu et al. 2003) and various DMD patients (Takeshima et al. 2001; van Deutekom et al. 2001; Aartsma-Rus et al. 2002, 2003; De Angelis et al. 2002). AC can be used to skip one or more exons selected from DMD exons 2, 8, 11, 17, 19, 23, 29, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 55, and 59. See Aartsma-Rus et al. 2002, incorporated herein by reference. ACs can be used to skip one or more exons selected from DMD exons 8, 11, 43, 44, 45, 50, 51, 53, and 55. Approximately 75% of all DMD patients result from skipping these exons. Skipping an exon adjacent to an out-of-frame deletion or an in-frame exon containing a nonsense mutation can restore the reading frame and induce the synthesis of BMD-like dystrophin in treated cells (van Deutekom et al. 2001; Aartsma-Rus et al. 2003). ACs hybridizing to their target sequence in the target DMD pre-mRNA can induce skipping of one or more exons. ACs can induce the expression of a re-spliced ​​target protein containing an active fragment of dystrophin. Non-limiting examples of ACs of exon 52 are described in U.S. Patent Application Publication No. 2019 / 0365918, which is incorporated by reference in its entirety for all purposes. The compound may include cargoes that target the EP, cCPP, and DMD genes.

[0590] Cyclic cell-penetrating peptides (cCPPs) conjugated to cargo moieties Cyclic cell-penetrating peptides (cCPPs) can be conjugated to cargo moieties.

[0591] The cargo moiety may be conjugated to the cCPP via a linker. The cargo moiety may comprise a therapeutic moiety. The therapeutic moiety may comprise an oligonucleotide, a peptide, or a small molecule. The oligonucleotide may comprise an antisense oligonucleotide. The cargo moiety is conjugated to the linker at the terminal carbonyl group to form the following structure:

[0592] [ka] wherein EP is an exocyclic peptide, M, AA SC , Cargo, 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’ The - can be independently replaced with one or more amino acids, such as, for example, glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0593] The endosomal escape vehicle (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP) and a linker, conjugated to a cargo and having the formula (C):

[0594] [ka] or its protonated form. In addition, in the formula, R1, R2, and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group; R4 is H or an amino acid side chain; EP is an exocyclic peptide as defined herein; Cargo is a moiety 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 of 2 to 20.

[0595] R1, R2, R3, R4, EP, cargo, m, n, x', y, q, and z' are as defined herein.

[0596] The EEV can be conjugated to a cargo, the EEV-conjugate having the structure of formula (Ca) or (Cb):

[0597] [ka]

[0598] [ka] or a protonated form thereof, wherein EP, m, and z are as defined above in formula (C).

[0599] The EEV can be conjugated to a cargo, the EEV-conjugate having the formula (Cc):

[0600] [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.

[0601] The EEV may be conjugated to an oligonucleotide cargo, the EEV-oligonucleotide conjugate having the structure of formula (C-1), (C-2), (C-3), or (C-4):

[0602] [ka]

[0603] [ka]

[0604] [ka] may include:

[0605] The EEV can be conjugated to an oligonucleotide cargo, the EEV conjugate having the structure:

[0606] [ka] is selected from.

[0607] 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. The control sequence does not contain certain substituted amino acid residues (such as, but not limited to, arginine, phenylalanine, and / or glycine) in the modified sequence, but is otherwise identical.

[0608] As used herein, cytoplasmic delivery efficiency refers to the ability of a cCPP to cross the cell membrane and enter the cytoplasm of a cell. The cytoplasmic delivery efficiency of a cCPP does not necessarily depend on the receptor or cell type. Cytoplasmic delivery efficiency may refer to absolute cytoplasmic delivery efficiency or relative cytoplasmic delivery efficiency.

[0609] Absolute cytoplasmic delivery efficiency is the ratio of the cytosolic concentration of cCPP (or cCPP-cargo conjugate) to the concentration of cCPP (or cCPP-cargo conjugate) in the growth medium. Relative cytoplasmic delivery efficiency refers to the concentration of cCPP in the cytosol compared to the concentration of a control cCPP in the cytosol. Quantification can be achieved by fluorescently labeling the cCPP (e.g., with FITC dye) and measuring fluorescence intensity using techniques well known in the art.

[0610] The 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 the relative cytoplasmic delivery efficiency, the cell type may be incubated in the presence of the cCPP for a specific period (e.g., 30 minutes, 1 hour, 2 hours, etc.), and then the amount of cCPP internalized by the cells 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, and the amount of the control cCPP internalized by the cells is quantified.

