CN117897176A - Compositions and methods for modulating tissue distribution of intracellular therapeutic agents - Google Patents

Abstract

Provided herein are compounds comprising a cyclic cell penetrating peptide, a therapeutic moiety, and a regulatory peptide, wherein 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 an intracellular therapeutic agent using the foregoing compounds.

Description

Compositions and methods for modulating tissue distribution of intracellular therapeutic agents

Cross Reference to Related Applications

The disclosure of each of U.S. provisional patent application number 63/186,664 filed on day 5 and 10 of 2021, U.S. provisional patent application number 63/214,085 filed on day 6 and 23 of 2021, U.S. provisional patent application number 63/239,671 filed on day 9 and 1 of 2021, U.S. provisional patent application number 63/290,960 filed on day 12 and 17 of 2021, U.S. provisional patent application number 63/298,565 filed on day 11 of 2022, U.S. provisional patent application number 63/268,577 filed on day 25 of 2022, and U.S. provisional patent application number 63/362,295 filed on day 3 and 31 of 2022 is hereby incorporated by reference in its entirety.

Background

Biological agents such as proteins, peptides and nucleic acids are promising approaches for the treatment of a variety of diseases and disorders. In particular, oligonucleotides such as antisense compounds are very widely used in therapy, as these compounds can be synthesized with any nucleotide sequence directed against virtually any target gene or genomic segment. However, the plasma membrane presents significant challenges in both drug discovery and therapy, particularly for therapeutic agents such as biological agents. For example, a major problem with the use of oligonucleotide-based biologics in therapy is their limited ability to gain access to 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 (e.g., by covalent attachment of cholesterol molecules). However, intracellular delivery efficiency is still low.

A potential strategy to disrupt membrane barriers and deliver therapeutic agents, such as biologies, into cells is to attach them to "Cell Penetrating Peptides (CPPs)". CPPs that enter the cell via endocytosis must be expelled from the endocytic vesicle in order to reach the cytosol. Unfortunately, endosomal membranes have proven to be an important barrier to the delivery of these CPPs to the cytoplasm; a generally negligible portion of the peptide escapes into the cell interior (see, e.g., el-Sayed, A et al AAPS j.,2009, volume 11: pages 13-22; varkouhi, A K et al, j. Controlled release,2011, volume 151: pages 220-228; apeebaum, J S et al chem. Biol.,2012, volume 19: pages 819-830). Cyclic CPP (cCPP) with improved properties have been described for intracellular delivery of cargo parts (U.S. patent publication Nos. 2017/0190743 and 2017/0355730).

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

Disclosure of Invention

Described herein are compositions and methods for modulating tissue distribution and/or retention of intracellular therapeutic agents. It has been found that when a compound comprising a Cell Penetrating Peptide (CPP) linked to a Therapeutic Moiety (TM) further comprises an Exocyclic Peptide (EP) as described herein, the compound has an altered tissue distribution and/or retention. EP is typically a peptide containing lysine. EP has been identified as a previously known "nuclear localization signal" (NLS) in the art, such as the nuclear localization sequence of the SV40 viral large T antigen, the smallest functional unit of which is the seven amino acid sequence PKKKRKV. Inclusion of EP in CPP-TM compounds can, for example, alter the expression level, activity, or function of TM 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 and modulates tissue distribution and/or retention of the compound in CNS tissue.

In an embodiment, there is provided a compound comprising the following moieties:

(a) Cell Penetrating Peptide (CPP);

(b) A Therapeutic Moiety (TM); and

(C) An Exocyclic Peptide (EP), wherein the tissue distribution or retention of the compound is modulated compared to a compound comprising CPP and TM but no EP.

In one embodiment, the EP is conjugated to the CPP. In one embodiment, the EP is conjugated to 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 not an Antisense Compound (AC).

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

In embodiments, the compound comprises AC that modulates splicing of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7a, and exon 7b of CD 33.

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.

In embodiments, pharmaceutical compositions comprising a compound described herein and a pharmaceutically acceptable carrier are provided.

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

The present disclosure also relates to a method of 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 embodiments, the compound is administered intrathecally to the subject and the compound modulates tissue distribution or retention of the therapeutic agent in a Central Nervous System (CNS) tissue. In embodiments, the compounds modulate tissue distribution or retention of the therapeutic agent in muscle tissue.

The present disclosure also relates to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a compound of the present disclosure. In embodiments, 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 spatially blocks mRNA. In embodiments, administration of the compound modulates splicing of the target pre-mRNA.

Drawings

FIG. 1 shows modified nucleotides used in the antisense oligonucleotides described herein.

FIGS. 2A-2D provide structures of morpholino subunit monomers for use in synthesizing phosphorodiamidate-linked morpholino oligomers. FIG. 2A provides the structure of adenine morpholino monomer. Fig. 2B provides the structure of the cytosine morpholino monomer. Fig. 2C provides the structure of guanine morpholino monomers. FIG. 2D provides the structure of thymine morpholino monomer.

Figures 3A-3D illustrate conjugation chemistry for attaching AC to a cyclic cell penetrating peptide. FIG. 3A shows amide bond formation between a peptide having a carboxylic acid group or having a TFP activating ester and a primary amine residue at the 5' end of AC. FIG. 3B shows conjugation of secondary or primary amine modified AC at 3' to peptide-TFP ester via amide linkage. Figure 3C shows conjugation of peptide-azide to 5' cyclooctyne modified AC via copper-free azide-alkyne cycloaddition. FIG. 3D illustrates another exemplary conjugation between a 3 'modified cyclooctyne AC or a 3' modified azide AC and a CPP containing a linker-azide or linker-alkyne/cyclooctyne moiety via copper-free azide-alkyne cycloaddition or copper-catalyzed azide-alkyne cycloaddition (click reaction), respectively.

FIG. 4 shows conjugation chemistry for joining AC and CPP with an additional linker pattern containing a polyethylene glycol (PEG) moiety.

Fig. 5A-5C show the structures of three conjugates used in the examples. Fig. 5A shows the structure of an oligonucleotide conjugate comprising a PMO oligonucleotide and a binding group. FIG. 5B shows the structure of a cell penetrating peptide-oligonucleotide conjugate comprising a PMO oligonucleotide, a binding group, a cyclic cell penetrating peptide, and a nuclear localization signal (PMO-NLS-EEV). FIG. 5C shows the structure of a conjugate comprising a binding group, a polyethylene glycol (PEG) linker, and a cyclic cell penetrating peptide-CPP 12-PEG4-dk (LSR).

Fig. 6 shows representative structures of cyclic peptide-linker conjugates of the present disclosure.

Fig. 7 shows another representative structure of a cyclic peptide-linker conjugate of the present disclosure.

FIG. 8 provides a general schematic of an antigen-degrading construct. The hash box indicates the optional linker sequence. 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 may be located at any suitable position in the construct (N-terminal, C-terminal, or internal to the construct as shown).

Figure 9 illustrates the direct effect of the degradation moiety on the degradation of the target antigen.

FIG. 10 illustrates the indirect effect of the degradation moiety on the degradation of the target antigen.

FIG. 11 shows conjugation of an exemplary CPP and an exemplary AC, as described in example 3.

Figures 12A-12D show the level of exon 23 correction in the muscle tissue of MDX mice after five days of treatment with the indicated compounds. Results for the diaphragm (fig. 12A), heart (fig. 12B), tibialis anterior (fig. 12C), and triceps (fig. 12D) are shown.

Figures 13A-13C show the level of dystrophin expression in muscle tissue of MDX mice after five days of treatment with the indicated compounds. Results for the diaphragm (fig. 13A), heart (fig. 13B), and tibialis anterior (fig. 13C) are shown.

FIGS. 14A-14B are schematic diagrams of synthetic compounds EEV-PMO-CD33-1 (FIG. 14A) and EEV-PMO-CD33-2 (FIG. 14B).

Fig. 15 shows the 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 cerebellum, cortex, hippocampus and olfactory bulb of rat brain.

FIGS. 16A-16C 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 spinal cord, DRG and CSF of rat brain.

Detailed Description

Compounds of formula (I)

Endosome escape carrier (EEV)

Provided herein are Endosomal Escape Vehicles (EEVs) that can be used to transport cargo across a cell membrane, e.g., deliver cargo to the cytosol or nucleus of a cell. The cargo may comprise a large molecule, such as a peptide or oligonucleotide, or a small molecule. EEV may comprise a Cell Penetrating Peptide (CPP), for example, a cyclic cell penetrating peptide (cCPP) conjugated to an Exocyclic Peptide (EP). EP is interchangeably referred to as a regulatory peptide (MP). The EP may comprise a sequence of Nuclear Localization Signals (NLS). The EP may be coupled to the cargo. EP's may be coupled to cCPP. The EP may be coupled to cargo and cCPP. The coupling between the EP, cargo, cCPP, or combinations thereof may be non-covalent or covalent. The EP may be attached to the N-terminus of cCPP by a peptide bond. The EP may be attached to the C-terminal end of cCPP by a peptide bond. EP may be attached to cCPP by a side chain of an amino acid in cCPP. EP may be attached to cCPP through the side chain of lysine, which may be conjugated to the side chain of glutamine in cCPP. The EP may be conjugated to the 5 'or 3' end of the oligonucleotide cargo. The EP may be coupled to a linker. The exocyclic peptide may be conjugated to the amino group of the linker. The EP may be coupled to the linker via the C-terminal ends of EP and cCPP via cCPP and/or a side chain on the EP. For example, an EP may comprise a terminal lysine, which may then be coupled to a glutamine-containing cCPP via an amide linkage. When EP contains a terminal lysine and the side chain of lysine is available for attachment cCPP, the C-terminus or N-terminus can be attached to a linker on the cargo.

Cyclic exopeptides

The Exocyclic Peptide (EP) may comprise 2 to 10 amino acid residues, for example 2,3, 4,5,6,7, 8, 9 or 10 amino acid residues, including all ranges and values therebetween. An EP may comprise 6 to 9 amino acid residues. An EP may comprise 4 to 8 amino acid residues.

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 generic species of natural amino acids because it has a structure similar to that of a natural amino acid, thereby mimicking the structure and reactivity of a natural amino acid. The unnatural amino acid can be a modified amino acid and/or amino acid analog that is not one of the 20 common naturally occurring amino acids or the rare natural amino acid selenocysteine or pyrrolysine. The unnatural amino acid can also be a D-isomer of the natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, 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 are listed in table 1 along with their abbreviations used herein. For example, the amino acid may be A, G, P, K, R, V, F, H, nal or citrulline.

The EP may comprise at least one positively charged amino acid residue, e.g. at least one lysine residue and/or at least one amino acid residue comprising a side chain comprising a guanidine group or a protonated form thereof. An EP may comprise 1 or 2 amino acid residues comprising a side chain comprising a guanidine group or a protonated form thereof. The amino acid residue comprising a guanidine-containing side chain may be an arginine residue. Protonated forms may refer to salts thereof throughout the disclosure.

The EP may comprise at least two, at least three or at least four or more lysine residues. An EP may comprise 2,3 or 4 lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group including, for example, trifluoroacetyl (-COCF) ₃ ) Allyloxycarbonyl (Alloc), 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl (Dde) or (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene-3) -methylbutyl (ivDde). The amino group on the side chain of each lysine residue may be substituted with a trifluoroacetyl group (-COCF) ₃ ) And (3) substitution. Protecting groups may be included to effect amide conjugation. The protecting group may be removed after conjugation of EP to cCPP.

An EP may comprise at least 2 amino acid residues with hydrophobic side chains. Amino acid residues having hydrophobic side chains may be selected from valine, proline, alanine, leucine, isoleucine and methionine. The amino acid residue having a hydrophobic side chain may be valine or proline.

The EP may comprise 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 comprise at least two, at least three or at least four or more lysine residues and/or arginine residues.

EP may comprise 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、KKKKR、KBKBK、RKKKKG、KRKKKG、KKRKKG、KKKKRG、RKKKKB、KRKKKB、KKRKKB、KKKKRB、KKKRKV、RRRRRR、HHHHHH、RHRHRH、HRHRHR、KRKRKR、RKRKRK、RBRBRB、KBKBKB、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV Or PKKKRKG, wherein B is β -alanine. The amino acids in EP may have D or L stereochemistry.

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

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

An EP may comprise an amino acid sequence identified in the art as a Nuclear Localization Sequence (NLS). An 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. EP may consist of NLS comprising the amino acid sequence PKKKRKV. The EP may comprise a polypeptide selected from NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK And RKCLQAGMNLEARKTKK amino acid sequence NLS. EP may be prepared from a composition selected from NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK amino acid sequence NLS composition

All exocyclic sequences may also contain N-terminal acetyl groups. Thus, for example, an EP may have the following structure: ac-PKKKRKV.

Cell Penetrating Peptide (CPP)

The Cell Penetrating Peptide (CPP) may comprise from 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). cCPP are capable of penetrating cell membranes. The Exocyclic Peptide (EP) may be conjugated to cCPP and the resulting construct may be referred to as an Endosomal Escape Vector (EEV). cCPP can direct cargo (e.g., a Therapeutic Moiety (TM), such as an oligonucleotide, peptide, or small molecule) to penetrate a cell membrane. cCPP can deliver cargo to the cytosol of a cell. cCPP can deliver cargo to the cell site where the target (e.g., pre-mRNA) is located. To conjugate cCPP to a cargo (e.g., peptide, oligonucleotide, or small molecule), at least one bond or lone pair of electrons on cCPP may be replaced.

The total number of amino acid residues in cCPP is in the range of 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. cCPP may comprise from 6 to 13 amino acid residues. cCPP disclosed herein may comprise from 6 to 10 amino acids. By way of example, cCPP comprising 6-10 amino acid residues may have a structure according to any one of formulas I-a to I-E:

Wherein the method comprises the steps of AA₁、AA₂、AA₃、AA₄、AA₅、AA₆、AA₇、AA₈、AA₉ And AA (alpha) ₁₀ Is an amino acid residue.

CCPP may comprise from 6 to 8 amino acids. cCPP may comprise 8 amino acids.

CCPP may be natural or unnatural amino acids. The term "unnatural amino acid" refers to an organic compound that is a generic species of natural amino acids because it has a structure similar to that of a natural amino acid, thereby mimicking the structure and reactivity of a natural amino acid. The unnatural amino acid can be a modified amino acid and/or amino acid analog that is not one of the 20 common naturally occurring amino acids or the rare natural amino acid selenocysteine or pyrrolysine. The unnatural amino acid can also be a D-isomer of the natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, 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 are listed in table 1 along with their abbreviations used herein.

TABLE 1 amino acid abbreviations

* Single letter abbreviations: when shown in uppercase letters herein, it means an L-amino acid form, and when shown in lowercase letters herein, it means a D-amino acid form.

CCPP may comprise from 4 to 20 amino acids, wherein: (i) At least one amino acid has a side chain comprising a guanidino group or a protonated form thereof; (ii) At least one amino acid having no side chain or having a chain comprising Or a side chain of a protonated form thereof; and (iii) at least two amino acids independently have side chains comprising aromatic or heteroaromatic groups.

At least two amino acids may have no side chains or have a chain comprising Or a side chain of a protonated form thereof. As used herein, when a side chain is not present, the amino acid has two hydrogen atoms on the carbon atom linking the amine and the carboxylic acid (e.g., -CH ₂-).

The amino acid without a side chain may be glycine or β -alanine.

CCPP may comprise 6 to 20 amino acid residues that form cCPP, wherein: (i) At least one amino acid may be glycine, beta-alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid has a guanidine group, Or a side chain of a protonated form thereof.

CCPP may comprise 6 to 20 amino acid residues that form cCPP, wherein: (i) At least two amino acids may independently be glycine, beta-alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid has a guanidine group, Or a side chain of a protonated form thereof.

CCPP may comprise 6 to 20 amino acid residues that form cCPP, wherein: (i) At least three amino acids may independently be glycine, beta-alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aromatic or heteroaromatic group; and (iii) at least one amino acid may have a guanidine group, Or a side chain of a protonated form thereof.

Glycine and related amino acid residues

CCPP may comprise (i) 1,2,3, 4, 5, or 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 2 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 4 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 5 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3,4, or 5 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 or 4 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof.

CCPP may comprise (i) 1,2, 3,4,5 or 6 glycine residues. cCPP may comprise (i) 2 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3,4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues. cCPP may comprise (i) 2 or 3 glycine residues. cCPP may comprise (i) 1 or 2 glycine residues.

CCPP may comprise (i) 3,4, 5, or 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 4 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 5 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3,4, or 5 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 or 4 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof.

CCPP may comprise at least three glycine residues. cCPP may comprise (i) 3,4, 5 or 6 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3,4 or 5 glycine residues. cCPP can comprise (i) 3 or 4 glycine residues

In embodiments, none of the glycine, β -alanine, or 4-aminobutyric acid residues in cCPP are contiguous. Two or three glycine, beta-alanine, or 4-aminobutyric acid residues may be contiguous. The two glycine, β -alanine or 4-aminobutyric acid residues may be contiguous.

In embodiments, none of the glycine residues cCPP are contiguous. Each glycine residue in cCPP may be separated by an amino acid residue that is not glycine. Two or three glycine residues may be contiguous. The two glycine residues may be contiguous.

Amino acid side chains with aromatic or heteroaromatic groups

CCPP may comprise (ii) 2,3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2,3 or 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group.

CCPP may comprise (ii) 2,3, 4,5 or 6 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2,3 or 4 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic group.

The aromatic group may be a 6 to 14 membered aryl group. Aryl groups may be phenyl, naphthyl or anthracenyl, each of which is optionally substituted. Aryl groups may be phenyl or naphthyl, each of which is optionally substituted. The heteroaromatic group may be a 6 to 14 membered heteroaryl group having 1,2 or 3 heteroatoms selected from N, O and S. Heteroaryl may be pyridinyl, quinolinyl or isoquinolinyl.

Amino acid residues having a side chain comprising an aromatic or heteroaromatic group may each independently be bis (Gao Naiji alanine), gao Naiji alanine, naphthylalanine, phenylglycine, bis (homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3- (3-benzothienyl) -alanine, 3- (2-quinolinyl) -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 acids having side chains comprising aromatic or heteroaromatic groups may each be independently selected from:

Wherein the H at the N-terminal and/or H at the C-terminal is replaced by a peptide bond.