[0611] Relative cytoplasmic delivery efficiency is calculated by the IC of cCPPs with modified sequences for intracellular targets 50 Measure the IC of cCPP with modified sequences 50 can be determined by comparing with a control sequence (as described herein).

[0612] 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%, 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 The relative cytoplasmic delivery efficiency of the cCPP may be in the range of 310%, about 320%, 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 cyclic (FfΦRrRrQ).

[0613] 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).

[0614] The cCPPs of the present disclosure may increase cytoplasmic delivery efficiency by 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 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, about 20 fold, about 21 fold, about 22 fold, about 23 fold, about 24 fold, about 25 fold, about 26 fold, about 27 fold, about 28 fold, about 29 fold, about 30 fold, about 31 fold, about 32 fold, about 33 fold, about 34 fold, about 35 fold, about 36 fold, about 37 fold, about 38 fold, about 39 fold, about 40 fold, about 41 fold, about 42 fold, about 43 fold, about 44 fold, about The improvement may be about 0.0 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).

[0615] 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 appreciated by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0616] Modification of the compounds described herein includes the addition, removal, or movement of various components as described for each compound. Similarly, if one or more chiral centers are present in the molecule, the chirality of the molecule can be changed. Furthermore, the synthesis of the compounds can include the 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.

[0617] Starting materials and reagents used in preparing the compounds and compositions of the present disclosure are available from Aldrich Chemical Corporation (Milwaukee, Wisconsin), Acros Organics (Morris Plains, New Jersey), Fisher Scientific (Pittsburgh, Pennsylvania), Sigma (St. Louis, Missouri), Pfizer (New York, New York), GlaxoSmithKline (Raleigh, North Carolina), Merck (Whitehouse Station, New Jersey), Johnson & Johnson (New Brunswick, New Jersey), Abe These compounds are available from commercial suppliers such as Pharma (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 are commercially available 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), or are prepared by methods known to those skilled in the art according to procedures described in references such asOther materials, such as the pharmaceutical carriers disclosed herein, can be obtained from commercial sources.

[0618] 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.

[0619] The compounds of the present disclosure can be prepared by solid-phase peptide synthesis in which the α-N-terminal amino acid is protected with an acid or base protecting group. Such protecting groups should have the properties of being stable to the conditions of peptide bond formation while being easily removable without disrupting the growing peptide chain or racemizing any of the chiral centers contained therein. Suitable protecting groups include 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 synthesizing 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-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl, 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.

[0620] 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 those that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions and insoluble in the media used. Solid supports for the synthesis of α-C-terminal carboxypeptides are 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 a 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-terminus of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in approximately 3-fold molar excess, and 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 solid-phase synthesis, the polypeptide is removed from the resin and deprotected, either sequentially or in a single operation. Removal and deprotection of the polypeptide can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent containing 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, for example, transesterification with methanol followed by aminolysis or direct transamidation. The protected peptide can be purified at this point or carried directly to the next step. Removal of 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 on weakly basic resins (acetate form), hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., Amberlite XAD), silica gel adsorption chromatography, ion exchange chromatography on carboxymethylcellulose, partition chromatography on e.g., Sephadex G-25, LH-20, or countercurrent distribution, high-performance liquid chromatography (HPLC), particularly reverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packings.

[0621] Methods for synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited by the method for synthesizing the AC. In embodiments, provided herein are compounds having reactive phosphorus groups useful for forming internucleoside linkages, including, for example, phosphodiester and phosphorothioate internucleoside linkages. Methods for preparing and / or purifying precursors or antisense compounds are not limited to 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.

[0622] Oligomerization of modified and unmodified nucleosides can be routinely carried out by following literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and / or for 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).

[0623] The antisense compounds provided herein can be easily and routinely produced through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art can additionally or alternatively be used. 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.

[0624] Methods for purifying and analyzing oligonucleotides are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. Such synthesis and analysis methods can be performed in multi-well plates. The method of the present invention is not limited by the method of oligomer purification.

[0625] How to use Also provided herein are methods of using the compounds or compositions described herein. Also provided herein are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of any of the compounds or compositions described herein. The compounds of the compositions can be used to treat any disease or condition amenable to treatment with the therapeutic moieties disclosed herein.

[0626] Also provided herein are methods for treating cancer in a subject. The methods include administering to the subject an effective amount of one or more of the compounds or compositions described herein, or pharmaceutically acceptable salts thereof. The compounds and compositions described herein, or pharmaceutically acceptable salts thereof, are useful for treating cancer in humans, e.g., pediatric and geriatric populations, and in animals, e.g., veterinary applications. The methods of the present disclosure can optionally include identifying a patient who is or may be in need of cancer treatment. Examples of cancer types treatable by the compounds and compositions described herein include bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, and testicular cancer. Further examples include cancers and / or tumors of the anus, bile duct, bone, bone marrow, intestine (including colon and rectum), eye, gallbladder, kidney, mouth, larynx, esophagus, stomach, testicle, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, blood cells (including lymphocytes and other immune system cells). Further examples of cancers treatable by the compounds and compositions described herein include carcinoma, Kaposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (such as acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous), and lymphoma (Hodgkin's and non-Hodgkin's), and multiple myeloma.

[0627] The methods for treating or preventing cancer described herein may further include treatment with one or more additional agents (e.g., anticancer agents or ionizing radiation). One or more additional agents and compounds and compositions or pharmaceutically acceptable salts thereof described herein can be administered in any order, including simultaneous administration and administration separated by a time interval of up to several days. The methods may also include more than one administration of one or more additional agents and / or compounds and compositions or pharmaceutically acceptable salts thereof described herein. Administration of one or more additional agents and compounds and compositions or pharmaceutically acceptable salts thereof described herein may be by the same route or different routes. When treating with one or more additional agents, the compounds and compositions or pharmaceutically acceptable salts thereof described herein can be combined into a pharmaceutical composition containing one or more additional agents.

[0628] For example, the compounds and compositions described herein, or pharmaceutically acceptable salts thereof, can be combined with additional anticancer agents, such as 13-cis-retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, accutane, actinomycin-D, adriamycin, adrsil, agrilin, Ala-Cort, aldesleukin, alemtuzumab, alitretinoin, alkaban-AQ, alkeran, all-trans retinoic acid, alpha interferon, alkaban-AQ ... Tretamine, amethopterin, amifostine, aminoglutethimide, anagrelide, anandrone, anastrozole, arabinosylcytosine, Aranesp, Aredia, aromasin, arsenic trioxide, asparaginase, ATRA, Avastin, BCG, BCNU, bevacizumab, bexarotene, bicalutamide, BiCNU, blenoxane, bleomycin, bortezomib, busulfan, busulfex, C225, leucovorin calcium, Campas, Camptosar, camptothecin-11, capecitabine, Carlac, carboplatin, Carmustine, Carmustine Wafer, Casodex, CCNU, CDDP, CeeNU, Cervidine, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine Liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, DaunoXome, Decadron, Delta-Cortef, Del Tazone, Denileukin diftitox, Depocyte, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposome, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol,Etopophos, etoposide, etoposide phosphate, Evulexin, Evista, Exemestane, Fairston, Faslodex, Femara, filgrastim, floxuridine, Fludara, fludarabine, Fluoroplex, fluorouracil, fluorouracil (cream), fluoxymesterone, flutamide, folinic acid, FUDR, fulvestrant, G-CSF, gefitinib, gemcitabine, gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, mechlorethamine, mechlorethamine hydrochloride, medralone, medrol, Megrez, megestrol, megestrol acetate, melphalan, mercaptopurine, mesna, Menex, methotrexate, methotrexate sodium, methylprednisolone, Mirocel, Retinol Trozole, Neosar, Neulasta, Numega, Nepgen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide Acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orpred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG-Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-Asparaginase, Phenylanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelon, Procarbazine, Procrit, Proleukin, Carmustine Implant with Prolifegroprolife 20, Purinethol, raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alpha-2a), Rubex, rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solumedrol, STI-571, streptozocin, tamoxifen, Targetretin, Taxol, Taxotere, Temodar, temozolomide, teniposide, TESPA, thalidomide, Thalomid, TheraCys, thioguanine, thioguanine Tabloid, thiophosphoamide,Thioplex, thiotepa, TICE, Toposar, topotecan, toremifene, trastuzumab, tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, Bepcid, Besanoid, Viadur, vinblastine, vinblastine sulfate, Vincasar Pfs, vincristine, vinorelbine, vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, zoledronic acid, Zometa, Gliadel Wafer, Gleevec, GM-CSF, goserelin, granulocyte colony-stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, hexamethylmelamine, HMM, Hycamtin, Hydrea, hydrocortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortone phosphate, hydroxyurea, ibritumomab, ibritumomab tiuxetan, idamycin, idarubicin, Ifex, IFN-α, ifosfamide, IL The pharmaceutical composition may include IL-2, IL-11, imatinib mesylate, imidazole carboxamide, interferon α, interferon α-2b (PEG conjugate), interleukin 2, interleukin-11, Intron A (interferon α-2b), leucovorin, leukeran, leukine, leuprolide, leulocristine, leustatin, liposomal Ara-C, Liquid Pred, lomustine, L-PAM, L-sarcolysin, methycortene, mitomycin, mitomycin-C, mitoxantrone, M-prednisole, MTC, MTX, mustagen, mustine, mutamycin, Myleran, Iressa, irinotecan, isotretinoin, hydrolase, lanacort, L-asparaginase, and LCR. Additional anticancer agents may also include biologics such as antibodies.