The amino acid residues having a side chain comprising an aromatic or heteroaromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, gao Naiji alanine, bis (homophenylalanine), bis- (Gao Naiji alanine), 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-benzothienyl alanine, 4-phenylphenylalanine, 3, 4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5, 6-pentafluorophenylalanine, homophenylalanine, β -homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthryl) -alanine. The amino acid residues having a side chain comprising an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain comprising an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, homophenylalanine, gao Naiji alanine, bis (Gao Naiji alanine) or bis (Gao Naiji alanine), each of which is optionally substituted with one or more substituents. Amino acid residues having a side chain comprising an aromatic group may each independently be residues 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 comprising an aromatic group may be a residue of phenylalanine. The at least two amino acid residues having a side chain comprising an aromatic group may be residues of phenylalanine. Each amino acid residue having a side chain comprising an aromatic group may be a residue of phenylalanine.

In embodiments, none of the amino acids having side chains comprising aromatic or heteroaromatic groups are contiguous. The two amino acids having side chains comprising aromatic or heteroaromatic groups may be contiguous. Two contiguous amino acids may have opposite stereochemistry. Two contiguous amino acids may have the same stereochemistry. Three amino acids having side chains containing aromatic or heteroaromatic groups may be contiguous. The three contiguous amino acids may have the same stereochemistry. The three contiguous amino acids may have alternating stereochemistry.

The amino acid residue comprising an aromatic or heteroaromatic group may be an L-amino acid. The amino acid residue comprising an aromatic or heteroaromatic group may be a D-amino acid. The amino acid residue comprising an aromatic or heteroaromatic group may be a mixture of D-amino acids and L-amino acids.

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

While not 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 comprising aromatic or heteroaromatic groups) may improve the cytoplasmic delivery efficiency of cCPP relative to amino acids having lower hydrophobicity values. Each hydrophobic amino acid may independently have a hydrophobicity value that is greater than glycine. Each hydrophobic amino acid can independently be a hydrophobic amino acid having a hydrophobicity value greater than alanine. Each hydrophobic amino acid independently can have a hydrophobicity value greater than or equal to phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 2 lists the hydrophobicity values for various amino acids as reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S.A., 1984; vol. 81: pages 140-144), engleman et al (Ann. Rev. Of Biophys. Chem., 1986; 1986, vol. 15: pages 321-353), kyte and Doolittle (J. Mol. Biol. 1982; vol. 157: pages 105-132), hoop and Woods (Proc. Natl. Acad. Sci. U.S.A., 1981; vol. 6: pages 3824-3828), and Janin (Nature, 1979; vol. 277: pages 491-492), each of which is incorporated herein by reference in its entirety. Hydrophobicity can be measured using the hydrophobicity scale reported by Engleman et al.

TABLE 2 amino acid hydrophobicity

The size of the aromatic or heteroaromatic groups may be selected to improve the cytoplasmic delivery efficiency of cCPP. While not wishing to be bound by theory, it is believed that larger aromatic or heteroaromatic groups on the amino acid side chains may improve cytoplasmic delivery efficiency compared to otherwise identical sequences with smaller hydrophobic amino acids. The size of the hydrophobic amino acid may 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 of the side chain (SASA), or a combination thereof. The size of the hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, and the larger hydrophobic amino acid has side chains with a molecular weight of at least about 90g/mol, or at least about 130g/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. The hydrophobic amino acid may have a side chain with SASA greater than or equal to alanine, or greater than or equal to glycine. The larger hydrophobic amino acid may have a side chain with a SASA greater than alanine or greater than glycine. The hydrophobic amino acid may have an aromatic or heteroaromatic group with a SASA greater than or equal to about piperidine-2-carboxylic acid, greater than or equal to about tryptophan, greater than or equal to about phenylalanine, or greater than or equal to about naphthylalanine. First hydrophobic amino acid (AA _H1 ) May have a SASA of at least about At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about At least about/> At least about/> At least about/> At least about/> Or at least about/> Is a side chain of (c). Second hydrophobic Amino Acid (AA) _H2 ) May have a SASA of at least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about At least about/> At least about/> At least about/> Or at least about/> Is a side chain of (c). AA (AA) _H1 And AA (alpha) _H2 May have a side chain of at least about/> At least about/> At least about/> At least about/> At least about At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> Greater than about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> At least about/> Greater than about/> At least about/> At least about At least about/> At least about/> Or at least about/> is a combination of SASA. AA (AA) _H2 May be a hydrophobic amino acid residue having a side chain SASA less than or equal to AA _H1 Is a SASA of the hydrophobic side chain of (C). By way of example and not limitation, cCPP having a Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to cCPP which is otherwise identical to that having a Phe-Arg motif; cCPP having a Phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to otherwise identical cCPP having a Nal-Phe-Arg motif; and the Phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to otherwise identical cCPP with the Nal-Phe-Arg motif.

As used herein, "hydrophobic surface area" or "SASA" refers to the surface area of an amino acid side chain that is accessible to a solvent (reported in square angstroms; ). SASA can be calculated using the "rolling ball" algorithm developed by Shrake & Rupley (J Mol biol. Vol. 79, phase 2: pages 351-371), which is incorporated herein by reference in its entirety for all purposes. This algorithm uses solvent "spheres" of specific radius to probe the molecular surface. Typical values for spheres are/> Which approximates the radius of a water molecule.

The SASA values for some of the side chains are shown in table 3 below. The SASA values described herein are based on the theoretical values set forth in Table 3 below, as reported by Tien et al (PLOS ONE (11): e80635.https:// doi.org/10.1371/journ.fine.fine.0080635), which is incorporated herein by reference in its entirety for all purposes.

TABLE 3 amino acid SASA values

Amino acid residues having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof

Guanidine, as used herein, refers to the following structure:

As used herein, the protonated form of guanidine refers to the following structure:

Guanidine-displacing groups refer to functional groups on the side chains of amino acids that will be positively charged at physiological pH or above, or that reproduce guanidine the hydrogen bonding of the groups gives and accepts activity.

The guanidine replacement group facilitates cell permeation and delivery of therapeutic agents while reducing toxicity associated with the guanidine group or protonated form thereof. cCPP may comprise at least one compound having a guanidine or guanidine-containing group Amino acids in the side chains of the replacement groups. cCPP can comprise at least two compositions having a composition comprising guanidine or guanidine/> Amino acids in the side chains of the replacement groups. cCPP can comprise at least three compositions having a composition comprising guanidine or guanidine/> Amino acids substituted in side chains of groups

Guanidine or guanidine The group may be guanidine or guanidine/> Is an isostere of (2). Guanidine or guanidine/> The replacement group may be less basic than guanidine.

As used herein, guanidine replacement group refers to Or a protonated form thereof.

The present disclosure relates to cCPP comprising 4 to 20 amino acid residues, wherein: (i) At least one amino acid has a side chain comprising a guanidino group or a protonated form thereof; (ii) At least one amino acid residue having no side chain or having a chain comprising or a side chain of a protonated form thereof; and (iii) at least two amino acid residues independently have a side chain comprising an aromatic or heteroaromatic group.

At least two amino acid residues may have no side chains or have a chain comprising Or a side chain of a protonated form thereof. As used herein, when a side chain is absent, the amino acid residue has two hydrogen atoms on the carbon atom linking the amine and carboxylic acid (e.g., -CH ₂-).

CCPP may comprise at least one amino acid having a side chain comprising one of the following moieties: Or a protonated form thereof.

CCPP may comprise at least two amino acids, each independently having one of the following moieties: Or a protonated form thereof. At least two amino acids may have side chains comprising the same moiety selected from the group consisting of: or a protonated form thereof. At least one amino acid may have a nucleotide sequence comprising/> or a side chain of a protonated form thereof. At least two amino acids may have a nucleotide sequence comprising/> Or a side chain of a protonated form thereof. One, two, three or four amino acids may have a sequence comprising Or a side chain of a protonated form thereof. One amino acid may have a nucleotide sequence comprising/> Or a side chain of a protonated form thereof. Two amino acids may have a nucleotide sequence comprising/> Or a side chain of a protonated form thereof. Or a protonated form thereof may be attached to the end of the amino acid side chain. /(I) may be attached to the end of the amino acid side chain.

CCPP may comprise (iii) 2,3,4,5 or 6 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 2,3,4, or 5 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group, or a protonated form thereof. cCPP may comprise (iii) 2,3 or 4 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidino group or a protonated form thereof. cCPP may comprise (iii) two amino acid residues having a side chain comprising a guanidino group or a protonated form thereof. cCPP may comprise (iii) three amino acid residues having a side chain comprising a guanidino group or a protonated form thereof.

The amino acid residues may independently have side chains comprising non-contiguous guanidine groups, guanidine replacement groups, or protonated forms thereof. The two amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, which may be contiguous. The three amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, which may be contiguous. The four amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, which may be contiguous. Contiguous amino acid residues may have the same stereochemistry. Contiguous amino acids may have alternating stereochemistry.

The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be an L-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be a D-amino acid. The amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be an L-amino acid or a mixture of D-amino acids.

Each amino acid residue having a side chain comprising a guanidino group or a protonated form thereof may independently be an arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidino butyric acid, or residue of a protonated form thereof. Each amino acid residue having a side chain comprising a guanidine group or protonated form thereof can independently be an arginine residue or protonated form thereof.

Each amino acid having a side chain comprising a guanidine replacement group or protonated form thereof can independently be Or a protonated form thereof.

Without being bound by theory, it is hypothesized that the guanidine replacement group has a reduced basicity relative to arginine, and in some cases is uncharged at physiological pH (e.g., -N (H) C (O)), and is capable of sustaining bidentate hydrogen bond interactions with phospholipids on the plasma membrane, which is believed to promote efficient membrane binding and subsequent internalization. Removal of the positive charge is also believed to reduce cCPP's toxicity.

Those skilled in the art will appreciate that the N-terminus and/or C-terminus of the above-described unnatural aromatic hydrophobic amino acids form amide linkages upon incorporation into the peptides disclosed herein.

CCPP may comprise 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, wherein 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. Although the term "first amino acid" generally refers to the N-terminal amino acid of a peptide sequence by convention, as used herein, a "first amino acid" is used to distinguish the referred amino acid from another amino acid (e.g., "second amino acid") in cCPP, such that the term "first amino acid" may refer to or may refer to an amino acid located at the N-terminal end of a peptide sequence.

CCPP may comprise: the N-terminus of the second glycine forms a peptide bond with an amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the second glycine forms a peptide bond with an amino acid having a side chain comprising a guanidino group or protonated form thereof.

CCPP may comprise a first amino acid having a side chain comprising a guanidino group or a protonated form thereof, and a second amino acid having a side chain comprising a guanidino group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidino group or a protonated form thereof, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain comprising a guanidino group or a protonated form thereof.

CCPP may comprise residues of asparagine, aspartic acid, glutamine, glutamic acid or homoglutamine. cCPP may comprise residues of asparagine. cCPP may comprise residues of glutamine.

CCPP may 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-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthracenyl) -alanine.

While not wishing to be bound by theory, it is believed that the chirality of the amino acids in cCPP can affect cytoplasmic uptake efficiency. cCPP may comprise at least one D amino acid. cCPP may comprise one to fifteen D amino acids. cCPP may comprise one to ten D amino acids. cCPP may comprise 1,2, 3 or 4D amino acids. cCPP may comprise 2,3,4,5,6, 7 or 8 contiguous amino acids with alternating D and L chiralities. cCPP may comprise three contiguous amino acids with the same chirality. cCPP may comprise two contiguous amino acids having the same chirality. At least two amino acids may have opposite chiralities. At least two amino acids having opposite chiralities may be adjacent to each other. At least three amino acids may have alternating stereochemistry with respect to each other. At least three amino acids having alternating chiralities relative to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry relative to each other. At least four amino acids having alternating chiralities relative to each other may be adjacent to each other. At least two amino acids may have the same chirality. At least two amino acids having the same chirality may be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chiralities. At least two amino acids having opposite chiralities may be adjacent to at least two amino acids having the same chirality. Thus, adjacent amino acids in cCPP may have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D. The amino acid residues forming cCPP may all be L-amino acids. The amino acid residues forming cCPP may all be D-amino acids.

At least two amino acids may have different chiralities. At least two amino acids having 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 of the amino acid residues forming cCPP may be achiral. cCPP may comprise a 3,4 or 5 amino acid motif, wherein two amino acids having the same chirality may be separated by an achiral amino acid. cCPP may comprise the following sequence: D-X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L, wherein X is an achiral amino acid. The achiral amino acid may be glycine.

An amino acid having a side chain comprising:

Or a protonated form thereof, may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. An amino acid having a side chain comprising: /(I) Or a protonated form thereof, may be adjacent to at least one amino acid having a side chain comprising guanidine, or a protonated form thereof. An amino acid having a side chain comprising guanidine or a protonated form thereof can be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Two amino acids having side chains comprising: /(I) or protonated forms thereof, may be adjacent to each other. Two amino acids having side chains comprising guanidine or a protonated form thereof are adjacent to each other. cCPP may comprise at least two contiguous amino acids having a side chain which may comprise an aromatic or heteroaromatic group, and at least two non-contiguous amino acids having a side chain comprising: or a protonated form thereof. cCPP can comprise at least two contiguous amino acids having a side chain comprising an aromatic or heteroaromatic group and at least two amino acids having a chain comprising/> or a side chain of a protonated form thereof. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, e.g., any of the sequences described in the preceding paragraphs.

At least two amino acids having side chains comprising:

Or a protonated form thereof, with at least two amino acids having side chains comprising a guanidino group or a protonated form thereof.

CCPP can have the structure of formula (a):

Or a protonated form thereof,

Wherein:

R ₁、R₂And R is ₃ Each independently is H or an aromatic or heteroaromatic side chain of an amino acid;

R ₁、R₂And R is ₃ at least one of which is an aromatic or heteroaromatic side chain of an amino acid;

R ₄、R₅、R₆、R₇ Independently H or an amino acid side chain;

R ₄、R₅、R₆、R₇ is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N-dimethylarginine, 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, lysine, N-methyllysine, N-dimethyllysine, N-ethyllysine, N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N-dimethyllysine, β -homoarginine, 3- (1-piperidinyl) alanine;

AA (AA) _SC Is an amino acid side chain; and

Q is 1, 2, 3 or 4;

Wherein the cyclic peptide of formula (A) is not FfΦ RrRrQ.

CCPP may have the structure of formula (I):

Or a protonated form thereof,

Wherein:

R ₁、R₂And R is ₃ Amino acid residues which may each independently be H or have a side chain comprising an aromatic group;

R ₁、R₂And R is ₃ at least one of which is an aromatic or heteroaromatic side chain of an amino acid;

R ₄And R is ₆ Independently H or an amino acid side chain;

AA (AA) _SC Is an amino acid side chain;

q is 1, 2, 3 or 4; and

Each m is independently an integer of 0, 1, 2 or 3.

R ₁、R₂And R is ₃ May each independently be H, -alkylene-aryl or-alkylene-heteroaryl. R ₁、R₂And R is ₃ Can each independently be H, -C _1-3 Alkylene-aryl or-C _1-3 Alkylene-heteroaryl. R ₁、R₂And R is ₃ May each independently be H or-alkylene-aryl. R ₁、R₂And R is ₃ Can each independently be H or-C _1-3 An alkylene-aryl group. C _1-3 The alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₁、R₂And R is ₃ Can each independently be H, -C _1-3 alkylene-Ph or-C _1-3 Alkylene-naphthalenyl. R ₁、R₂And R is ₃ Can each independently be H, -CH ₂ ph or-CH ₂ a naphthyl group. R ₁、R₂And R is ₃ Can each independently be H or-CH ₂Ph.

R ₁、R₂And R is ₃ Can each independently be a 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-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthracenyl) -alanine.

R ₁ May be the side chain of tyrosine. R ₁ May be the side chain of phenylalanine. R ₁ May be the side chain of 1-naphthylalanine. R ₁ May be the side chain of 2-naphthylalanine. R ₁ May be the side chain of tryptophan. R ₁ May be the side chain of 3-benzothienyl alanine. R ₁ May be the side chain of 4-phenylphenylalanine. R ₁ May be the side chain of 3, 4-difluorophenylalanine. R ₁ may be the side chain of 4-trifluoromethylphenylalanine. R ₁ May be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R ₁ May be the side chain of homophenylalanine. R ₁ May be the side chain of beta-homophenylalanine. R ₁ May be the side chain of 4-tert-butyl-phenylalanine. R ₁ May be the side chain of 4-pyridylalanine. R ₁ Can be the side chain of 3-pyridylalanine. R ₁ May be the side chain of 4-methylphenylalanine. R ₁ May be the side chain of 4-fluorophenylalanine. R ₁ May be the side chain of 4-phenylalanine. R ₁ May be the side chain of 3- (9-anthryl) -alanine.

R ₂ May be the side chain of tyrosine. R ₂ May be the side chain of phenylalanine. R ₂ May be the side chain of 1-naphthylalanine. R ₁ May be the side chain of 2-naphthylalanine. R ₂ May be the side chain of tryptophan. R ₂ May be the side chain of 3-benzothienyl alanine. R ₂ May be the side chain of 4-phenylphenylalanine. R ₂ May be the side chain of 3, 4-difluorophenylalanine. R ₂ may be the side chain of 4-trifluoromethylphenylalanine. R ₂ May be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R ₂ May be the side chain of homophenylalanine. R ₂ May be the side chain of beta-homophenylalanine. R ₂ May be the side chain of 4-tert-butyl-phenylalanine. R ₂ May be the side chain of 4-pyridylalanine. R ₂ Can be the side chain of 3-pyridylalanine. R ₂ May be the side chain of 4-methylphenylalanine. R ₂ May be the side chain of 4-fluorophenylalanine. R ₂ May be the side chain of 4-phenylalanine. R ₂ May be the side chain of 3- (9-anthryl) -alanine.

R ₃ May be the side chain of tyrosine. R ₃ May be the side chain of phenylalanine. R ₃ May be the side chain of 1-naphthylalanine. R ₃ May be the side chain of 2-naphthylalanine. R ₃ May be the side chain of tryptophan. R ₃ May be the side chain of 3-benzothienyl alanine. R ₃ May be the side chain of 4-phenylphenylalanine. R ₃ May be the side chain of 3, 4-difluorophenylalanine. R ₃ may be the side chain of 4-trifluoromethylphenylalanine. R ₃ May be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R ₃ May be the side chain of homophenylalanine. R ₃ May be the side chain of beta-homophenylalanine. R ₃ May be the side chain of 4-tert-butyl-phenylalanine. R ₃ May be the side chain of 4-pyridylalanine. R ₃ Can be the side chain of 3-pyridylalanine. R ₃ May be the side chain of 4-methylphenylalanine. R ₃ May be the side chain of 4-fluorophenylalanine. R ₃ May be the side chain of 4-phenylalanine. R ₃ May be the side chain of 3- (9-anthryl) -alanine.