[0629] Many tumors and cancers have viral genomes present in tumor or cancer cells. For example, Epstein-Barr virus (EBV) is associated with several mammalian malignancies. The compounds disclosed herein can also be used alone or in combination with anticancer or antiviral agents, such as cancercyclovir, azidothymidine (AZT), or lamivudine (3TC), to treat patients infected with viruses capable of causing cell transformation and / or to treat patients with tumors or cancers associated with the presence of viral genomes in cells. The compounds disclosed herein can also be used in combination with virus-based treatments for neoplastic diseases.

[0630] Also described herein are methods for killing tumor cells in a subject. The methods include contacting tumor cells with an effective amount of a compound or composition described herein, and optionally irradiating the tumor cells with an effective amount of ionizing radiation. Also provided herein are methods for tumor radiotherapy. The methods include contacting tumor cells with an effective amount of a compound or composition described herein and irradiating the tumor with an effective amount of ionizing radiation. As used herein, the term "ionizing radiation" refers to radiation containing particles or photons that have sufficient energy to cause ionization or that can generate sufficient energy through nuclear interactions. An example of ionizing radiation is X-rays. An effective amount of ionizing radiation refers to a dose of ionizing radiation that, when administered in combination with a compound described herein, results in increased cell damage or death. Ionizing radiation can be administered according to methods known in the art, such as administering radiolabeled antibodies and radioisotopes.

[0631] The methods and compounds described herein are useful for both prophylactic and therapeutic treatments. As used herein, the term treatment or therapy includes prevention, delay of onset, attenuation, eradication, or delay of progression of signs or symptoms after onset, and prevention of recurrence. For prophylactic use, a therapeutically effective amount of the compounds and compositions described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject before onset (e.g., before overt signs of cancer), during early onset (e.g., at the onset of early signs and symptoms of cancer), or after cancer has been established. Prophylactic administration can occur from days to years before symptoms of infection appear. Prophylactic administration can be used, for example, in the chemopreventive treatment of subjects exhibiting precancerous lesions, subjects diagnosed with early-stage malignancies, and subgroups (e.g., familial, racial, and / or occupational) susceptible to particular cancers. Therapeutic treatment includes administering a therapeutically effective amount of the compounds and compositions described herein, or a pharmaceutically acceptable salt thereof, to a subject after cancer has been diagnosed.

[0632] In some examples of the methods of treating cancer or tumor in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can act as an inhibitor of Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or a combination thereof.

[0633] The subject matter of the present disclosure also relates to a method for treating a subject having a metabolic disorder or condition. An effective amount of one or more compounds or compositions disclosed herein can be administered to a subject having a metabolic disorder and in need of treatment. In some examples, the metabolic disorder can include type II diabetes. In some examples of the method for treating a metabolic disorder in a subject, the compound or composition administered to the subject can include a therapeutic moiety, which can include a targeting moiety that can act as an inhibitor of PTP1B. In one particular example of this method, the subject is obese, and the method can include treating the subject for obesity by administering a composition disclosed herein.

[0634] The subject matter of the present disclosure also relates to a method for treating a subject having an immune disorder or condition, wherein an effective amount of one or more compounds or compositions disclosed herein is administered to a subject having an immune disorder and in need of treatment. In some examples of the method for treating an immune disorder in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can act as an inhibitor against Pin1.

[0635] The presently disclosed subject matter also relates to methods for treating a subject having an inflammatory disorder or condition. An effective amount of one or more compounds or compositions disclosed herein can be administered to a subject having an inflammatory disorder and in need of treatment thereof.

[0636] The subject matter of the present disclosure also relates to a method for treating a subject with cystic fibrosis. An effective amount of one or more compounds or compositions disclosed herein can be administered to a subject with cystic fibrosis and in need of treatment. In some examples of the method for treating cystic fibrosis in a subject, the compound or composition administered to the subject can include a therapeutic moiety, which can include a targeting moiety that can act as an inhibitor against CAL PDZ.

[0637] The compounds disclosed herein can be used to detect or diagnose a disease or condition in a subject. For example, a cCPP can include a targeting moiety and / or a detectable moiety that can interact with a target, e.g., a tumor.

[0638] In some embodiments, the disease is associated with insulin resistance. In some embodiments, the disease is diabetes. In some embodiments, the target gene is a PTP.

[0639] In some embodiments, the disease is a CNS disorder. In some embodiments, the disease is Alzheimer's disease (AD) (Zhao et al. Gerontology 2019;65:323-331). In some embodiments, the target gene is the CD33 gene. The CD33 gene is located on human chromosome 19q13.33 and encodes a 67-kDa transmembrane glycoprotein. Human CD33 preferentially binds α-2,6-linked sialic acid. CD33 is expressed exclusively on immune cells. CD33 is an inhibitory receptor that recruits inhibitory proteins such as SHP phosphatases via its immunoreceptor tyrosine-based inhibition motif (ITIM). CD33 is also involved in the adhesion process in immune cells or malignant cells, inhibition of cytokine release by monocytes, growth and survival of immune cells through inhibition of proliferation, and induction of apoptosis. Polymorphisms in CD33 are involved in regulating AD susceptibility. rs3865444C is an allele associated with an increased risk of AD in European, Chinese, and North American populations due to increased CD33 expression. Skipping exon 2 of CD33 results in decreased expression of CD33 and increased expression of D2-CD33, a CD33 isoform lacking the ligand-binding domain. Expression of D2-CD33 is associated with a decreased risk of developing AD. In some embodiments, the ACs of the present disclosure are used to skip an exon of CD33 selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7a, and exon 7b. In some embodiments, the exon is exon 2. In some embodiments, an AC hybridizing to its target sequence in a target CD33 pre-mRNA induces skipping of one or more exons. In some embodiments, the AC induces expression of a re-spliced ​​target protein comprising an inactive fragment of CD33.

[0640] In some embodiments, the disease is cancer (Laszlo et al. Oncotarget. 2016 Jul 12;7(28):43281-43294.). In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, or melanoma. In some embodiments, the target gene is the CD33 gene. Each of the aforementioned cancers expresses CD33. In some embodiments, the AC of the present disclosure is used to skip exons of CD33. In some aspects, the exon is selected from exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7a, and exon 7b. In some embodiments, the target gene is Myc, STAT3, MDM4, ERRB4, BCL2L1, GLDC, PKM2, MCL1, MDM2, BRCA2, IL5R, FGFR1, MSTR1, USP5, or CD33.

[0641] In some embodiments, compounds comprising AC and CPP that target the CD33 gene are provided herein. Non-limiting examples of the aforementioned compounds are shown below, which can be further modified to conjugate with the exocyclic peptides (EPs) described herein. Antisense oligonucleotides are underlined.

[0642] ENTR-0036:

[0643] [ka]

[0644] ENTR-0081:

[0645] [ka]

[0646] ENTR-0087:

[0647] [ka]

[0648] ENTR-0085:

[0649] [ka]

[0650] ENTR-0179:

[0651] [ka]

[0652] In some embodiments, the disease is an inflammatory disease or an autoimmune disease. In some embodiments, the target gene is NLRP3 or CD6.

[0653] In some embodiments, the disease is osteogenesis imperfecta (Wang and Marini, J. Clin Invest., 1996, 97, 448-454).

[0654] In some embodiments, the disease is cystic fibrosis (Friedman et al., J. Biol. Chem., 1999, 274, 36193-36199).