R ₄ May be H, -alkylene-aryl, -alkylene-heteroaryl. R ₄ Can be H, -C _1-3 Alkylene-aryl or-C _1-3 Alkylene-heteroaryl. R ₄ May be H or-alkylene-aryl. R ₄ can be H or-C _1-3 An alkylene-aryl group. C _1-3 The alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₄ Can be H, -C _1-3 alkylene-Ph or-C _1-3 Alkylene-naphthalenyl. R ₄ May be H or the side chain of an amino acid in Table 1 or Table 3. R ₄ May be H or an amino acid residue having a side chain comprising an aromatic group. R ₄ Can be H, -CH ₂ ph or-CH ₂ a naphthyl group. R ₄ Can be H or-CH ₂Ph.

R ₅ May be H, -alkylene-aryl, -alkylene-heteroaryl. R ₅ Can be H, -C _1-3 Alkylene-aryl or-C _1-3 Alkylene-heteroaryl. R ₅ May be H or-alkylene-aryl. R ₅ can be H or-C _1-3 An alkylene-aryl group. C _1-3 The alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₅ Can be H, -C _1-3 alkylene-Ph or-C _1-3 Alkylene-naphthalenyl. R ₅ May be H or the side chain of an amino acid in Table 1 or Table 3. R ₄ May be H or an amino acid residue having a side chain comprising an aromatic group. R ₅ Can be H, -CH ₂ ph or-CH ₂ a naphthyl group. R ₄ Can be H or-CH ₂Ph.

R ₆ May be H, -alkylene-aryl, -alkylene-heteroaryl. R ₆ Can be H, -C _1-3 Alkylene-aryl or-C _1-3 Alkylene-heteroaryl. R ₆ May be H or-alkylene-aryl. R ₆ can be H or-C _1-3 An alkylene-aryl group. C _1-3 The alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₆ Can be H, -C _1-3 alkylene-Ph or-C _1-3 Alkylene-naphthalenyl. R ₆ May be H or the side chain of an amino acid in Table 1 or Table 3. R ₆ May be H or an amino acid residue having a side chain comprising an aromatic group. R ₆ Can be H, -CH ₂ ph or-CH ₂ a naphthyl group. R ₆ Can be H or-CH ₂Ph.

R ₇ May be H, -alkylene-aryl, -alkylene-heteroaryl. R ₇ Can be H, -C _1-3 Alkylene-aryl or-C _1-3 Alkylene-heteroaryl. R ₇ May be H or-alkylene-aryl. R ₇ can be H or-C _1-3 An alkylene-aryl group. C _1-3 The alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₇ Can be H, -C _1-3 alkylene-Ph or-C _1-3 Alkylene-naphthalenyl. R ₇ May be H or the side chain of an amino acid in Table 1 or Table 3. R ₇ May be H or an amino acid residue having a side chain comprising an aromatic group. R ₇ Can be H, -CH ₂ ph or-CH ₂ a naphthyl group. R ₇ Can be H or-CH ₂Ph.

R ₁、R₂、R₃、R₄、R₅、R₆And R is ₇ One, two or three of them may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆And R is ₇ One of them may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆And R is ₇ Two of which may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆And R is ₇ Three of (B) may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆And R is ₇ At least one of them may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆And R is ₇ Not more than four of them may be-CH ₂Ph.

R ₁、R₂、R₃And R is ₄ One, two or three of them are-CH ₂Ph.R₁、R₂、R₃And R is ₄ One of them is-CH ₂Ph.R₁、R₂、R₃And R is ₄ Two of them are-CH ₂Ph.R₁、R₂、R₃And R is ₄ Three of (B) are-CH ₂Ph.R₁、R₂、R₃And R is ₄ At least one of them is-CH ₂Ph.

R ₁、R₂、R₃、R₄、R₅、R₆And R is ₇ One, two or three of them may be H. R ₁、R₂、R₃、R₄、R₅、R₆And R is ₇ One of which may be H. R ₁、R₂、R₃、R₄、R₅、R₆And R is ₇ Both of which are H. R ₁、R₂、R₃、R₅、R₆And R is ₇ Three of which may be H. R ₁、R₂、R₃、R₄、R₅、R₆And R is ₇ At least one of which may be H. R ₁、R₂、R₃、R₄、R₅、R₆And R is ₇ Not more than three of them may be-CH ₂Ph.

R ₁、R₂、R₃And R is ₄ One, two or three of them are H. R ₁、R₂、R₃And R is ₄ One of them is H. R ₁、R₂、R₃And R is ₄ Both of which are H. R ₁、R₂、R₃And R is ₄ Three of which are H. R ₁、R₂、R₃And R is ₄ At least one of which is H.

R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of 3-guanidino-2-aminopropionic acid. R ₄、R₅、R₆And R is ₇ At least one of them may be a side chain of 4-guanidino-2-aminobutyric acid. R ₄、R₅、R₆And R is ₇ at least one of which may be a side chain of arginine. R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of homoarginine. R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of N-methyl arginine. R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of N, N-dimethylarginine. R ₄、R₅、R₆And R is ₇ at least one of which may be a side chain of 2, 3-diaminopropionic acid. R ₄、R₅、R₆And R is ₇ At least one of them may be a side chain of 2, 4-diaminobutyric acid or lysine. R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of N-methyllysine. R ₄、R₅、R₆And R is ₇ At least one of them may be a side chain of N, N-dimethyllysine. R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of N-ethyl lysine. R ₄、R₅、R₆And R is ₇ at least one of them may be N, N, N-trimethyllysine, side chains of 4-guanidinophenylalanine. R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of citrulline. R ₄、R₅、R₆And R is ₇ At least one of them may be a side chain of N, N-dimethyl lysine, beta-homoarginine. R ₄、R₅、R₆And R is ₇ At least one of which may be a side chain of 3- (1-piperidinyl) alanine.

R ₄、R₅、R₆And R is ₇ at least two of which may be side chains of 3-guanidino-2-aminopropionic acid. R ₄、R₅、R₆And R is ₇ At least two of (a) may be side chains of 4-guanidino-2-aminobutyric acid. R ₄、R₅、R₆And R is ₇ At least two of which may be side chains of arginine. R ₄、R₅、R₆And R is ₇ at least two of which may be homoarginine side chains. R ₄、R₅、R₆And R is ₇ At least two of which may be side chains of N-methyl arginine. R ₄、R₅、R₆And R is ₇ At least two of which may be side chains of N, N-dimethylarginine. R ₄、R₅、R₆And R is ₇ At least two of which may be side chains of 2, 3-diaminopropionic acid. R ₄、R₅、R₆And R is ₇ At least two of them may be side chains of 2, 4-diaminobutyric acid or lysine. R ₄、R₅、R₆And R is ₇ At least two of which may be side chains of N-methyl lysine. R ₄、R₅、R₆And R is ₇ at least two of which may be side chains of N, N-dimethyllysine. R ₄、R₅、R₆And R is ₇ At least two of which may be side chains of N-ethyl lysine. R ₄、R₅、R₆And R is ₇ at least two of (a) may be the side chain of N, N, N-trimethyllysine, 4-guanidinophenylalanine. R ₄、R₅、R₆And R is ₇ at least two of which may be side chains of citrulline. R ₄、R₅、R₆And R is ₇ At least two of (a) may be side chains of N, N-dimethyl lysine, beta-homoarginine. R ₄、R₅、R₆And R is ₇ At least two of which may be side chains of 3- (1-piperidinyl) alanine.

R ₄、R₅、R₆And R is ₇ At least three of (2) may be side chains of 3-guanidino-2-aminopropionic acid. R ₄、R₅、R₆And R is ₇ At least three of (2) amino butyric acid may be a side chain of 4-guanidino-2-aminobutyric acid. R ₄、R₅、R₆And R is ₇ At least three of (a) may be side chains of arginine. R ₄、R₅、R₆And R is ₇ At least three of (a) may be the side chain of homoarginine. R ₄、R₅、R₆And R is ₇ at least three of (a) may be side chains of N-methylarginine. R ₄、R₅、R₆And R is ₇ at least three of (a) may be side chains of N, N-dimethylarginine. R ₄、R₅、R₆And R is ₇ at least three of (2) diaminopropionic acid may be a side chain of 2, 3-diaminopropionic acid. R ₄、R₅、R₆And R is ₇ at least three of (a) may be side chains of 2, 4-diaminobutyric acid or lysine. R ₄、R₅、R₆And R is ₇ At least three of (2) may be side chains of N-methyllysine. R ₄、R₅、R₆And R is ₇ At least three of (2) may be side chains of N, N-dimethyllysine. R ₄、R₅、R₆And R is ₇ At least three of (a) may be side chains of N-ethyl lysine. R ₄、R₅、R₆And R is ₇ At least three of (a) may be N, N, N-trimethyllysine, side chains of 4-guanidinophenylalanine. R ₄、R₅、R₆And R is ₇ At least three of (a) may be side chains of citrulline. R ₄、R₅、R₆And R is ₇ at least three of (a) may be side chains of N, N-dimethyl lysine and beta-homoarginine. R ₄、R₅、R₆And R is ₇ At least three of (2) may be side chains of 3- (1-piperidinyl) alanine.

AA (AA) _SC May be the side chain of asparagine, glutamine or a homoglutamine residue. AA (AA) _SC May be a side chain of a glutamine residue. cCPP can also contain and AA _SC (e.g., residues of asparagine, glutamine or homoglutamine). Thus cCPP may also comprise linkers conjugated to asparagine, glutamine or homoglutamine residues. cCPP may also comprise a linker conjugated to the glutamine residue.

Q may be 1,2 or 3.q may be 1 or 2.q may be 1.q may be 2.q may be 3.q may be 4.

M may be 1-3.m may be 1 or 2.m may be 0 and m may be 1.m may be 2.m may be 3.

CCPP of formula (a) may have the structure of formula (I):

Or a protonated form thereof, wherein AA _SC、R₁、R₂、R₃、R₄、R₆ M and q are as defined herein

CCPP of formula (A) may have the structure of formula (I-a) or formula (I-b):

Or a protonated form thereof, wherein AA _SC、R₁、R₂、R₃、R₄ And m is as defined herein.

CCPP of formula (A) may have the structure of formula (I-1), (I-2), (I-3) or (I-4):

/>

Or a protonated form thereof, wherein AA _SC And m is as defined herein.

CCPP of formula (A) may have the structure of formula (I-5) or (I-6):

Or a protonated form thereof, wherein AA _SC As defined herein.

CCPP of formula (A) may have the structure of formula (I-1):

Or a protonated form thereof, wherein AA _SC and m is as defined herein. /(I)

CCPP of formula (a) may have the structure of formula (I-2):

Or a protonated form thereof, wherein AA _SC And m is as defined herein.

CCPP of formula (A) may have the structure of formula (I-3):

Or a protonated form thereof, wherein AA _SC And m is as defined herein.

CCPP of formula (A) may have the structure of formula (I-4):

Or a protonated form thereof, wherein AA _SC And m is as defined herein.

CCPP of formula (a) may have the structure of formula (I-5):

Or a protonated form thereof, wherein AA _SC And m is as defined herein.

CCPP of formula (A) may have the structure of formula (I-6):

Or a protonated form thereof, wherein AA _SC And m is as defined herein.

CCPP may comprise one of the following sequences: FGFGRGR; gfFGrGr, ff Φ GRGR; ffFGRGR; or FfΦ GrGr. cCPP may have one of the following sequences: FGF phi; gfFGrGrQ, ff Φ GRGRQ; ffFGRGRQ; or FfΦ GrGrQ.

The present disclosure also relates to cCPP having the structure of formula (II):

/>

Wherein:

AA (AA) _SC Is an amino acid side chain;

R ^1a、R^1bAnd R is ^1c Each independently is a 6 to 14 membered aryl or a 6 to 14 membered heteroaryl;

R ^2a、R^2b、R^2cAnd R is ^2d Independently an amino acid side chain;

R ^2a、R^2b、R^2cAnd R is ^2d at least one of which is Or a protonated form thereof;

R ^2a、R^2b、R^2cAnd R is ^2d At least one of which is guanidine or a protonated form thereof;

Each n "is independently an integer of 0,1, 2,3,4, or 5;

Each n' is independently an integer of 0, 1, 2 or 3; and

If n' is 0, R ^2a、R^2b、R^2bOr R is ^2d is not present.

R ^2a、R^2b、R^2cAnd R is ^2d at least two of which may be Or a protonated form thereof. R ^2a、R^2b、R^2cAnd R is ^2d two or three of them may be/> Or a protonated form thereof. R ^2a、R^2b、R^2cAnd R is ^2d One of them may be Or a protonated form thereof. R ^2a、R^2b、R^2cAnd R is ^2d At least one of (a) may be/> Or a protonated form thereof, and R ^2a、R^2b、R^2cAnd R is ^2d the remainder of (c) may be guanidine or a protonated form thereof. R ^2a、R^2b、R^2cAnd R is ^2d at least two of which may be Or a protonated form thereof, and R ^2a、R^2b、R^2cAnd R is ^2d The remainder of (c) may be guanidine or a protonated form thereof.

All R ^2a、R^2b、R^2cAnd R is ^2d May be /> Or a protonated form thereof. R ^2a、R^2b、R^2cAnd R is ^2d At least one of (a) may be/> Or a protonated form thereof, and R ^2a、R^2b、R^2cAnd R is ^2d the remainder of (c) may be guanidine or a protonated form thereof. R ^2a、R^2b、R^2cAnd R is ^2d At least two of (a) may be/> Or a protonated form thereof, and R ^2a、R^2b、R^2cAnd R is ^2d The remainder of (c) is guanidine or a protonated form thereof.

R ^2a、R^2b、R^2cAnd R is ^2d Independently of each other, can be a side chain of 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homolysine, serine, homoserine, threonine, allothreonine, histidine, 1-methylhistidine, 2-aminobutyric acid, aspartic acid, glutamic acid or homoglutamic acid.

AA (AA) _SC May be wherein t may be an integer from 0 to 5. AA (AA) _SC May be wherein t may be an integer from 0 to 5.t may be 1 to 5.t is 2 or 3.t may be 2.t may be 3.

R ^1a、R^1bAnd R is ^1c each independently can be a 6 to 14 membered aryl. R ^1a、R^1bAnd R is ^1c may each independently be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O or S. R ^1a、R^1bAnd R is ^1c Each independently selected from phenyl, naphthyl, anthracenyl, pyridinyl, quinolinyl, or isoquinolinyl. R ^1a、R^1bAnd R is ^1c Each independently selected from phenyl, naphthyl or anthracenyl. R ^1a、R^1bAnd R is ^1c each independently may be phenyl or naphthyl. R ^1a、R^1bAnd R is ^1c Each independently selected from pyridinyl, quinolinyl, or isoquinolinyl.

Each n' may independently be 1 or 2. Each n' may be 1. Each n' may be 2. At least one n' may be 0. At least one n' may be 1. At least one n' may be 2. At least one n' may be 3. At least one n' may be 4. At least one n' may be 5.

Each n "may independently be an integer from 1 to 3. Each n "may independently be 2 or 3. Each n "may be 2. Each n "may be 3. At least one n "may be 0. At least one n "may be 1. At least one n "may be 2. At least one n "may be 3.

Each n "may independently be 1 or 2, and each n' may independently be 2 or 3. Each n "may be 1 and each n' may independently be 2 or 3. Each n "may be 1 and each n' may be 2. Each n "is 1 and each n' is 3.

CCPP of formula (II) may have the structure of formula (II-1):

Wherein R is ^1a、R^1b、R^1c、R^2a、R^2b、R^2c、R^2d、AA_SC N' and n "are as defined herein.

CCPP of formula (II) may have the structure of formula (IIa):

Wherein R is ^1a、R^1b、R^1c、R^2a、R^2b、R^2c、R^2d、AA_SC- And n' is as defined herein.

CCPP of formula (II) may have the structure of formula (IIb):

Wherein R is ^2a、R^2b、AA_SC- And n' is as defined herein.

CCPP can have the structure of formula (IIb):

Or a protonated form thereof,

Wherein:

AA (AA) _SC And n' is as defined herein.

CCPP of formula (IIa) has one of the following structures:

/>

wherein AA is _SC And n is as defined herein.

CCPP of formula (IIa) has one of the following structures:

/>

wherein AA is _SC And n is as defined herein

CCPP of formula (IIa) has one of the following structures:

/>

wherein AA is _SC And n is as defined herein.

CCPP of formula (II) may have the following structure:

cCPP of formula (II) may have the following structure:

cCPP can have the structure of formula (III):

Wherein:

AA (AA) _SC Is an amino acid side chain;

R ^1a、R^1bAnd R is ^1c Each independently is a 6 to 14 membered aryl or a 6 to 14 membered heteroaryl;

R ^2aAnd R is ^2c Each independently is H, Or a protonated form thereof;

R ^2bAnd R is ^2d Each independently guanidine or protonated form thereof;

Each n "is independently an integer from 1 to 3;

Each n' is independently an integer from 1 to 5; and

Each p' is independently an integer from 0 to 5.

CCPP of formula (III) may have the structure of formula (III-1):

Wherein:

AA (AA) _SC、R^1a、R^1b、R^1c、R^2a、R^2c、R^2b、R^2d N ', n ", and p' are as defined herein.

CCPP of formula (III) may have the structure of formula (IIIa):

Wherein:

AA (AA) _SC、R^2a、R^2c、R^2b、R^2d N ', n ", and p' are as defined herein.

In the formulae (III), (III-1) and (IIIa), R ^aAnd R is ^c may be H. R ^aAnd R is ^c May be H and R ^bAnd R is ^d each independently guanidine or protonated form thereof. R ^a may be H. R ^b may be H. p' may be 0.R ^aAnd R is ^c May be H and each p' may be 0.

In the formulae (III), (III-1) and (IIIa), R ^aAnd R is ^c May be H, R ^bAnd R is ^d each independently may be guanidine or a protonated form thereof, n "may be 2 or 3, and each p' may be 0.

P' may be 0.p' may be 1.p' may be 2.p' may be 3.p' may be 4.p' may be 5.

CCPP can have the following structure:

cCPP of formula (a) may be selected from:

CPP sequence
	(FfΦRrRrQ)
(FfΦCit-r-Cit-rQ)
	(FfΦGrGrQ)
(FfFGRGRQ)
	(FGFGRGRQ)
(GfFGrGrQ)
	(FGFGRRRQ) or
(FGFRRRRQ)

CCPP of formula (a) may be selected from:

CPP sequence
	FΦRRRRQ
fΦRrRrQ
	FfΦRrRrQ
FfΦCit-r-Cit-rQ
	FfΦGrGrQ
FfΦRGRGQ
	FfFGRGRQ
FGFGRGRQ
	GfFGrGrQ
FGFGRRRQ or
	FGFRRRRQ

AA (AA) _SC Can be conjugated to a linker.

In embodiments cCPP is selected from:

Φ=l-naphthylalanine; Ω=l-norleucine

In embodiments, cCPP is not selected from:

CPP sequence
	FΦRRRQ
FΦRRRC
	FΦRRRU
RRRΦFQ
	RRRRΦF
FΦRRRR
	FφrRrRq
FφrRrRQ
	FΦRRRRQ
fΦRrRrQ
	RRFRΦRQ
FRRRRΦQ
	rRFRΦRQ
RRΦFRRQ
	CRRRRFWQ
FfΦRrRrQ
	FFΦRRRRQ
RFRFRΦRQ
	URRRRFWQ
CRRRRFWQ
	FΦRRRRQK
FΦRRRRQC
	fΦRrRrRQ
FΦRRRRRQ
	RRRRΦFDΩC
FΦRRR
	FWRRR
RRRΦF
	RRRWF

Φ=l-naphthylalanine; Ω=l-norleucine

Joint

CCPP of the present disclosure may be conjugated to a linker. The connector may connect cargo to cCPP. The linker may be attached to the side chain of the amino acid of cCPP and the cargo may be attached at the appropriate position on the linker.

The linker may be any suitable moiety that can conjugate cCPP to one or more additional moieties, such as a cyclic Exopeptide (EP) and/or cargo. Prior to conjugation with cCPP and one or more additional moieties, the linker has two or more functional groups, each of which is capable of independently forming a covalent bond with cCPP and one or more additional moieties. If the cargo is an oligonucleotide, the linker may be covalently bound to the 5 'end of the cargo or the 3' end of the cargo. The linker may be covalently bound to the 5' end of the cargo. The linker may be covalently bound to the 3' end of the cargo. If the cargo is a peptide, the linker may be covalently bound to the N-terminus or the C-terminus of the cargo. The linker may be covalently bound to the backbone of the oligonucleotide or peptide cargo. The linker may be any suitable moiety that conjugates cCPP described herein with cargo such as oligonucleotides, peptides, or small molecules.

The linker may comprise a hydrocarbon linker.

The linker may comprise a cleavage site. The cleavage site may be a disulfide or caspase cleavage site (e.g., val-Cit-PABC).

The joint may comprise: (i) One or more D or L amino acids, each of which is optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) One or more- (R) ^1-J-R² ) z "-subunit, wherein R ¹And R is ² Each independently selected from the group consisting of alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR ³、-NR³ C (O) -, S and O, wherein R ³ 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; (viii) - (R) ^1- J) z "-or- (J-R) ¹ ) z "-, wherein R ¹ Each independently at each occurrence is alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR ³、-NR³ 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; or (ix) the linker may comprise one or more of (i) to (x).

The linker may comprise one or more D or L amino acids and/or- (R) ^1-J-R² ) z "-, wherein R ¹And R is ² each independently of the other is alkylene, each J is independently C, NR ³、-NR³ C (O) -, S and O, wherein R ⁴ independently selected from H and alkyl, and z "is an integer from 1 to 50; or a combination thereof.

The linker may comprise- (OCH) ₂CH₂)_z' - (e.g. as spacer), wherein z' is an integer from 1 to 23, such as 2,3, 4,5,6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23."- (OCH) ₂CH₂ ) z' "may also be referred to as polyethylene glycol (PEG).

The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise- (OCH) ₂CH₂)_z' -and peptides, wherein z' is an integer from 1 to 23. The peptide may comprise 2 to 10 amino acids. The linker may also contain a Functional Group (FG) capable of undergoing a click chemistry reaction. FG may be azide or alkyne and forms triazole when the cargo is conjugated to the linker.

The linker may comprise (i) a beta alanine residue and a lysine residue; (ii) - (J-R) ¹ ) z'; or (iii) combinations thereof. Each R ¹ Can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR ³、-NR³ C (O) -, S or O, wherein R ³ is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z "may be an integer from 1 to 50. Each R ¹ May be alkylene and each J may be O.

The linker may comprise residues of (i) beta-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or a combination thereof; and (ii) - (R) ^1- J) z "-or- (J-R) ¹ ) z. Each R ¹ Can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR ³、-NR³ C (O) -, S or O, wherein R ³ is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z "may be an integer from 1 to 50. Each R ¹ May be alkylene and each J may be O. The linker may comprise glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or a combination thereof.

The linker may be a trivalent linker. The joint may have the following structure:

Wherein A is ₁、B₁And C ₁ may independently be a hydrocarbon linker (e.g., NRH- (CH) ₂)_n -COOH), PEG linker (e.g., NRH- (CH) ₂O)_n -COOH, wherein R is H, methyl or ethyl) or one or more amino acid residues, and Z is independently a protecting group. The linker may also incorporate cleavage sites, including disulfides [ NH ] ₂-(CH₂O)_n-S-S-(CH₂O)_n -COOH ] or caspase cleavage site (Val-Cit-PABC).

The hydrocarbon may be a glycine or beta-alanine residue.

The linker may be divalent and connect cCPP to the cargo. The linker may be bivalent and connects cCPP to the Exocyclic Peptide (EP).

The linker may be trivalent and connects cCPP to the cargo and EP.

The linker may be divalent or trivalent C ₁-C₅₀ Alkylene wherein 1 to 25 methylene groups are optionally and independently substituted with-N (H) -, -N (C) ₁-C₄ Alkyl) -, -N (cycloalkyl) -, -O-, -C (O) O-, -S (O) ₂-、-S(O)₂N(C₁-C₄ Alkyl) -, S (O) ₂ N (cycloalkyl) -, -N (H) C (O) -, -N (C) ₁-C₄ Alkyl) C (O) -, -N (cycloalkyl) C (O) -, -C (O) N (H) -, -C (O) N (C) ₁-C₄ alkyl), -C (O) N (cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl, or cycloalkenyl substitution. The linker may be divalent or trivalent C ₁-C₅₀ Alkylene wherein 1-25 methylene groups are optionally and independently replaced by-N (H) -, -O-, -C (O) N (H) -, or a combination thereof.

The joint may have the following structure:

wherein: each AA is independently an amino acid residue; * Is with AA _SC And AA _SC Is a side chain of the amino acid residue cCPP; 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 may be an integer from 1 to 5. x may be an integer from 1 to 3. x may be 1.y may be an integer from 2 to 4. y may be 4.z may be an integer from 1 to 5. z may be an integer from 1 to 3. z may be 1. Each AA may be independently selected from glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminocaproic acid.

CCPP may be attached to the cargo by a joint ("L"). The linker may be conjugated to the cargo via a binding group ("M").

The joint may have the following structure:

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; each AA is independently an amino acid residue; * Is with AA _SC And AA _SC is a side chain of the amino acid residue cCPP; and M is a binding group as defined herein.

The joint may have the following structure:

Wherein: 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 with AA _SC And AA _SC is a side chain of the amino acid residue cCPP; and M is a binding group as defined herein.

The joint may have the following structure:

Wherein: 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 with AA _SC And AA _SC is a side chain of the amino acid residue cCPP; and M is a binding group as defined herein.

The joint may have the following structure:

wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; and z' is an integer from 1 to 23; * Is with AA _SC And AA _SC Is a side chain of the amino acid residue cCPP.

X may be an integer from 1 to 10, such as 1,2,3, 4, 5,6,7,8, 9, or 10, including all ranges and subranges therebetween.

X' may be an integer from 1 to 23, such as 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' may be an integer from 5 to 15. x' may be an integer from 9 to 13. x' may be an integer from 1 to 5. x' may be 1.

Y may be an integer from 1 to 5, such as 1,2, 3,4 or 5, including all ranges and subranges therebetween. y may be an integer from 2 to 5.y may be an integer from 3 to 5.y may be 3 or 4.y may be 4 or 5.y may be 3.y may be 4.y may be 5.

Z may be an integer from 1 to 10, such as 1,2,3, 4, 5,6,7,8, 9, or 10, including all ranges and subranges therebetween.

Z' may be an integer from 1 to 23, such as 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' may be an integer from 5 to 15. z' may be an integer from 9 to 13. z' may be 11.

As discussed above, the linker or M (where M is part of the linker) may be covalently bound to any suitable location on the cargo. The linker or M (where M is part of the linker) may be covalently bound 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) may be covalently bound to the N-terminus or the C-terminus of the peptide cargo. The linker or M (where M is part of the linker) may be covalently bound to the backbone of the oligonucleotide or peptide cargo.

The linker may be bound to a side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker may be attached to the side chain of the lysine on cCPP.

The linker may be bound to a side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the peptide cargo, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker may be bound to the side chain of lysine on the peptide cargo.

The joint may have the following structure:

Wherein the method comprises the steps of

M is a group that conjugates L with cargo such as oligonucleotides;

AA (AA) _s Is the side chain or terminal of the amino acid on cCPP;

Each AA (AA) _x independently an amino acid residue;

o is an integer of 0 to 10; and

P is an integer from 0 to 5.

The joint may have the following structure:

Wherein the method comprises the steps of

M is a group that conjugates L with cargo such as oligonucleotides;

AA (AA) _s Is the side chain or terminal of the amino acid on cCPP;

Each AA (AA) _x independently an amino acid residue;

o is an integer of 0 to 10; and

P is an integer from 0 to 5.

M may include alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted. M may be selected from:

Wherein R is alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl.

M may be selected from:

Wherein: r ¹⁰ Is alkylene, cycloalkyl or Wherein a is 0 to 10.

M may be R¹⁰ May be/> and a is 0 to 10.M may be/>

M may be a heterobifunctional crosslinker, e.g Which is disclosed in Williams et al curr.protoc Nucleic Acid chem.2010,42,4.41.1-4.41.20, incorporated herein by reference in its entirety.

M may be-C (O) -.

AA (AA) _s May be the side chain or the terminal of the amino acid on cCPP. AA (AA) _s Non-limiting examples of (a) include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). AA (AA) _s May be AA as defined herein _SC.

Each AA (AA) _x are independently natural or unnatural amino acids. One or more AA _x May be a natural amino acid. One or more AA _x May be an unnatural amino acid. One or more AA _x may be a beta-amino acid. The beta-amino acid may be beta-alanine.

O may be an integer from 0 to 10, such as 0, 1,2,3, 4, 5, 6, 7, 8, 9, and 10.o may be 0, 1,2 or 3.o may be 0.o may be 1.o may be 2.o may be 3.

P may be 0 to 5, for example 0,1,2,3,4 or 5.p may be 0.p may be 1.p may be 2.p may be 3.p may be 4.p may be 5.

The joint may have the following structure:

wherein M, AA is a single-phase alternating-current power supply _s Each- (R) ^1-J-R² ) z "-, o and z" are as defined herein; r may be 0 or 1.

R may be 0.r may be 1.

The joint may have the following structure:

wherein M, AA is a single-phase alternating-current power supply _s Each of o, p, q, r and z "may be as defined herein.

Z "may 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 "may be an integer from 5 to 20. z "may be an integer from 10 to 15.

The joint may have the following structure:

Wherein:

m, AA A _s And o is as defined herein.

Other non-limiting examples of suitable linkers include:

/>

wherein M and AA _s As defined herein.

Provided herein are compounds comprising cCPP and AC complementary to a target in a pre-mRNA sequence, the compounds further comprising L, wherein the linker is conjugated to AC through a binding group (M), wherein M is

Provided herein are compounds comprising cCPP and a cargo comprising an Antisense Compound (AC), such as an antisense oligonucleotide, complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein the linker is conjugated to AC through a binding group (M), wherein M is selected from the group consisting of:

Wherein: r ¹ Is alkylene, cycloalkyl or/> Wherein t' is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, wherein R ¹ Is/> And t' is 2.

The joint may have the following structure:

wherein AA is _s As defined herein, and m' is 0 to 10.

The linker may have the formula:

the linker may have the formula:

Wherein the "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

The linker may have the formula:

Wherein the "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

The linker may have the formula:

Wherein the "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

The linker may have the formula:

Wherein the "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

The linker may have the formula:

Linker c may be covalently bound to any suitable position on the cargo. The linker may be covalently bound to the 3 'end of the cargo or the 5' end of the oligonucleotide cargo. The linker may be covalently bound to the backbone of the cargo.

The linker may be bound to a side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker may be attached to the side chain of the lysine on cCPP.

CCPP-linker conjugates

CCPP may be conjugated to a linker as defined herein. The linker may be associated with AA of cCPP as defined herein _SCConjugation.

The linker may comprise- (OCH) ₂CH₂)_z' -subunits (e.g. as spacers), 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."- (OCH) ₂CH₂)_z' "also known as PEG. cCPP-linker conjugates can have a structure selected from table 4:

Table 4: exemplary cCPP linker conjugates

Ring (FfΦ -4gp-r-4 gp-rQ) -PEG 4-K-NH2
	Ring (FfΦ -Cit-r-Cit-rQ) -PEG 4-K-NH2
Ring (FfPhi-Pia-r-Pia-rQ) -PEG 4-K-NH2
	Ring (FfΦ -Dml-r-Dml-rQ) -PEG 4-K-NH2
Ring (FfΦ -Cit-r-Cit-rQ) -PEG 12-OH
	Ring (fΦR-Cit-R-Cit-Q) -PEG 12-OH

The linker may comprise- (OCH) ₂CH₂)_z' -subunits and peptide subunits, wherein z' is an integer from 1 to 23. The peptide subunit may comprise 2 to 10 amino acids. cCPP-linker conjugates can have a structure selected from table 5:

Table 5: exemplary cCPP linker conjugates

Ac-PKKKKRKV-Lys (cyclo [ FfΦ -R-R-Cit-rQ ]) -PEG 12-K(N3)-NH2
	Ac-PKKKKRKV-Lys (cyclo [ FfΦ -Cit-R-R-rQ ]) -PEG 12-K(N3)-NH2
Ac-PKKKKRKV-K (Ring (FfΦR-cit-R-cit-Q)) -PEG 12-K(N3)-NH2
	Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -B-k (N) 3)-NH2
Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG2-k (N) 3)-NH2
	Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG4-k (N) 3)-NH2
Ac-PKKKKRKV-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG12-k (N 3)-NH2
	Ac-pkkkrkv-PEG2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG12-k (N 3)-NH2
Ac-rrv-PEG2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG12-OH
	Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-r-Q ]) -PEG12-k (N) 3)-NH2
Ac-PKKK-Cit-KV-PEG2-Lys (cyclo [ FfΦ -Cit-r-Cit-r-Q ]) -PEG12-k (N 3)-NH2
	Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-r-Q ] -PEG12-K (N) 3)-NH2

CCPP-linker conjugates can have the structure shown in figure 1 (e.g., compound 1a, compound 1b, compound 2a, or compound 3 a) or the sequences listed in table 4.

CCPP-linker conjugates can have the sequences as set forth in table 5.

CCPP-linker conjugate may be Ac-PKKKKRKV-K (cyclo [ FfΦ GrGrQ ])-PEG 12-K (N) ₃)-NH₂ . EEVs comprising a cyclic cell penetrating peptide (cCPP), a linker, and an Exocyclic Peptide (EP) are provided. The EEV may have the structure of formula (B):

Or a protonated form thereof,

Wherein:

R ₁、R₂And R is ₃ Each independently is H or an aromatic or heteroaromatic side chain of an amino acid;

R ₄And R is ₆ 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 1-4; and

Z' is an integer from 1 to 23.

R ₁、R₂、R₃、R₄、R₇ EP, m, q, y, x ', z' are as described herein.

N may be 0.n may be 1.n may be 2.

EEVs may have the structure of formula (B-a) or (B-B):

Or a protonated form thereof, wherein EP, R ¹、R²、R³、R⁴ M and z' are as defined above in formula (B).

EEVs may have the structure of formula (B-c):

Or a protonated form thereof, wherein EP, R ¹、R²、R³、R⁴ and m is 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; z is an integer from 1 to 10.

EEVs may have the structure of formula (B-1), (B-2), (B-3) or (B-4):

/>

/>

or a protonated form thereof, wherein EP is as defined above in formula (B).

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

The EEV may comprise cCPP of the formula:

The EEV may have the formula: ac-PKKKRKV-miniPEG-Lys (loop (FfFGRGRQ) -miniPEG2-K (N3).

The EEV may be:

EEVs may be

EEV may be Ac-P-K (Tfa) -K (Tfa) -K (Tfa) -R-K (Tfa) -V-miniPEG-K (cyclo (Ff-Nal-GrGrQ) -PEG12-OH.

EEVs may be

EEV may be Ac-P-K-K-K-R-K-V-miniPEG-K (ring (Ff-Nal-GrGrQ) -PEG12-OH.

EEVs may be

EEVs may be

EEVs may be

EEVs may be

EEVs may be

EEVs may be

The EEV may be:

EEVs may be

EEVs may be

EEVs may be

EEVs may be

EEVs may be selected from

Ac-rr-miniPEG-Dap [ cyclo (FfΦ -Cit-r-Cit-rQ) ] -PEG12-OH

Ac-frr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-rfr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-rbfbr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-rrr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-rbr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-rbrbr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-hh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-hbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-hbhbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-rbhbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-hbrbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH

Ac-rr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-frr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-rfr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-rbfbr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-rrr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-rbr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-rbrbr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-hh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-hbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-hbhbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-rbhbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-hbrbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH

Ac-KKKK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KGKK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KKGK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KKK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG2-K (N3) -NH2

Ac-KGK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KBK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KBKBK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KR-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KBR-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PKKKRKV-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PKKKRKV-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PGKKRKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PKGKRKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PKKGRKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PKKKGKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PKKKRGV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-PKKKRKG-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KKKRK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2

Ac-KKRK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2 and

Ac-KRK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2.

EEVs may be selected from

EEVs may be selected from

EEVs may be selected from

EEVs may be selected from

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₂-K(N₃)-NH₂

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₂-OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₂-K(N₃)-NH₂

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₂-OH

The cargo may be a protein and the EEV may be selected from:

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂-OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Ac-rr-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-rr-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-rr-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-rr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-rr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-rr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂-OH

Ac-rr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Ac-rrr-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-rrr-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-rrr-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-rrr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-rrr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-rrr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂-OH

Ac-rrr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Ac-rhr-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-rhr-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-rhr-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-rhr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-rhr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-rhr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂-OH

Ac-rhr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Ac-rbr-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-rbr-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-rbr-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-rbr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-rbr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-rbr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂-OH

Ac-rbr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Ac-rbrbr-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-rbrbr-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-rbrbr-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-rbrbr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-rbrbr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-rbrbr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂-OH

Ac-rbrbr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Ac-rbhbr-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-rbhbr-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-rbhbr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂-OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Ac-hbrbh-PEG ₂ -K (cyclo [ Ff-Nal-GrGrQ ]) -PEG ₁₂-OH

Ac-hbrbh-PEG ₂ -K (cyclo [ Ff-Nal-Cit-r-Cit-rQ ]) -PEG ₁₂-OH

Ac-hbrbh-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂-OH

Ac-hbrbh-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂-OH

Ac-hbrbh-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂-OH

Ac-hbrbh-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH and

Ac-hbrbh-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂-OH

Wherein b is beta-alanine and the exocyclic sequence may be D or L stereochemistry.