[0655] In some embodiments, the disease is merosin-deficient congenital muscular dystrophy type 1A (MDC1A). MDC1A is an autosomal recessive neuromuscular disease characterized by neonatal onset of muscle weakness, hypotonia, hypomyelinating neuropathy, and mild brain abnormalities. Splice site mutations are estimated to affect approximately 40% of MDC1A patients. The causative mutation is located in the LAMA2 gene, which encodes the α2 chain of the laminin-211 (or merosin) heterotrimeric protein complex (LAMA2), which is expressed in the basement membrane of muscle and Schwann cells. In MDC1A, laminin-211 loses its proper interaction with receptors such as integrin α7β1 and dystroglycan, resulting in apoptosis and degeneration of muscle cells and Schwann cells, which leads to fibrosis and loss of muscle function. In some embodiments, the AC hybridizes to a LAMA2 target pre-mRNA. To date, the development of therapeutic strategies for MDC1A has primarily focused on preventing fibrosis and apoptosis. The degree of LAMA2 deficiency is highly correlated with clinical severity in patients and mouse models. Lack of functional Lama2 leads to the development of severe muscle atrophy and hindlimb paralysis in mice. Therefore, restoring LAMA2 expression holds great potential for the treatment of MDC1A. It has previously been demonstrated that muscle-specific overexpression of laminin-211 in merosin-deficient mice improved muscle pathology but not the associated paralysis, indicating that correction of peripheral neuropathy requires restoration of Lama2 beyond skeletal muscle. In some embodiments, AC restores proper splicing to the gene.

[0656] Some embodiments can use antisense compounds to alter the ratio of long to short forms of bcl-x pre-mRNA. See U.S. Patent Nos. 6,172,216, 6,214,986, and Taylor et al., Nat. Biotechnol. 1999, 17, 1097-1100, each of which is incorporated herein by reference. The number of genes and gene products involved in apoptosis is increasing. One of these is bcl-2, an intracellular membrane protein that has been shown to block or delay apoptosis. Overexpression of bcl-2 has been associated with hyperplasia, autoimmunity, and resistance to apoptosis, including that induced by chemotherapy (Fang et al., J. Immunol. 1994, 153, 4388-4398). A family of bcl-2-related genes has been described. All bcl-2 family members share two highly conserved domains, BH1 and BH2. These family members include, but are not limited to, A-1, mcl-1, bax, and bcl-x. bcl-x was isolated at low stringency using a bcl-2 cDNA probe due to its sequence homology with bcl-2. Bcl-x has been shown to function as a bcl-2-independent regulator of apoptosis (Boise et al., Cell, 1993, 74, 597-608). Two isoforms of bcl-x have been reported in humans. Bcl-xl (long) contains highly conserved BH1 and BH2 domains. When transfected into IL-3-dependent cell lines, bcl-xl inhibited apoptosis during growth factor withdrawal in a manner similar to bcl-2. In contrast, the bcl-x short isoform, bcl-xs, which is produced by alternative splicing and lacks the 63 amino acid region of exon 1 containing the BH1 and BH2 domains, antagonizes the anti-apoptotic effects of either bcl-2 or bcl-xl. As described by Boise et al., Cell, 1993, 74:597-608, the bcl-x transcript can be divided into regions described by those skilled in the art as follows:Nucleotides 1-134, 5'-untranslated region (5'-UTR); nucleotides 135-137, translation initiation codon (AUG); nucleotides 135-836, coding region, of which nucleotides 135-509 are the shorter exon 1 of the bcl-xs transcript and nucleotides 135-698 are the longer exon 1 of the bcl-xl transcript; nucleotides 699-836, exon 2; nucleotides 834-836, stop codon; and nucleotides 837-926, 3'-untranslated region (3'-UTR). Between exon 1 and exon 2 (between nucleotides 698 and 699), an intron is spliced ​​from the pre-mRNA when the mature bcl-xl(long) mRNA transcript is produced. Alternative splicing from positions 509 to 699 produces a bcl-xs (short) mRNA transcript that is 189 nucleotides shorter than the long transcript, encoding a protein product (bcl-xs) that is 63 amino acids shorter than bcl-xl. Thus, nucleotide position 698 is sometimes referred to in the art as the "5' splice site," and position 509 is sometimes referred to as the "cryptic 5' splice site," and nucleotide 699 is sometimes referred to as the "3' splice site." In some embodiments, the AC hybridizes to a sequence containing the cryptic 5' splice site of the bcl-x pre-mRNA, thereby inhibiting production of the short isoform and increasing the ratio of bcl-xl to bcl-xs isoforms.

[0657] In some embodiments, the AC promotes skipping of specific exons containing premature stop codons. See Wilton et al., Neuromuscul. Disord., 1999, 9, 330-338, incorporated herein by reference.

[0658] In some embodiments, the AC counteracts or corrects aberrant splicing in a target pre-mRNA. See U.S. Pat. No. 5,627,274 and WO 94 / 26887 (each of which is incorporated herein by reference, which disclose compositions and methods for counteracting aberrant splicing in pre-mRNA molecules containing mutations using antisense oligonucleotides that do not activate RNAse H).