In embodiments, provided herein are TMs conjugated to two CPPs. Non-limiting examples of structures of TMs conjugated to two CPPs are provided below. For illustrative purposes only, the TM in the illustrated structure is AC. Other TMs, such as therapeutic polypeptides, may also be used. Exemplary antisense oligonucleotides are underlined. The antisense oligonucleotide sequences shown below are for illustrative purposes only and may be replaced with another antisense oligonucleotide sequence depending on the target of interest. In embodiments, provided herein are TMs conjugated with three CPPs. Non-limiting examples of structures of TMs conjugated to three CPPs are provided below. For illustrative purposes only, the TM in the illustrated structure is AC. Other TMs, such as therapeutic polypeptides, may also be used. Underlined indicates antisense oligonucleotides. The antisense oligonucleotide sequences shown below are for illustrative purposes only and may be replaced with another antisense oligonucleotide sequence depending on the target of interest.

Goods (e.g. freight)

A Cell Penetrating Peptide (CPP), such as a cyclic cell penetrating peptide (e.g., cCPP), may be conjugated to the cargo. The cargo may be a Therapeutic Moiety (TM). The cargo may be conjugated to the terminal carbonyl group of the linker. At least one atom of the cyclic peptide may be replaced by a cargo or at least one lone pair may form a bond with the cargo. The cargo may be conjugated to cCPP via a linker. Goods can pass through the connector and AA _SC conjugation. At least one atom of cCPP may be replaced with a therapeutic moiety or at least one lone pair of cCPP forms a bond with a therapeutic moiety. The hydroxyl group on the amino acid side chain of cCPP may be replaced by a bond to the cargo. The hydroxyl group on the glutamine side chain of cCPP can be replaced by a bond to the cargo. The cargo may be conjugated to cCPP via a linker. Goods can pass through the connector and AA _SCConjugation.

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, oligonucleotide or small molecule. The cargo may be a peptide sequence or a non-peptide based therapeutic agent. The cargo may be an antibody or antigen binding fragment thereof, including but not limited to scFv or nanobody.

The cargo may comprise one or more additional amino acids (e.g., K, UK, TRV); a linker (e.g., a bifunctional linker LC-SMCC); coenzyme A; coumaryl aminopropionic acid phosphate (pCAP); 8-amino-3, 6-dioxaoctanoic acid (miniPEG); l-2, 3-diaminopropionic acid (Dap or J); l-beta-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.

The cargo may comprise any of those listed in table 6, or derivatives or combinations thereof.

TABLE 6 exemplary cargo portions

* PCAP, phosphocoumaryl aminopropionic acid; omega, norleucine; u, 2-aminobutyric acid; D-pThr is D-phosphothreonine and Pip is L-piperidine-2-carboxylate.

Detectable moiety

The compound may comprise a detectable moiety. The detectable moiety may be attached to a Cell Penetrating Peptide (CPP) at an amino group, a carboxylate group, or a side chain of any amino acid of the CPP (e.g., at a side chain of an amino group, carboxylate group, or any amino acid in cCPP). The detectable moiety may be attached to the cyclic cell penetrating peptide at the side chain of any of the amino acids in cCPP (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, chelate 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 other reagents.

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

The detectable moiety may contain a luminophore, such as a fluorescent label or a near infrared label. Examples of suitable luminophores include, but are not limited to, metalloporphyrins; benzoporphyrin; azabenzoporphyrins; naphthalene porphyrin; a phthalocyanine; polycyclic aromatic hydrocarbons such as diimine, pyrene; azo dyes; xanthene dyes; dipyrromethene boron, azadipyrromethene boron, cyanine dyes, metal ligand complexes such as bipyridine, bipyridine-like, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine (acridine), Oxazine derivatives, such as dibenzo/> an oxazine; aza-rotaene, squaric acid; 8-hydroxyquinoline, polymethine, luminescent nanoparticles such as quantum dots, nanocrystals; a quinolone; terbium complexes; an inorganic phosphor; ionophores, such as crown ether ancillary or derivatized dyes; or a combination thereof. Specific examples of suitable luminophores include, but are not limited to, octaethylporphyrin Pd (II); octaethylporphyrin Pt (II); tetraphenylporphyrin Pd (II); tetraphenylporphyrin Pt (II); meso-tetraphenylporphyrin tetrabenzoporphin Pd (II); meso-tetraphenylmethyl benzoporphyrin Pt (II); pd (II) octaethylporphyrin; pt (II) octaethylporphyrin; meso-tetrakis (pentafluorophenyl) porphyrin Pd (II); meso-tetrakis (pentafluorophenyl) porphyrin Pt (II); tris (4, 7-diphenyl-1, 10-phenanthroline) Ru (II) (Ru (dpp) ₃ ) ; tris (1, 10-phenanthroline) Ru (II) (Ru (phen) ₃ ) Tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate (Ru (bpy) ₃ ) ; erythrosine B; fluorescein; fluorescein Isothiocyanate (FITC); eosin; ((N-methyl-benzoimidazol-2-yl) -7- (diethylamino) -coumarin) iridium (III);

Benzothiazol) ((benzothiazol-2-yl) -7- (diethylamino) -coumarin) -2- (acetylacetonate); lumogen dye; macroflex fluorescent red; macrolex fluorescent yellow; texas red; rhodamine B; rhodamine 6G; thiorhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromocresol red; bromothymol blue; cy2; cy3; cy5; cy5.5; cy7; 4-nitrophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; a calcium magnesium reagent; bromoxylenol; phenol red; neutral red; nitrooxazine; 3,4,5, 6-tetrabromophenolphthalein; congo red; fluorescein; eosin; 2',7' -dichlorofluorescein; 5 (6) -carboxyfluorescein; carboxynaphthofluorescein; 8-hydroxypyrene-136-trisulfonic acid; semi-naphthorhodamine (semi-naphthorhodafluor); semi-naphthofluorescein; tris (4, 7-diphenyl-1, 10-phenanthroline) ruthenium (II) dichloride; (4, 7-diphenyl-1, 10-phenanthroline) ruthenium (II) tetraphenylboron; platinum (II) octaethylporphyrin; dialkyl carbocyanines; dioctadecyl epoxy carbocyanine; fluorenylmethoxy carbonyl chloride; 7-amino-4-methylcoumarin (Amc); green Fluorescent Protein (GFP); and derivatives or combinations thereof.

The detectable moiety may include rhodamine B (Rho), fluorescein Isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green Fluorescent Protein (GFP), or derivatives or combinations thereof.

The detectable moiety may be attached to the Cell Penetrating Peptide (CPP) at any amino group, carboxylate group, or side chain of an amino acid (e.g., at the side chain of an amino group, carboxylate group, or any amino acid in cCPP) of the Cell Penetrating Peptide (CPP).

Therapeutic moiety

The disclosed compounds may comprise a therapeutic moiety. The cargo may comprise a therapeutic moiety. The detectable moiety may be linked to the therapeutic moiety or the detectable moiety may be used as the therapeutic moiety. A therapeutic moiety refers to a group that will reduce one or more symptoms of a disease or disorder when administered to a subject. The therapeutic moiety may include a peptide, protein (e.g., an enzyme, an antibody, or a fragment thereof), a small molecule, or an oligonucleotide.

Therapeutic moieties may include a variety of drugs, including antagonists (e.g., enzyme inhibitors) and agonists (e.g., transcription factors that result in increased expression of the desired gene product (although antagonistic transcription factors may also be used as understood by those skilled in the art)), all of which are included. In addition, therapeutic moieties include those agents that are capable of producing direct toxicity to healthy and/or unhealthy cells in vivo and/or are capable of inducing toxicity. In addition, the therapeutic moiety is capable of inducing and/or eliciting an immune system against a potential pathogen.

The therapeutic moiety may include, for example, an anticancer agent, an antiviral agent, an antimicrobial agent, an anti-inflammatory agent, an immunosuppressant, an anesthetic agent, or any combination thereof.

The therapeutic moiety may include an anticancer agent. Examples of anticancer agents include 13-cis retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, ackutane, actinomycin-D, adriamycin, adrucil, agrylin, ala-Cort, aldesleukin, alemtuzumab, alisretin acid (Alitretinoin), alkaban-AQ, alkeran, all-trans retinoic acid, interferon-alpha, altretamine, methotrexate, aminophosptine, aminoglutethimide, anagrelide (ANAGRELIDE), anandron, anastrozole, cytarabine (Arabinosylcytosine), aranesp, aredia, arimidex, aromascin, arsenic trioxide, asparaginase, ATRA, avastin, BCG, BCNU, bevacizumab, salrotene (Bexarotene), bicalutamide (Bicalutamide), biCNU, blenoxane, bevacizumab bleomycin, bortezomib, busulfan, busulfex, C225, calcium folinate, campath, camptosar, camptothecin-11, capecitabine, carac, carboplatin, carmustine tablet, casodex, CCNU, CDDP, ceeNU, cerubidine, cetuximab, chlorambucil (Chlorambucil), cisplatin, nervofactor (Citrovorum Factor), cladribine, cortisone, cosmegen, CPT-11, cyclophosphamide, citrovorum Factor, cytarabine (Citrovorum Factor), cytarabine liposome, citrovorum Factor-Citrovorum Factor, dacarbazine, citrovorum Factor, alfadaplatin (Citrovorum Factor), daunorubicin hydrochloride, daunorubicin liposome, citrovorum Factor-Citrovorum Factor, dinium (Citrovorum Factor), citrovorum Factor, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexasone, dexrazoxane (Dexrazoxane), DHAD, DIC, diodex, docetaxel (docetaxel), doxil, doxorubicin (doxorubicin), doxorubicin liposome, droxia, DTIC, DTIC-Dome, duralone, efudex, eligard, ellence, eloxatin, elspar, emcyt, epirubicin (Epirubicin), alfuzosin (epoetin alfa), erbitux, erwinia L-asparaginase (erwinia L-ASPARAGINASE), estramustine (Estramustine), ethyol, etopophos, etoposide, eulexin, evista, exemestane, fareston, faslodex, femara, feegestatin, fluorouridine, fludarabine, fluoroplex, fluorouracil (cream), fluoromethyltestosterone (Fluoxymesterone), flusterine (Flutamide), folinic acid, FUDR, fluxwell fulvestrant, G-CSF, gefitinib, gemcitabine, gemtuzumab ozogamicin, gemzar, gleevec, lupron, lupron depot, matulane, maxidex, nitrogen mustard (Mechlorethamine), nitrogen mustard hydrochloride, medralone, medrol, megace, megestrol acetate, melphalan, mercaptopurine, mesna, mesnex, methotrexate, sodium methotrexate, methylprednisolone, medralone, medrol, megace, furazoles, medralone, medrol, megace, nilutamide (Medralone, medrol, megace), nitrogen mustard, medralone, medrol, megace, octreotide (Medralone, medrol, megace), octreotide acetate, medralone, medrol, megace, oxaliplatin, paclitaxel, pamidronate, medralone, medrol, megace interferon, pegapase, febufaxine, PEG-INTRON, PEG-L-asparaginase, phenylalanine nitrogen mustard, medralone, medrol, megace-AQ, prednisolone, prednisone, prelone, procarbazine, PROCRIT, proleukin, prolifeprospan containing A carmustine implant, purinethol, raloxifene, rheumatrex, rituxan, rituximab, roveron-A (interferon alpha-2A), rubex, rubomycin hydrochloride, sandostatin, sandostatin LAR, sargramostim, solu-Cortef, solu-Medrol, STI-571, streptozotocin (Streptozocin), tamoxifen, TARGRETIN, TAXOL, TAXOTERE, TEMODAR, temozolomide, teniposide, TESPA, thalidomide, thalomid, theraCys, thioguanine Tabloid, thiophosphamide, thioplex, thiotepa, TICE, toposar, topotecan, toremifene, trastuzumab, retinoic acid, trexall, trisenox, TSPA, VCR, velban, velcade, vePesid, vesanoid, viadur, vinblastine sulfate, VINCASAR PFS, vincristine, vinorelbine tartrate, VLB, VP-16, vumon, xeloda, zanosar, zevalin, zinecard, zoladex zoledronic acid, zometa, GLIADEL WAFER, glivc, GM-CSF, goserelin, granulocyte colony stimulating factor, halotestin, herceptin, hexadrol, hexalen, altretamine, HMM, hycamtin, hydrea, hydrocortisone acetate (Hydrocort Acetate), hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone, hydroxyurea, temozolomab (Ibritumomab), temozolomide tazitan, idamycin, idarubicin (Idarubicin), ifex, IFN-alpha, ifosfamide, IL 2, IL-11, eosin ma tinib mesylate, imidazole carboxamide, interferon alpha-2 b (PEG conjugate), interleukin 2, interleukin 11, intron A (interferon alpha-2 b), leucovorin, leukeran, leukine, leuprolide, leurocristine, leustatin, leuprolide, liposome Ara-C, liquid pred, lomustine (Lomustine), L-PAM, L-Sarcolysin, meticorten, mitomycin-C, mitoxantrone, M-Prednisol, MTC, MTX, mustargen, mustine, mutamycin, myleran, iressa, irinotecan, isotretinoin (Isotretinoin), kidrolase, lanacort, L-asparaginase and LCR. The therapeutic moiety may also include a biological drug, such as, for example, an antibody.

Therapeutic moieties may include antiviral agents such as ganciclovir, azidothymidine (AZT), lamivudine (3 TC), and the like.