[0659] In some embodiments, the disease is proximal spinal muscular atrophy (SMA). SMA is a genetic neurodegenerative disorder characterized by the loss of spinal motor neurons. SMA is an early-onset autosomal recessive disease and is currently the leading cause of death in infants. SMA is caused by the loss of both copies of survival of motor neuron 1 (SMN1), a protein that is part of a multiprotein complex thought to be involved in the biogenesis and recycling of snRNPs. A nearly identical gene, SMN2, resides in an overlapping region on chromosome 5q13. SMN1 and SMN2 potentially encode the same protein, but SMN2 contains a translationally silent mutation at position +6 of exon 7, which results in inefficient inclusion of exon 7 in the SMN2 transcript. Thus, the predominant form of SMN2 is a truncated form lacking exon 7, which is unstable and inactive (Cartegni and Drainer, Nat., 2002, 30, 377-384). In some embodiments, the AC targets intron 6, exon 7, or intron 7 of SMN2. In some embodiments, the AC modulates splicing of SMN2 pre-mRNA. In some embodiments, modulation of splicing results in increased exon 7 inclusion.

[0660] In some embodiments, the target gene is the beta-globin gene. See Sierakowska et al. 1996, incorporated herein by reference. In some embodiments, the target gene is the cystic fibrosis transmembrane conductance regulator gene. See Friedman et al. 1999, incorporated herein by reference. In some embodiments, the target gene is the BRCA1 gene. In some embodiments, the target gene is the eIF4E gene. In some embodiments, the target gene is a gene involved in the pathogenesis of Duchenne muscular dystrophy, spinal muscular atrophy, or Steinert myotonic dystrophy. In some embodiments, the target gene is the DMD gene. In some embodiments, the target gene is BRCA1. In some embodiments, the target gene is a gene encoding a muscle structural protein. In some embodiments, the target gene is a gene involved in neuromuscular disorders (NMD). In some embodiments, the target gene is a gene involved in cancer.

[0661] In some embodiments, the target gene is a gene that undergoes alternative splicing. In some embodiments, the compounds and methods of the present invention can be used to preferentially increase the proportion of a protein isoform by pre...

Claims

1. (a) Formula (I): 【Chemistry 1】 (In the formula, one of R 1 , R 2 , and R 3 is H; two of R 1 , R 2 , and R 3 are —CH 2 Ph; R 4 and R 6 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. or a protonated form thereof; (b) a compound having the following structure: 【Chemistry 2】 (In the formula, x′ is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * is the attachment point to AA SC ; M is a bonding group. and a linker comprising (c) an exocyclic peptide (EP) containing 2 to 10 amino acid residues and containing at least one lysine residue and / or at least one arginine residue; (d) a therapeutic moiety (TM).

2. The compound of claim 1 , wherein the therapeutic moiety is a protein, polypeptide, small molecule, or oligonucleotide other than an antisense compound.

3. The compound of claim 1 , wherein the therapeutic moiety is an antisense compound (AC).

4. M is, 【Chemistry 3】 2. The compound of claim 1 ,

5. The compound of claim 1, wherein M is -C(O).

6. The compound of claim 1 , wherein z is 11.

7. 2. The compound of claim 1, wherein x is 1.

8. The compound of claim 1, wherein the EP comprises 4 to 8 amino acid residues.

9. The compound of claim 1 , wherein the EP comprises 1, 2, 3, or 4 lysine residues.

10. 2. The compound of claim 1, wherein the exocyclic peptide comprises one of the following sequences: PKKKRKV, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRK, KRR, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG.

11. The compound of claim 1, wherein the exocyclic peptide has the structure: Ac-PKKKRKV-.

12. The compound of claim 1, wherein q is 1.

13. The compound of claim 1, wherein m is 1.

14. The compound of claim 1, wherein the cCPP is selected from FGFGRGRQ, GfFGrGrQ, FGFGRRRQ, and FGFRRRRQ.

15. The compound of claim 1 having a structure selected from the following: (a) Ac-PKKKRKV-miniPEG 2 -K(cyclo(GfFGrGrQ))-PEG 12 -OH; Ac-PKKKRKV-miniPEG 2 -K(cyclo[FGFKRKRQ])-PEG 12 -OH; Ac-PKKKRKV-miniPEG 2 -K(cyclo[FGFRGRGQ])-PEG 12 -OH; Ac-PKKKRKV-miniPEG 2 -K (cyclo[FGFGRGRGRQ])-PEG 12 -OH; Ac-PKKKRKV-miniPEG 2 -K(cyclo[FGFGRrRQ])-PEG 12 -OH; Ac-PKKKRKV-miniPEG 2 -K(cyclo[FGFGRRRQ])-PEG 12 -OH; or Ac-PKKKRKV-miniPEG 2 -K(cyclo[FGFRRRRQ])-PEG 12 -OH; or (b) Ac-KKKRKG-miniPEG 2 -K (cyclo[FGFGRGRQ])-PEG 12 -OH; Ac-KKKRK-miniPEG 2 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; Ac-KKRKK-PEG 4 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; Ac-KRKKK-PEG 4 -K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-KKKKR-PEG 4 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; Ac-RKKKKK-PEG 4 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; or Ac-KKKRK-PEG 4 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; or (c) Ac-PKKKRKV-PEG 2 -K(cyclo[FGFGRGRQ])-PEG 2 -K(N 3 )-NH 2 ; Ac-PKKKRKV-PEG 2 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; Ac-PKKKRKV-PEG 2 -K(cyclo[GfFGrGrQ])-PEG 2 -K(N 3 )-NH 2 ; or Ac-PKKKRKV-PEG 2 -K(cyclo[GfFGrGrQ])-PEG 12 -OH; or (d) Ac-PKKKRKV-PEG 2 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; Ac-PKKKRKV-PEG 2 -K(cyclo[GfFGrGrQ])-PEG 12 -OH; Ac-PKKKRKV-PEG 2 -K(cyclo[FGFGRRRQ])-PEG 12 -OH; or Ac-PKKKRKV-PEG 2 -K(cyclo[FGFRRRRQ])-PEG 12 -OH; or (e) Ac-PKKKRKV-miniPEG 2 -K(cyclo[FGFGRGRQ])-PEG 12 -OH; or Ac-PKKKRKV-miniPEG 2 -K(cyclo[GfFGrGrQ])-PEG 12 -OH.

16. (a) Formula (I): 【Chemistry 4】 (In the formula, R 1 , R 2 and R 3 are CH 2 Ph; R 4 and R 6 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. or a protonated form thereof; (b) a compound having the following structure: 【Chemistry 5】 (In the formula, x′ is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * is the attachment point to AA SC ; M is a bonding group. and a linker comprising (c) an exocyclic peptide (EP) containing 2 to 10 amino acid residues and containing at least one lysine residue and / or at least one arginine residue; (d) a therapeutic moiety (TM).

17. The compound of claim 16, wherein the cCPP is selected from FfFGRGRQ and FfFGrGrQ.

18. The compound of claim 16, having a structure selected from the following: Ac-PKKKRKV-miniPEG 2 -K (cyclo(FfFGRGRQ)-miniPEG 2 -K (N 3 ); Ac-PKKKRKV-PEG 2 -K(cyclo[FfFGRGRQ])-PEG 12 -OH; Ac-KKKK-miniPEG 2 -K(cyclo(FfFGrGrQ))-miniPEG 2 -K(N 3 )-NH 2 ; Ac-KKKK-miniPEG 2 -K(cyclo(FfFGrGrQ))-miniPEG 2 -K(N 3 )-NH 2 ; Ac-KBKBK-miniPEG 2 -K(cyclo(FfFGrGrQ))-miniPEG 2 -K(N 3 )-NH 2 ; Ac-PKKKRKV-miniPEG 2 -K(cyclo(FfFGrGrQ))-miniPEG 2 -K(N 3 )-NH 2 ; Ac-KGKK-miniPEG 2 -K(cyclo(FfFGrGrQ))-miniPEG 2 -K(N 3 )-NH 2 ; or Ac-KGKK-miniPEG 2 -K(cyclo(FfFGrGrQ))-miniPEG 2 -K(N 3 )-NH 2 .

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

20. A pharmaceutical composition comprising a compound described in claim 1 for treating a disease or disorder in a subject in need of such treatment, wherein the treatment comprises administering the composition to the subject.

21. The pharmaceutical composition of claim 20, wherein the administration comprises subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, or intrasternal administration.

22. The pharmaceutical composition of claim 20, wherein the administration comprises intrathecal administration.

23. 21. The pharmaceutical composition of claim 20, wherein the disease or disorder is a central nervous system disorder, a neuromuscular disorder, or a musculoskeletal disorder.

24. 21. The pharmaceutical composition of claim 20, wherein the disease is Duchenne muscular dystrophy.

25. 21. The pharmaceutical composition of claim 20, wherein the disease or disorder comprises a neuroinflammatory disease selected from Alzheimer's disease or Parkinson's disease.

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