The therapeutic moiety may include an antibacterial agent, such as dapsone acetate (acedapsone); sulfadiazine sodium (acetosulfone sodium); aminomycin (alamecin); alexidine (alexidine); penicillium chloramidine (amdinocillin); chloramidine penicillin diester (amdinocillin pivoxil); a Mi Huan element (amicycline); amifloxacin (amifloxacin); amifloxacin mesylate; amikacin (amikacin); amikacin sulfate; aminosalicylic acid; sodium aminosalicylate; amoxicillin; bleomycin (amphomycin); ampicillin (ampicillin); ampicillin sodium; apaxillin sodium (APALCILLIN SODIUM); apramycin (apramycin); aspartyl (aspartocin); astemicin sulfate (astromicin sulfate); bleomycin (avilamycin); a Fu Meisu (avoparcin); azithromycin (azithromycin); azlocillin (azlocillin); azlocillin sodium; bammoxillin hydrochloride; bacitracin (bacitracin); methylene disalicylate bacitracin; bacitracin zinc; babolmycin (bambermycins); calcium benalamate (benzoylpas calcium); erythromycin (berythromycin); betamycin sulfate (betamicin sulfate); biapenem (biapenem); binicamycin (biniramycin); bensalsalate hydrochloride (biphenamine hydrochloride); magnesium sulfate bis pyrithione (bispyrithione magsulfex); buticacin (butikacin); butirox sulfate (butirosin sulfate); patulin sulfate (capreomycin sulfate); carbodol (carbadox); carbenicillin disodium (carbenicillin disodium); carbenicillin Lin Yinman sodium (carbenicillin indanyl sodium); carbenicillin sodium phenyl ester (carbenicillin phenyl sodium); carbenicillin potassium (carbenicillin potassium); sodium capromorphan (carumonam sodium); cefaclor (cefaclor); cefadroxil (cefadroxil); cefamandole (cefamandole); cefamandole nafate (cefamandole nafate); cefamandole nafate (cefamandole sodium); cefprozil (cefaparole); ceftriaxone (cefatrizine); ceffluxazole sodium (cefazaflur sodium); cefazolin (cefazolin); cefazolin sodium; cefbuperazone (cefbuperazone); cefdinir (cefdinir); cefepime (cefepime); cefepime hydrochloride; ceftiofur (cefetecol); cefixime (cefixime); cefmenoxime hydrochloride (cefmenoxime hydrochloride); cefmetazole (cefmetazole); cefmetazole sodium; cefnesirimate sodium (cefonicid monosodium); cefonicid sodium; cefoperazone sodium (cefoperazone sodium); cefradine (ceforanide); cefotaxime sodium (cefotaxime sodium); cefotetan (ceftetan); cefotetan disodium; cefotiam hydrochloride (cefotiam hydrochloride); cefoxitin (cefoxitin); cefoxitin sodium; cefazene (cefpimizole); cefazedone sodium; cefpiramide (cefpiramide); cefpiramide sodium; cefpirome sulfate; cefpodoxime proxetil (cefpodoxime proxetil); cefprozil (cefprozil); cefradine (cefroxadine); cefsulodin sodium (cefsulodin sodium); ceftazidime (ceftazidime); ceftibuten (ceftibuten); ceftizoxime sodium (ceftizoxime sodium); ceftriaxone sodium (ceftriaxone sodium); cefuroxime (cefuroxime); cefuroxime axetil (cefuroxime axetil); cefuroxime axetil Xin Pi (cefuroxime pivoxetil); cefuroxime sodium (cefuroxime sodium); cefazetidine sodium (CEPHACETRILE SODIUM); cefalexin (cephalexin); cefalexin hydrochloride; cefalexin (cephaloglycin); ceftiofur (cephaloridine); cefalotin sodium (cephalothin sodium); cefpirome sodium (CEPHAPIRIN SODIUM); cefradine (cephradine); sitagliptin hydrochloride (cetocycline hydrochloride); acetylchloramphenicol (cetophenicol); chloramphenicol (chloramphenicol); chloramphenicol palmitate; chloramphenicol pantothenate complex (chloramphenicol pantothenate complex); chloramphenicol sodium succinate (chloramphenicol sodium succinate); chlorhexidine phosphoramidate (chlorhexidine phosphanilate); chloroxylenol (chloroxylenol); aureomycin bisulfate (chlortetracycline bisulfate); aureomycin hydrochloride; cinnoxacin (cinoxacin); ciprofloxacin (ciprofloxacin); ciprofloxacin hydrochloride; sirolimus (cirolemycin); clarithromycin (clarithromycin); clinafloxacin hydrochloride (clinafloxacin hydrochloride); clindamycin (clindamycin); clindamycin hydrochloride; clindamycin palmitate hydrochloride; clindamycin phosphate; clofazimine (clofazimine); benzathine o-clopenicillin (cloxacillin benzathine); cloxacillin sodium (cloxacillin sodium); chlorohydroxyquinoline (cloxyquin); colistin sodium methane sulfonate (colistimethate sodium); colistin sulfate (colistin sulfate); -coumaramycin (coumermycin); coumaramycin sodium; cyclohexacillin (cyclacillin); cycloserine (cycloserine); dapoxetine (dalfopristin); dapsone (dapsone); daptomycin (daptomycin); demeclocycline (demeclocycline); demeclocycline hydrochloride; norcyclophilin (demecycline); lufenin (denofungin); diammine veratrole li (diaveridine); dicloxacillin (dicloxacillin); dicloxacillin sodium; streptomycin sulfate (dihydrostreptomycin sulfate); dithiooxine (dipyrithione); dirithromycin (dirithromycin); doxycycline (doxycycline); doxycycline calcium; doxycycline phosphate complex (doxycycline fosfatex); doxycycline hydrochloride; qu Kesha sodium star (droxacin sodium); enoxacin (enoxacin); epiicillin (epicillin); differential tetracycline hydrochloride (EPITETRACYCLINE HYDROCHLORIDE); erythromycin; vinegar stearin erythromycin; erythromycin estolate; erythromycin ethylsuccinate; erythromycin glucoheptonate; erythromycin lactobionate; erythromycin propionate; erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxacin (fleroxacin); flucloxacillin (floxacillin); fluorodeuteroalanine (fludalanine); flumequine (flumequine); fosfomycin (fosfomycin); fosfomycin trometamol (fosfomycin tromethamine); furoxicillin (fumoxicillin); furazolium chloride (furazolium chloride); furazolium tartrate (furazolium tartrate); sodium fusidate (fusidate sodium); fusidic acid; gentamicin sulfate (GENTAMICIN SULFATE); glamoran (gloximonam); bacitracin (gramicidin); haloprogin (haloprogin); sea taxilin (hetacillin); potassium pentasil; sea kexetine (hexedine); with bafloxacin (ibafloxacin); imipenem (imipenem); isoconazole (isoconazole); isoppamixin (isepamicin); isoniazid (isoniazid); cross-linked Saccharomycin (josamycin); kanamycin sulfate (KANAMYCIN SULFATE); kitasamycin (kitasamycin); levofuraltadone (levofuraltadone); left-hand potassium Pi Xilin (levopropylcillin potassium); erythromycin (lexithromycin); lincomycin (lincomycin); lincomycin hydrochloride; lomefloxacin (lomefloxacin); lomefloxacin hydrochloride; lomefloxacin mesylate; chlorocarbon cephalosporin (loracarbef); sulfamoron (mafenide); meclocycline (meclocycline); methyl chlorocyclosulfosalicylate; potassium dihydrogen phosphate (megalomicin potassium phosphate) of megamycin; mequindox (mequidox); meropenem (meropenem); metacycline (METHACYCLINE); metacycline hydrochloride; urotropin (methenamine); hippuric acid urotropine; urotropine mandelate; methicillin sodium (METHICILLIN SODIUM); metiprine (metioprim); metronidazole hydrochloride; metronidazole phosphate; mezlocillin (mezlocillin); mezlocillin sodium; minocycline (minocycline); minocycline hydrochloride; milbemycin hydrochloride (MIRINCAMYCIN HYDROCHLORIDE); monensin (monensin); sodium monensin; nafcillin sodium (NAFCILLIN SODIUM); sodium naphthyridine (nalidixate sodium); nalidixic acid; natamycin (natainycin); darkmycin (nebramycin); neomycin palmitate (neomycin palmitate); neomycin sulfate; neomycin undecylenate; netilmicin sulfate (NETILMICIN SULFATE); neutral mycin (neutramycin); nifuradine (nifuiradene); nifuratel (nifuraldezone); nifuratel (nifuratel); nifurone (nifuratrone); nifurazid (nifurdazil); nifuramide (nifurimide); nifuratel (nifiupirinol); nifuraquazole (nifurquinazol); nifurazol (nifurthiazole); nitrocyclosporin (nitrocycline); nitrofurantoin (nitrofurantoin); niter (nitromide); norfloxacin (norfloxacin); sodium novobiocin (novobiocin sodium); ofloxacin (ofloxacin); omeprazole (onnetoprim); oxacillin (oxacillin); oxacillin sodium; oxime monan (oximonam); sodium oxime monan; Quinic acid (oxolinic acid); oxytetracycline (oxytetracycline); oxytetracycline calcium; oxytetracycline hydrochloride; patadine (paldimycin); parachlorophenol (parachlorophenol); -guaifenesin (paulomycin); pefloxacin (pefloxacin); pefloxacin mesylate; penciclin (PENAMECILLIN); benzathine G (PENICILLIN G benzathine); penicillin G potassium; procaine penicillin G (PENICILLIN G procaine); penicillin G sodium; penicillin V; benzathine V (PENICILLIN V benzathine); haibamingpenicillin V (PENICILLIN V hydroabamine); penicillin V potassium; sodium pendazole (pentizidone sodium); phenyl aminosalicylate; piperacillin sodium (PIPERACILLIN SODIUM); pibenzollin sodium (pirbenicillin sodium); pizoxillin sodium (PIRIDICILLIN SODIUM); pirlimycin hydrochloride (PIRLIMYCIN HYDROCHLORIDE); pimpimicin hydrochloride (PIVAMPICILLIN HYDROCHLORIDE); pimpimicin pamoate (PIVAMPICILLIN PAMOATE); pimpimicin phenylpropionate (PIVAMPICILLIN PROBENATE); polymyxin sulfate B (polymyxin B sulfate); pofemycin (porfironmycin); priecarin (propikacin); pyrazinamide (pyrazinamide); zinc pyrithione (pyrithione zinc); quetiapine acetate (quindecamine acetate); quinupristin (quinupristin); racemic thiamphenicol (racephenicol); lei Mo Latin (ramoplanin); ranitimycin (ranimycin); relomycin (relomycin); rapamicin (repromicin); rifabutin (rifabutin); rifamestane (rifametane); li Fuke shake (rifamexil); li Fumi t (rifamide); rifampin (rifampin); rifapentine (rifapentine); rifaximin (rifaximin); rolicycline (rolitetracycline); rilycycline nitrate (rolitetracycline nitrate); luo Shami stars (rosaramicin); butyric acid Luo Shami star (rosaramicin butyrate); luo Shami Star propionate (rosaramicin propionate); luo Shami Star sodium phosphate (rosaramicin sodium phosphate); luo Shami Star stearate (rosaramicin stearate); roxacin (rosoxacin); roxarsone (roxarsone); roxithromycin (roxithromycin); mountain bike (sancycline); sodium sanfeipenem (SANFETRINEM SODIUM); sha Moxi forest (sarmoxicillin); sha Pixi forest (sarpicillin); secoifungin (scopafungin); sisomicin (sisomicin); sisomicin sulfate; sparfloxacin (sparfloxacin); spectinomycin hydrochloride (spectinomycin hydrochloride); spiramycin (spiramycin); stavomycin hydrochloride (STALLIMYCIN HYDROCHLORIDE); stavycin (steffimycin); streptomycin sulfate; -isoniazid (streptonicozid); sulfabenzene (sulfabenz); sulfanilamide (sulfabenzamide); sulfacetamide (sulfacetamide); sodium sulfacetamide; sulfaxetine (sulfacytine); sulfadiazine (sulfadiazine); sulfadiazine sodium; sulfadoxine (sulfadoxine); sulfalin (sulfalene); sulfamethazine (sulfamerazine); sulfamoxypyrimidine (sulfameter); sulfadimidine (sulfamethazine); sulfamethyldiazole (sulfamethizole); sulfamethod/> Azole (sulfamethoxazole); sulfamonomethoxine (sulfamonomethoxine); sulfadizole (sulfamoxole); zinc aminobenzenesulfonate (sulfanilate zinc); sulfanitenpyram (sulfanitran); sulfasalazine (sulfasalazine); sulfoisothiazole (sulfasomizole); sulfathiazole (sulfathiazole); sulfapyrazole (sulfazamet); sulfanide/> azole (sulfisoxazole); sulfanide acetate/> Azole (sulfisoxazole acetyl); sulfanide/> azoldiethanolamine (sulfisboxazolediolamine); sulfocolistin (sulfomyxin); thioppenem (sulopenem); sultacillin (sultamricillin); sodium oxacillin (suncillin sodium); phthaloxacillin hydrochloride (TALAMPICILLIN HYDROCHLORIDE); teicoplanin (teicoplanin); temafloxacin hydrochloride (temafloxacin hydrochloride); temoxicillin (temocillin); tetracyclines (TETRACYCLINE); tetracycline hydrochloride; a tetracycline phosphate complex; tetraoxaprine (tetroxoprim); thiamphenicol (thiamphenicol); potassium thiophenyl penicillin (THIPHENCILLIN POTASSIUM); ticarcillin Lin Jia sodium phenyl (TICARCILLIN CRESYL sodium); ticarcillin disodium (TICARCILLIN DISODIUM); tecavist Lin Shanna (TICARCILLIN MONOSODIUM); tenatone (ticlatone); chlorination Qiao Duo (tiodonium chloride); tobramycin (tobramycin); tobramycin sulfate (tobramycin sulfate); tosufloxacin (tosufloxacin); trimethoprim (trimethoprim); trimethoprim sulfate; triple sulfadiazine (trisulfapyrimidines); vinegar bamboo peach mycomycin (troleandomycin); propylspectinomycin sulfate (trospectomycin sulfate); brevudine (tyrothricin); vancomycin (vancomycin); vancomycin hydrochloride; virginiamycin (VIRGINIAMYCIN); or geldanamycin (zorbamycin).

The therapeutic moiety may include an anti-inflammatory agent.

The therapeutic moiety may include dexamethasone (Dex).

The therapeutic moiety may comprise a therapeutic protein. For example, some people have certain enzyme deficiencies (e.g., lysosomal storage diseases). Such enzymes/proteins can be delivered to human cells by linking them to the cyclic cell penetrating peptides (cCPP) disclosed herein. The disclosed cCPP has been tested with proteins (e.g., GFP, PTP1B, actin, calmodulin, troponin C) and shown to function.

The therapeutic moiety may be an anti-infective agent. The term "anti-infective agent" refers to an agent capable of killing, inhibiting or slowing the growth of an infectious agent. The term "infectious agent" refers to a pathogenic microorganism, such as a bacterium, virus, fungus, and an intracellular or extracellular parasite. Anti-infective agents are useful in the treatment of infectious diseases, as infectious diseases are caused by infectious agents.

The infectious agent may be a gram negative bacterium. The gram-negative bacterium may be a genus selected from the group consisting of 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 bacteria may be a genus selected from the group consisting of Lactobacillus, azobium, streptococcus, pediococcus, protobacter, bacillus, enterococcus, staphylococcus, clostridium, vibrio, 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 of the following species: nocardia stellate, nocardia Brazilian and nocardia guinea pigs.

The infectious agent may be a fungus. The fungus may be from the genus Mucor (Mucor). The fungus may be from the genus cryptococcus (Crytococcus). The fungus may be from the genus Candida (Candida). The fungus can be selected from Mucor racemosus (Mucor racemosus), candida albicans (Candida albicans), cryptococcus neoformans (Crytococcus neoformans) or Aspergillus fumigatus (Aspergillus fumingatus).

The infectious agent may be a protozoa. The protozoa may be Plasmodium (Plasmodium) (e.g., plasmodium falciparum (p.falciparum), plasmodium vivax (p.vivax), plasmodium ovale (p.ovale), or Plasmodium malariae (p.malarial)). Protozoa cause malaria.

Exemplary organisms include bacillus, bartonella, bordetella, borrelia, brucella, campylobacter, chlamydia, chlamydophila, clostridium, corynebacterium, enterococcus, escherichia, franciscensis, haemophilus, helicobacter, legionella, leptospira, listeria, mycobacterium, mycoplasma, neisseria, pseudomonas, rickettsia, salmonella, shigella, staphylococcus, streptococcus, treponema, ureaplasma, vibrio and yersinia.

The infectious agent may be a parasite. The parasite may be of the genus Cryptosporidium. The parasite may be an endoparasite. The endoparasites may be heartworms, tapeworms or plaids. The parasite may be an ectoparasite. The parasite causes a disease selected from acanthamoeba disease (acanthamoebiasis), babesia disease (babesiosis), ciliate sachalinensis (balantidiasis), blastocyst disease (blastocystosis), coccidiosis (coccidiosis), amoeba disease (amoebiasis), giardiasis (giardiasis), isospora disease (isosporiasis), sporosporidiosis (cystosporiasis), leishmaniasis (LEISHMANIASIS), primary amoeba meningoeni, malaria, nasosporium disease (rhinosporidiosis), toxoplasmosis (toxoplasmosis), trichomoniasis (trichomoniasis), trypanosomiasis (trypanomiasis), chagas disease (CHAGAS DISEASE) or scab disease (scabies).

The infectious agent may be a virus. Non-limiting examples of viruses 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.

The anti-infective agent may be an antiviral agent. Non-limiting examples of antiviral agents include nucleoside or nucleotide reverse transcriptase inhibitors such As Zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d 4T), lamivudine (3 TC), emtricitabine, abacavir succinate, elvucitabine, adefovir dipivoxil, lobucavir (BMS-180194), lodenosine (lodenosine) (FddA) and tenofovir, including tenofovir dipivoxil and tenofovir dipivoxil fumarate; non-nucleoside reverse transcriptase inhibitors such as nevirapine, delavirdine, efavirenz, itraconazole and rilpivirine; protease inhibitors such as ritonavir, telanavir (tipranavir), saquinavir, nelfinavir, indinavir (indinavir), amprenavir (amprenavir), fusionavir (fosamprenavir), atazanavir (atazanavir), lopinavir (lopinavir), darunavir (darunavir) (TMC-114), lasinavir (lasinavir) and bocanavir (brecanavir) (VX-385); cell entry inhibitors such as CCR5 antagonists (e.g., maraviroc (maraviroc), velcro (vicriviroc), INCB9471 and TAK-652) and CXCR4 antagonists (AMD-11070); fusion inhibitors such as enfuvirtide (enfuvirtide); integrase inhibitors such as raltegravir, BMS-707035, and etiquevir; tat inhibitors such as didehydrocortisone a (dCA); maturation inhibitors such as berivimat; immunomodulators, such as levamisole; other antiviral agents such as hydroxyurea, ribavirin, interleukin 2 (IL-2), interleukin 12 (IL-12), tablet Sha Fu (pensafuside), peramivir, zanamivir, oseltamivir phosphate, balofloxacin Sha Weima Boxidate,

The anti-infective agent may be an antibiotic. Non-limiting examples of antibiotics include aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, and tobramycin; carbacephem (cabecephems) such as chlorocarba-cephem; carbapenems such as ertapenem, imipenem/cilastatin and meropenem; cephalosporins such as cefadroxil, cefazolin, cefalexin, cefaclor, cefamandole, cefalexin, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftizoxime, ceftriaxone and cefepime; macrolides such as azithromycin, clarithromycin, dirithromycin, erythromycin and vinegared marcomycin; a monocyclic lactam; penicillins, such as amoxicillin, ampicillin, carbenicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin and ticarcillin; polypeptides such as bacitracin, colistin and polymyxin B; quinolones such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin; sulfonamides, e.g. sulfamide, sulfacetamide, sulfamethoxazole, sulfasalazine, sulfaisox Azole and trimethoprim-sulfamethoxazole/> An azole; tetracyclines such as 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 acetonide, betamethasone dipropionate, difluocortone, fluticasone, cortisone, hydrocortisone, mometasone, methylprednisolone, beclomethasone dipropionate, clobetasol, prednisone, prednisolone, methylprednisolone, betamethasone, budesonide, and dexamethasone. The anti-infective agent may be a non-steroidal anti-inflammatory agent. Non-limiting examples of non-steroidal anti-inflammatory agents include celecoxib, nimesulide, rofecoxib, meclofenamic acid sodium, flunixin, fluprofen, flurbiprofen, sulindac, meloxicam, piroxicam, etodolac, fenoprofen, fenbufen, ketoprofen, suprofen, diclofenac, sodium bromfenac, phenylbutazone, thalidomide, and indomethacin.

The anti-infective agent may be an antifungal agent. Non-limiting examples of antifungal agents include amphotericin B, caspofungin (), fluconazole (fluconazole), flucytosine (), itraconazole (itraconazole), ketoconazole (ketoconazole), amorolfine (), butenafine (), naftifine (), terbinafine (terbinafine), neoconazole (elubiol), econazole (), itraconazole (itraconazole), isoconazole (), imidazole, miconazole (), fluconazole (), clotrimazole (), enconazole (), oxiconazole (), tioconazole (), econazole (), fluconazole (, fluconazole,) terconazole (), butoconazole (), thiabendazole (), voriconazole (), saperconazole (), sertaconazole (), fenticonazole (), posaconazole (), bibenzozole (), flutrozole (), nystatin (), pimaricin (), natamycin (), tolnaftate (), sulfamuron (), dapsone (), griseofulvin (), potassium iodide, gentian violet (), cyclopiroside (), cyclopisiformine (), chlorpropynyl iodine (), undecylenate, silver sulfadiazine (), and the like, undecylenic acid, undecylenic acid alkanolamides and carbol-fuchsin (Carbol-Fuchsin).

The therapeutic moiety may be an analgesic or a pain relieving agent. Non-limiting examples of analgesics or pain relievers include aspirin, acetaminophen, ibuprofen, naproxen, procaine, lidocaine, tetracaine, dibucaine (dibucaine), benzocaine (benzocaine), 2- (diethylamino) ethyl p-butylaminobenzoate HCI, mepivacaine (mepivacaine), pirocaine (piperocaine) and dyclonine (dyclonine)

The therapeutic moiety may be an antibody or antigen binding fragment. Antibodies and antigen binding fragments may be derived from any suitable source, including humans, mice, camelids (e.g., camels, alpacas, llamas), rats, ungulates, or non-human primates (e.g., monkeys, rhesus monkeys).

It will also be appreciated that the cargo described herein comprising an anti-infective agent and other therapeutic moiety includes possible salts thereof, wherein pharmaceutically acceptable salts are of course particularly relevant for therapeutic applications. Salts include acid addition salts and basic salts. Examples of acid addition salts are hydrochloride, fumarate, oxalate and the like. Examples of basic salts are those in which the (remaining) counter ion may be selected from alkali metals such as sodium and potassium, alkaline earth metals such as calcium, potassium and ammonium ions [ ] ⁺N(R′)₄ wherein R' independently represents optionally substituted C _1-6 -alkyl, optionally substituted C _2-6 -alkenyl, optionally substituted aryl or optionally substituted heteroaryl).

The therapeutic moiety may be an oligonucleotide. The oligonucleotide may be an Antisense Compound (AC). Oligonucleotides may include, for example, but are not limited to, antisense oligonucleotides, small interfering rnas (sirnas), micrornas (mirnas), ribozymes, immunostimulatory nucleic acids, antagomir, antimir, microrna mimics, supermir, ul adaptors, CRISPR mechanisms, and aptamers. The term "antisense oligonucleotide" or simply "antisense" is intended 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. publication No. 2019/0365918, U.S. publication No. US2020/0040336, U.S. patent No. 9499818, and U.S. patent No. 9447417, each of which is incorporated herein by reference in its entirety for all purposes.

The therapeutic moiety may be used to treat any of the following diseases: neuromuscular disorders, pompe disease, β -mediterranean poor blood, kobe's dystrophy, duchenne muscular dystrophy, becker muscular dystrophy, diabetes, alzheimer's disease, cancer, cystic fibrosis, merosin deficient congenital muscular dystrophy type 1A (MDC 1A), proximal Spinal Muscular Atrophy (SMA), huntington's disease-like 2 (HDL 2), myotonic dystrophy, spinocerebellar ataxia, spinal and Bulbar Muscular Atrophy (SBMA), dentate nuclear pallidolupulus atrophy (DRPLA), amyotrophic lateral sclerosis, frontotemporal dementia, fragile X syndrome, fragile X mental retardation 1 (FMR 1), fragile X mental retardation 2 (FMR 2), fragile XE mental retardation (FRAXE), friedreich's ataxia (FRDA), fragile X-related/ataxia syndrome (fxs), straighter's syndrome, ocular muscular dystrophy or non-linear muscular dystrophy, myotonic syndrome (jd), or jd-myotonic syndrome (jd), myotonic syndrome, or myotonic syndrome (jd-myotonic syndrome). The therapeutic moiety may 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, renal cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, or melanoma. The therapeutic moiety may be used to treat an ocular disorder. Non-limiting examples of ocular disorders include ametropia, macular degeneration, cataracts, diabetic retinopathy, glaucoma, amblyopia, or strabismus.

The therapeutic moiety may include a targeting moiety. The targeting moiety may comprise, for example, an amino acid sequence that can target one or more enzyme domains. The targeting moiety may include an inhibitor against an enzyme that may 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 may be an Antisense Compound (AC) described in U.S. publication No. 2019/0365918, which is incorporated herein by reference in its entirety. For example, the targeting moiety may comprise any of the sequences listed in table 7.

TABLE 7 exemplary targeting moieties

/>

* Fpa, sigma: l-4-fluorophenylalanine; pip, Θ: l-homoproline; nle, Ω: l-norleucine; phg, ψ:

L-phenylglycine; f ₂ pmp, Λ: l-4- (phosphonodifluoromethyl) phenylalanine; dap: l-2, 3-diaminopropionic acid; nal, Φ': l-beta-naphthylalanine; pp, θ: l-pipecolic acid; sar, xi: sarcosine; tm, trimesic acid.

The targeting moiety and the cell penetrating peptide may overlap. That is, the residues forming the cell penetrating peptide may also be part of the sequence forming the targeting moiety, and vice versa.

The therapeutic moiety may be attached to the cell penetrating peptide at the amino group, carboxylate group, or side chain of any amino acid of the cell penetrating peptide (e.g., at the amino group, carboxylate group, or side chain of any amino acid of cCPP). The therapeutic moiety may be attached to the detectable moiety.

The therapeutic moiety can comprise a targeting moiety that can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, pin1, grb2 SH2, CAL PDZ, etc., or a combination thereof.

Ras is a protein encoded by the Ras gene in humans. Normal Ras proteins play an important role 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, and once it is turned on, ras recruits and activates proteins necessary for growth factor and other receptor signaling. Mutant forms of Ras have been associated with a variety of cancers, including lung cancer, colon cancer, pancreatic cancer, and a variety of leukemias.

Protein-tyrosine phosphatase 1B (PTP 1B) is a prototype member of the PTP superfamily and plays a number of roles during eukaryotic cell signaling. PTP1B is a negative regulator of the insulin signaling pathway and is considered a promising potential therapeutic target, particularly for the treatment of type II diabetes. PIP1B is also associated with the development of breast cancer.

Pin1 is an enzyme that binds to a subset of proteins and plays a role in regulating protein function for post-phosphorylation control. Pin1 activity can regulate the outcome of proline-directed kinase signaling and thus can regulate cell proliferation and cell survival. Deregulation of Pin1 can play a role in a variety of diseases. Up-regulation of Pin1 may be associated with certain cancers, while down-regulation of Pin1 may be associated with alzheimer's disease. Inhibitors of Pin1 may be of therapeutic interest for cancer and immune disorders.

Grb2 is an adaptor protein involved in signal transduction and cellular communication. The Grb2 protein contains an SH2 domain that binds tyrosine phosphorylation sequences. Grb2 is widely expressed and is essential for a variety of cellular functions. Inhibition of Grb2 function can impair the developmental process and can block transformation and proliferation of various cell types.

Recently, it has been reported that the activity of cystic fibrosis membrane conductance regulator (CFTR), a mutated chloride channel protein in Cystic Fibrosis (CF) patients, is down-regulated by CFTR-related 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 was shown to improve the activity of ΔPhe508-CFTR (the most common form of CFTR mutation) (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, vol.49: p.9907). Thus, disclosed herein are methods of treating a subject suffering from cystic fibrosis by administering an effective amount of a compound or composition disclosed herein. The compound or composition administered to the subject may comprise a therapeutic moiety, which may comprise a targeting moiety that may act as an inhibitor against CAL PDZ. Furthermore, one or more of the compositions disclosed herein may be administered with a molecule that corrects CFTR function.

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

Also disclosed herein are compositions comprising the compounds described herein.

Also disclosed herein are pharmaceutically acceptable salts and prodrugs of the disclosed compounds. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases according to the particular substituents found on the compound. It may be appropriate to administer the compounds as salts under conditions wherein the compounds disclosed herein are sufficiently basic or acidic to form stable, non-toxic acid or base 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, alpha-ketoglutaric acid, alpha-sugar phosphoric acid, maleic acid, toluenesulfonic acid, methanesulfonic acid, and the like. Thus, disclosed herein are hydrochlorides, nitrates, phosphates, carbonates, bicarbonates, sulfates, acetates, propionates, benzoates, succinates, fumarates, mandelates, oxalates, citrates, tartrates, malonates, ascorbates, alpha-ketoglutarates, alpha-sugar phosphates, maleates, tosylates and methanesulfonates. Pharmaceutically acceptable salts of the compounds may be obtained using standard methods well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid that provides a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids may also be prepared.

The therapeutic moiety may include a therapeutic polypeptide, oligonucleotide, or small molecule. Therapeutic polypeptides may include peptide inhibitors. Therapeutic polypeptides may include binding agents that specifically bind 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. Antigen binding fragments may include Fab fragments, F (ab') ₂ Fragments, fv fragments, minibodies, diabodies, nanobodies, single domain antibodies (dabs), single chain variable fragments (scFv), or multispecific antibodies.

The oligonucleotide may comprise an Antisense Compound (AC). AC may include a nucleotide sequence complementary to a target nucleotide sequence encoding a protein target of interest.

The Therapeutic Moiety (TM) may be conjugated to the chemically reactive side chain of the cCPP amino acid. Any amino acid side chain on cCPP that is capable of forming a covalent bond or that can be so modified can be used to link the TM to cCPP. The amino acid at cCPP may be a natural or unnatural amino acid. The chemically reactive side chain may include an amine group, carboxylic acid, amide, hydroxyl group, sulfhydryl group, guanidine group, phenol group, thioether group, imidazole group, or indole group. The cCPP amino acids conjugated to TM may include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, tryptophan, or analogs thereof. The amino acid on cCPP used for conjugation to the TM may be ornithine, 2, 3-diaminopropionic acid or an analogue thereof. The amino acid may be lysine or an analogue thereof. The amino acid may be glutamic acid or an analogue thereof. The amino acid may be aspartic acid or an analogue thereof. The side chain may be substituted with a bond to the TM or linker.

TM may include a therapeutic polypeptide and cCPP may be conjugated to a chemically reactive side chain of an amino acid of the therapeutic polypeptide. Any amino acid side chain on the TM that is capable of forming a covalent bond or that can be modified as such can be used to attach cCPP to the TM. The amino acid on the TM may be a natural or unnatural amino acid. The chemically reactive side chain may include an amine group, carboxylic acid, amide, hydroxyl group, sulfhydryl group, guanidine group, phenol group, thioether group, imidazole group, or indole group. The amino acid of TM conjugated to cCPP may include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, tryptophan, or analogs thereof. The amino acid on the TM used for conjugation cCPP may be ornithine, 2, 3-diaminopropionic acid or an analog thereof. The amino acid may be lysine or an analogue thereof. The amino acid may be glutamic acid or an analogue thereof. The amino acid may be aspartic acid or an analogue thereof. The side chain of the TM may be substituted with a bond or linker to cCPP.

TM may be an Antisense Compound (AC) comprising an oligonucleotide, wherein the 5 'or 3' end of the oligonucleotide is conjugated to a chemically reactive side chain of an amino acid of cCPP. AC may be chemically conjugated to cCPP through a moiety on the 5 'or 3' end of the AC. The chemically reactive side chain of cCPP may include an amine group, carboxylic acid, amide, hydroxyl group, sulfhydryl group, guanidine group, phenol group, thioether group, imidazole group, or indole group. The cCPP amino acid conjugated to AC may include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, or tryptophan. The amino acid cCPP conjugated to AC may include lysine or cysteine.

Non-limiting examples of unconjugated AC structures (i.e., prior to conjugation with CPP) are provided below. AC in the following structure refers to antisense oligonucleotides.

Non-limiting examples of linear CPPs include polyarginine (e.g., R ₉Or R is ₁₁ ) Antennapedia mutant sequence, HIV-TAT, penetratin, antp-3A (antp mutant), buforin II. Transporter, MAP (model amphiphilic peptide), K-FGF, ku70, prion, pVEC, pep-1, synB1, pep-7, HN-1, BGSC (biguanide-spermidine-cholesterol) and BGTC (biguanide-triaminoethylamine-cholesterol).

Oligonucleotides

The compound may include a cyclic cell penetrating peptide (cCPP) conjugated to an Antisense Compound (AC) as a therapeutic moiety. The AC may include antisense oligonucleotides, siRNA, mini RNA, antagomir, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, supermir, miRNA mimics, miRNA inhibitors, or combinations thereof.

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. Antisense oligonucleotides can include single stranded DNA or RNA complementary to a selected sequence (e.g., target gene mRNA).

Antisense oligonucleotides can modulate one or more aspects of protein transcription, translation, expression, and function via hybridization of the antisense oligonucleotide to a target nucleic acid. Hybridization of antisense oligonucleotides to their target sequences can inhibit the expression of the target protein. Hybridization of antisense oligonucleotides to their target sequences can inhibit expression of one or more target protein isoforms. Hybridization of antisense oligonucleotides to their target sequences can up-regulate expression of the target protein. Hybridization of antisense oligonucleotides to their target sequences can down-regulate expression of the target protein.

Antisense compounds can inhibit gene expression by binding to complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression by preventing translation of the complementary mRNA strand (by binding thereto) or by causing degradation of the target mRNA. Antisense DNA can be used to target specific complementary (coding or non-coding) RNAs. If binding occurs, the DNA/RNA hybrid can be degraded by RNase H. The antisense oligonucleotide can comprise about 10 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 25 nucleotides. The term also includes antisense oligonucleotides that may not be fully complementary to the desired target gene. Thus, the compounds disclosed herein can be used in situations where antisense sequences are found to have non-target specific activity, or where it is desirable to have one or more antisense sequences mismatched with the target sequence.

Antisense oligonucleotides have proven to be effective and targeted inhibitors of protein synthesis and are therefore useful for specifically inhibiting protein synthesis by targeting genes. The efficacy of antisense oligonucleotides in inhibiting protein synthesis has been well established.

Methods of generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeted to any polynucleotide sequence of interest. The selection of antisense oligonucleotide sequences 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 will reduce or inhibit specific binding to target mRNA in a host cell. Target regions of mRNA include those at or near AUG translation initiation codon and those sequences that are substantially complementary to the 5' region of mRNA. These secondary structural analysis and target site selection considerations may be performed, for example, using the 4 th edition of OLIGO primer analysis software (Molecular Biology Insights) and/or BLASTN 2.0.5 algorithm software (Altschul et al, nucleic Acids res.1997,25 (17): 3389-402).

RNA interfering nucleic acids

The therapeutic moiety may be an RNA interference (RNAi) molecule or a small interfering RNA molecule. RNA interference methods using RNAi or siRNA molecules can be used to disrupt expression of a gene or polynucleotide of interest.

Small interfering RNAs (sirnas) are RNA duplex, typically about 16 to about 30 nucleotides long, that bind to a cytoplasmic polyprotein complex called RNAi-induced silencing complex (RISC). RISC loaded with siRNA mediates degradation of homologous mRNA transcripts, so siRNA can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA acts through a natural mechanism that evolves through non-coding RNAs to control gene expression. A variety of RNAi agents, including siRNAs targeting clinically relevant targets, are currently under drug development, as described, for example, in de Fougerolles, A. Et al, nature Reviews 6:443-453 (2007).

Although the first described RNAi molecules were RNA: RNA hybrids comprising an RNA sense strand and an RNA antisense strand, it has been demonstrated that DNA sense strand: RNA antisense hybrid, RNA sense strand: DNA antisense hybrid and DNA: DNA hybrid are capable of mediating RNAi (Lamberton, J.S. and Christian, A.T. (2003) Molecular Biotechnology 24:111-119). RNAi molecules including any of these different types of double-stranded molecules can be used. Furthermore, it should 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, a sense strand and an antisense strand, such as small interfering RNAs (siRNAs); a double-stranded oligonucleotide comprising two separate strands joined together by a non-nucleotide linker; an oligonucleotide comprising a hairpin loop that forms a complement of a double stranded region, e.g., a shRNAi molecule, and an expression vector that expresses one or more polynucleotides capable of forming a double stranded polynucleotide alone or in combination with another polynucleotide.

As used herein, a "single stranded siRNA compound" is an siRNA compound consisting of a single molecule. It may comprise duplex regions formed by intra-strand pairing, for example, it may be or comprise a hairpin or disc handle structure. The single stranded siRNA compound may be antisense to the target molecule.

The single stranded siRNA compound may be long enough to be able to enter RISC and participate in RISC-mediated cleavage of target mRNA. The 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 in length. The single stranded siRNA is less than about 200, about 100 or about 60 nucleotides in length.

Hairpin siRNA compounds can have a duplex region equal to 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 may be equal to or less than about 200, about 100, or about 50 nucleotide pairs in length. The duplex region ranges in length 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. The hairpin may have a single stranded overhang or a terminal unpaired region. The length of the overhang may be about 2 to about 3 nucleotides. The overhang may be on the sense side of the hairpin or on the antisense side of the hairpin.

As used herein, a "double stranded siRNA compound" is an siRNA compound comprising more than one, and in some cases two strands, wherein inter-strand hybridization can form a region of duplex structure.

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

The sense strand of the double stranded siRNA compound may be equal to or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40 or about 60 nucleotides in length. It may be equal to or less than about 200, about 100, or about 50 nucleotides in length. The length may range from about 17 to about 25, from about 19 to about 23, and from about 19 to about 21 nucleotides.

The double stranded portion of the double stranded siRNA compound may be equal to or at least 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 30, about 40 or about 60 nucleotide pairs in length, which may be equal to or less than about 200, about 100 or about 50 nucleotide pairs in length. The length can range from about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21 nucleotide pairs.

The siRNA compound can be large enough to be cleaved by an endogenous molecule (e.g., by Dicer) to produce a smaller siRNA compound, such as an siRNA agent.

The sense strand and the antisense strand can be selected such that the double stranded siRNA compound comprises a single stranded or unpaired region at one or both ends of the molecule. Thus, a double stranded siRNA compound may contain paired sense and antisense strands to contain an overhang, such as one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3 nucleotides. An overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being interleaved. Some embodiments will have at least one 3' overhang. In embodiments, both ends of the siRNA molecule will have a 3' overhang. The overhang may be 2 nucleotides.

The duplex region may be about 15 to about 30, or about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides in length, for example, in the ssiRNA (cohesive overhang siRNA) compound range discussed above. ssiRNA compounds can be similar in length and structure to the natural Dicer processing products from long dsiRNA. Also included are embodiments wherein both chains of ssiRNA compounds are linked (e.g., covalently linked). Including hairpins or other single stranded structures that provide a double stranded region and 3' overhangs.

SiRNA compounds (including double stranded siRNA compounds and single stranded siRNA compounds) described herein can mediate silencing of a target RNA (e.g., mRNA, e.g., transcripts of a gene encoding a protein). For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene.

As used herein, the phrase "mediate RNAi" refers to the ability to silence target RNA in a sequence-specific manner. While not wishing to be bound by theory, it is believed that silencing uses RNAi machinery or methods and guide RNAs, e.g., ssiRNA compounds of about 21 to about 23 nucleotides.

SiRNA compounds that are "substantially complementary" to a target RNA (e.g., a target mRNA) can silence the production of a protein encoded by the target mRNA. siRNA compounds that are "substantially complementary" to the RNA encoding the protein of interest can silence the production of the protein of interest encoded by the mRNA. The siRNA compound can be "exactly complementary" to the target RNA, e.g., the target RNA and the siRNA compound anneal, e.g., form a hybrid consisting of only watson-crick base pairs in the exactly complementary region. A "substantially complementary" target RNA may include an internal region (e.g., at least about 10 nucleotides) that is precisely complementary to the target RNA. In embodiments, the siRNA compound specifically distinguishes single nucleotide differences. In this case, the siRNA compound only mediates RNAi if exact complementarity is found in the region of single nucleotide differences (e.g., within 7 nucleotides).

MicroRNA

The therapeutic moiety may be a microrna molecule. Micrornas (mirnas) are a highly conserved class of small RNA molecules that are transcribed from DNA in plant and animal genomes, but are not translated into proteins. Processed mirnas are single-stranded 17-25 nucleotide (nt) RNA molecules that are integrated into RNA-induced silencing complexes (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis, and differentiation. They are believed to play a role in the regulation of gene expression by binding to the 'he 3' -untranslated region of a particular mRNA. RISC mediates down-regulation of gene expression by translational inhibition, transcriptional cleavage, or both. RISC is also associated with transcriptional silencing in a variety of eukaryotic nuclei.

Antagomir

The therapeutic moiety may be antagomir. Antagomir are RNA-like oligonucleotides having various modifications directed against RNase protection and pharmacological properties such as enhanced tissue and cell uptake. They differ from normal RNAs in, for example, sugar, phosphorothioate backbones and complete 2 '-0-methylation of cholesterol moieties, for example, the 3' -end. Antagomir can be used to effectively quench endogenous mirnas by forming a duplex comprising an antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. One example of antagomir-mediated miRNA silencing is silencing of miR-122, described in Krutzfeldt et al, nature,2005,438:685-689, which is expressly incorporated herein by reference in its entirety. Antagomir RNA can be synthesized using standard solid phase oligonucleotide synthesis protocols. See U.S. patent application Ser. Nos. 11/502,158 and 11/657,341 (the disclosures of each of which are incorporated herein by reference).

Antagomir may include ligand conjugated monomer subunits and monomers for oligonucleotide synthesis. Monomers are described in U.S. application Ser. No. 10/916,185, filed 8/10/2004. antagomir may have a ZXY structure, such as described in PCT application No. PCT/US2004/07070 filed on 8 of 3/2004. antagomir may be complexed with an amphiphilic moiety. The amphiphilic moiety for use with oligonucleotide agents is described in PCT application No. PCT/US2004/07070, filed on 8/3/2004.

Aptamer

The therapeutic moiety may be an aptamer. Aptamers are nucleic acid or peptide molecules that bind with high affinity and specificity to a particular molecule of interest (Tuerk and Gold, science 249:505 (1990); ellington and Szostank, nature 346:818 (1990)). DNA or RNA aptamers have been successfully produced that bind to many different entities ranging 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). The aptamer may be RNA or DNA-based and may include a nuclear switch. The nuclear switch is a part of an mRNA molecule that can directly bind to a small target molecule, and its binding to the target affects the activity of the gene. Thus, mRNA containing a nuclear switch is directly involved in regulating its own activity, depending on the presence or absence of its target molecule. Typically, aptamers are engineered to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, by repeating several rounds of in vitro selection or equivalent SELEX (systematic evolution through exponentially enriched ligands). The aptamer may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other aptamers specific for the same target. In addition, the term "aptamer" also includes "secondary aptamers" that contain consensus sequences derived from comparing two or more known aptamers to a given target. The aptamer may be an "intracellular aptamer," or an "intermer," which specifically recognizes an intracellular target. See Famulok et al, chem biol.2001, month 10, 8 (10): 931-939; yoon and Rossi, adv Drug Deliv Rev.2018, month 9, 134:22-35, each of which is incorporated herein by reference.

Ribozyme

The therapeutic moiety may be a ribozyme. Ribozymes are complexes of RNA molecules with specific catalytic domains, which have endonuclease activity (Kim and Cech, proc NATL ACAD SCI U S A.12, 1987; 84 (24): 8788-92; forster and Symons, cell.1987, 24, 4, 1987; 49 (2): 211-20). For example, a large number of ribozymes accelerate the phosphotransesterification reaction with high specificity, typically cleaving only one of several phosphates in an oligonucleotide substrate (Cech et al, cell.1981, 12 months; volume 27, 3Pt 2: pages 487-496; michel and Westhof, J Mol biol.1990, 5 months; volume 216, 3: pages 585-610; reinhold-Hurek and Shub, nature.1992, 5 months, 14 days; 357 (6374): 173-6). This specificity is due to the need for the substrate to bind to the internal guide sequence ("IGS") of the ribozyme via specific base pairing interactions prior to chemical reaction.

At least six basic classes of naturally occurring enzymatic RNAs are currently known. Under physiological conditions, each can trans-catalyze the hydrolysis of RNA phosphodiester bonds (and thus can cleave other RNA molecules), typically, enzymatic nucleic acids act by first binding to the target RNA. Such binding occurs through a target binding moiety of the enzymatic nucleic acid that is held in close proximity to the enzymatic moiety of the molecule used to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds to the target RNA by complementary base pairing, and once bound to the correct site, acts enzymatically to cleave the target RNA. Strategic cleavage of such target RNAs would destroy their ability to direct the synthesis of the encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from the RNA to find another target and reproducibly bind and cleave the new target.

For example, the enzymatic nucleic acid molecule can be formed in a hammerhead, hairpin, delta hepatitis virus, group I intron or RNASEP RNA (binding to RNA guide sequence) or a neurospora VS RNA motif. Specific examples of hammerhead motifs are described by Rossi et al Nucleic Acids Res.1992, 9 months 11 months; 20 (17) 4559-65. Examples of hairpin motifs are described by the following documents: hampel et al (European patent application publication No. EP 0360257); hampel and Tritz, biochemistry 1989, 6, 13; 28 (12) 4929-33; hampel et al, nucleic Acids Res.1990, 1 month 25; 18 299-304 and U.S. Pat. No. 5631359. Examples of hepatitis viral motifs are described by Perrotta and Ben, biochemistry.1992, 12 months 1; 31 (47) 11843-52; an example of an RNaseP motif is described by Guerrier-Takada et al, cell.1983, month 12; 35 (3 Pt 2): 849-57; neurospora VS RNA ribozyme motifs are described by Collins (Saville and Collins, cell.1990, 5, 18; volume 61, 4: pages 685-696; saville and Collins, proc NATL ACAD SCI U S A.1991, 10, 1; volume 88, 19: pages 8826-8830; collins and Olive, biochemistry.1993, 23; 32 (l l): 2795-9); and examples of group I introns are described in U.S. Pat. No. 4,987,071. The enzymatic nucleotide molecules may have specific substrate binding sites complementary to one or more target gene DNA or RNA regions, and they have nucleotide sequences within or around the substrate binding sites that confer RNA cleavage activity to the molecule. Thus, the ribozyme construct need not be limited to the specific motifs mentioned herein.

Methods for producing ribozymes targeting polynucleotide sequences are known in the art. Ribozymes can be designed as described in International patent application publication No. WO 93/23569 and International patent application publication No. WO 94/02595, each specifically incorporated herein by reference, and synthesized as described herein for in vitro and in vivo testing.

The ribozyme activity can be increased by altering the length of the ribozyme binding arm or chemically synthesizing a ribozyme having the following modifications: modifications that prevent their degradation by serum ribonucleases (see, e.g., international patent application publication No. WO 92/07065; international patent application publication No. WO 93/15187; international patent application publication No. WO 91/03162; european patent application publication No. 92110298.4; U.S. patent 5334711; and International patent application publication No. WO 94/13688, which describe various chemical modifications that can be made to the sugar portion of an enzymatic RNA molecule), modifications that enhance their efficacy in cells and that eliminate stem Pi bases to shorten RNA synthesis time and reduce chemical requirements.

Immunostimulatory oligonucleotides

The therapeutic moiety may be an immunostimulatory oligonucleotide. Immunostimulatory oligonucleotides (ISS; single-or double-stranded) are capable of inducing an immune response when administered to a patient (which may be a mammal or other patient). ISS include, for example, certain palindromic structures leading to hairpin secondary structures (see Yamamoto s. Et al (1992) j. Immunol. 148:4072-4076), or CpG motifs, as well as other known ISS features (such as multiple G domains, see WO 96/11266).

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

Immunostimulatory nucleic acids are considered to be non-sequence specific when it is not desirable for them to specifically bind to a target polynucleotide and reduce its expression in order to elicit an immune response. Thus, certain immunostimulatory nucleic acids may include sequences corresponding to regions of a naturally occurring gene or mRNA, but they may still be considered non-sequence specific immunostimulatory nucleic acids.

The immunostimulatory nucleic acid or oligonucleotide may include at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. The immunostimulatory nucleic acid may include at least one CpG dinucleotide having a methylated cytosine. The nucleic acid may comprise a single CpG dinucleotide, wherein the cytosine in the CpG dinucleotide is methylated. The nucleotide may include the sequence 5'TAACGTTGAGGG'CAT 3'. The nucleic acid may comprise at least two CpG dinucleotides, wherein at least one cytosine in the CpG dinucleotide is methylated. Each cytosine in a CpG dinucleotide present in the sequence may be methylated. The nucleic acid may comprise a plurality of CpG dinucleotides, wherein at least one of the CpG dinucleotides comprises a methylated cytosine.

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

Decoy oligonucleotides

The therapeutic moiety may be a decoy oligonucleotide. Because transcription factors recognize their relatively short binding sequences, short oligonucleotides carrying the consensus binding sequence for a particular 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 the intracellular delivery of such "decoy oligonucleotides" which are then recognized and bound by the target factors. The bait occupies the DNA binding site of the transcription factor so that the transcription factor is then unable to bind to the promoter region of the target gene. Decoys may be used as therapeutic agents to inhibit expression of genes activated by transcription factors, or to up-regulate genes inhibited by binding of transcription factors. Examples 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.

Supermir

The therapeutic moiety may be supermir. supermir refers to single-, double-or partially double-stranded oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof, which have substantially the same nucleotide sequence as mirnas and are antisense to their targets, the term including oligonucleotides consisting of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, and which contain at least one functionally similar non-naturally occurring moiety. Such modified or substituted oligonucleotides have desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases. supermir may not include a sense strand. supermir may not self-hybridize to a significant extent. supermir may have a secondary structure, but it is essentially single-stranded under physiological conditions. Substantially single-stranded supermir is single-stranded to the extent that less than about 50% (e.g., less than about 40%, about 30%, about 20%, about 10%, or about 5%) of supermir is duplex with itself. supermir may include hairpin fragments, e.g., sequences, such as duplex regions that can self-hybridize at the 3' end and form duplex regions, e.g., duplex regions of at least about 1, about 2, about 3, or about 4, or less than about 8, about 7, about 6, or about 5 nucleotides. The duplex regions may be joined by a linker, such as a nucleotide linker, e.g., about 3, about 4, about 5, or about 6 dT, e.g., a modified dT. supermir can be duplex with shorter oligonucleotides (e.g., at one or both of the 3 'and 5' ends of supermir or at one end and non-terminal or intermediate) of, for example, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length.

MiRNA mimics

The therapeutic moiety may be a miRNA mimic. miRNA mimics represent a class of molecules useful for mimicking the gene silencing ability of one or more mirnas. Thus, the term "microrna mimetic" refers to a synthetic non-coding RNA that is capable of entering the RNAi pathway and regulating gene expression (i.e., a miRNA that is not obtained by purification from a source of endogenous miRNA). miRNA mimics may be designed as mature molecules (e.g., single-stranded) or as mimetic precursors (e.g., primary or pre-mirnas). miRNA mimics may include nucleic acids (modified or modified nucleic acids), including oligonucleotides, including but not limited to 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). Furthermore, miRNA mimics may include conjugates capable of affecting delivery, intracellular compartmentalization, stability, specificity, functionality, strand use, and/or potency. In one design, the miRNA mimic is a double-stranded molecule (e.g., having a duplex region between about 16 and about 31 nucleotides in length) and contains one or more sequences having identity to the mature strand of a given miRNA. Modifications may include 2' modifications (including 2' -0 methyl modifications and 2' f modifications) and internucleotide modifications (e.g., phosphorothioate modifications) on one or both strands of the molecule that enhance nucleic acid stability and/or specificity. In addition, the miRNA mimic may comprise an overhang. The overhang may comprise about 1 to about 6 nucleotides at the 3 'or 5' end of either strand, and may be modified to enhance stability or functionality. The miRNA mimic may comprise a duplex region of about 16 to about 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2 '-0-methyl modifications of nucleotides 1 and 2 (counted from the 5' end of the sense oligonucleotide), as well as all Cs and Us; antisense strand modifications can include 2' f modifications of all Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with 2 nucleotide 3' overhangs.

MiRNA inhibitors

The therapeutic moiety may be a miRNA inhibitor. The terms "antimir," "microrna inhibitor," "miR inhibitor," or "miRNA inhibitor" are synonymous and refer to an oligonucleotide or modified oligonucleotide that interferes with the ability of a particular miRNA. Typically, the inhibitor is a nucleic acid or modified nucleic acid in nature, including oligonucleotides, including RNA, modified RNA, DNA, modified DNA, locked Nucleic Acid (LNA), or any combination of the above.

Modifications include 2' modifications (including 2' -0 alkyl modifications and 2' f modifications) and internucleotide modifications (e.g., phosphorothioate modifications) that can affect delivery, stability, specificity, intracellular compartmentalization, or potency. Furthermore, miRNA inhibitors may include conjugates capable of affecting delivery, intracellular compartmentalization, stability, and/or potency. Inhibitors can take a variety of configurations, including single-stranded, double-stranded (RNA/RNA or RNA/DNA duplex) and hairpin designs, typically microrna inhibitors include one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (or strands) of the miRNA to be targeted, and further, miRNA inhibitors can include additional sequences located 5 'and 3' of the reverse complement of the mature miRNA. The additional sequence may be the reverse complement of the sequence adjacent to the mature miRNA in the primary miRNA from which the mature miRNA is derived, or the additional sequence may be any sequence (mixture with A, G, C or U). One or both of the additional sequences may be any sequence capable of forming a hairpin. The reverse complement sequence as a miRNA may flank the hairpin structure on the 5 'and 3' sides. When double-stranded, microRNA inhibitors can include mismatches between nucleotides on opposite strands. In addition, microrna inhibitors can be linked to a conjugate moiety to facilitate uptake of the inhibitor into cells. For example, the microrna inhibitor can be linked to cholesterol 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 WO2007/095387 and WO 2008/036825, each of which is incorporated herein by reference in its entirety. One of ordinary skill in the art can select the sequence of the desired miRNA from the database and design inhibitors useful in the methods disclosed herein.

Antisense Compounds (AC)

Therapeutic moieties include Antisense Compounds (ACs) capable of altering one or more aspects of translation or expression of a target gene. The principle behind antisense technology is that antisense compounds that hybridize to a target nucleic acid modulate gene expression activity, such as translation, by one of many antisense mechanisms. Antisense technology is an effective means for altering the expression of one or more specific gene products and thus may prove useful in many therapeutic, diagnostic and research applications.

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

Antisense compound hybridization sites

The antisense mechanism relies on hybridization of an antisense compound to a target nucleic acid.

AC can hybridize to a sequence of about 5 to about 50 nucleic acids in length, which can also be referred to as the length of AC. The length of the AC may be from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, or from about 45 to about 50 nucleic acids. The length of the AC may 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. The length of AC may be about 10 nucleic acids. The length of AC may be about 15 nucleic acids. The length of AC may be about 20 nucleic acids. The length of AC may be about 25 nucleic acids. The length of AC may be about 30 nucleic acids.

AC may be less than about 100% complementary to the target nucleic acid sequence. As used herein, the term "percent complementarity" refers to the number of nucleobases of AC that have nucleobase complementarity to the corresponding nucleobase of an oligomeric compound or nucleic acid divided by the total length of AC (the number of nucleobases). One skilled in the art recognizes that it is possible to include mismatches without eliminating the activity of the antisense compound. The AC may contain up to about 20% nucleotides that disrupt base pairing of the AC with the target nucleic acid. The AC may contain no more than about 15%, no more than about 10%, no more than 5% or no mismatches. The AC can be 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% complementary 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 an oligonucleotide region is calculated by dividing the number of complementary nucleobases in that region by the total number of nucleobase regions.

Incorporation of nucleotide affinity modifications may allow for a greater number of mismatches than unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences. One of ordinary skill in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, such as by determining the melting temperature (Tm). Tm or Δtm may be calculated by techniques familiar to those of ordinary skill in the art. For example, freier et al (Nucleic ACIDS RESEARCH,1997,25, 22:4429-4443) describe techniques that allow one of ordinary skill in the art to evaluate the ability of nucleotide modifications to increase the melting temperature of RNA to DNA duplex.

Antisense mechanism

AC according to the present disclosure may regulate one or more aspects of protein transcription, translation, and expression.

AC can regulate transcription, translation, or protein expression through steric blocking. The following review articles describe the mechanism of spatial blocking and its application, and are incorporated herein by reference in their entirety: roberts et al Nature Reviews Drug Discovery (2020) 19:673-694.

The antisense mechanism works by hybridization of the antisense compound to the target nucleic acid. AC can hybridize to its target sequence and down-regulate the expression of the target protein. AC may hybridize to its target sequence to down-regulate expression of one or more target protein isoforms. AC can hybridize to its target sequence to up-regulate expression of the target protein. AC may hybridize to its target sequence to increase expression of one or more target protein isoforms.

Efficacy of AC can be assessed by evaluating the antisense activity affected by its administration. As used herein, the term "antisense activity" refers to any detectable and/or measurable activity attributable to hybridization of an antisense compound to its target nucleic acid. Such detection and/or measurement may be direct or indirect. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of a target protein. Antisense activity can be assessed by detecting and/or measuring the amount of target nucleic acid.

Antisense compound design

The design of an AC according to the present disclosure will depend on the sequence targeted. Targeting AC to a specific target nucleic acid molecule can be a multi-step process. The process generally begins with the identification of a target nucleic acid whose expression is to be modulated. As used herein, the terms "target nucleic acid" and "nucleic acid encoding a target gene" include DNA encoding a selected target gene, RNA transcribed from such DNA (including pre-mRNA and mRNA), and cDNA derived from such RNA.

Those skilled in the art will be able to design, synthesize and screen antisense compounds of different nucleobase sequences to identify sequences that produce antisense activity. For example, antisense compounds can be designed that inhibit the expression of a target protein. Methods for designing, synthesizing and screening antisense compounds for antisense activity against a preselected target nucleic acid can be found, for example, in "ANTISENSE DRUG TECHNOLOGY, principles, strategies, and Applications," CRC Press, boca Raton, florida, edited by Stanley T.Crooke, which is incorporated by reference in its entirety for all purposes.

Antisense compounds comprising from about 8 to about 30 linked nucleosides are provided. Antisense compounds can comprise modified nucleosides, modified internucleoside linkages, and/or conjugation groups.

The antisense compound may be a "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 loop to limit conformational flexibility of the backbone and enhance the backbone geometry of the torsion angle γ. tc-DNA containing the homobases adenine and thymine forms very stable A-T base pairs with complementary RNA.

Nucleoside

Antisense compounds can comprise linked nucleosides. Some or all of the nucleosides can be modified nucleosides. One or more nucleosides can comprise a modified nucleobase. One or more nucleosides can comprise a modified sugar. Chemically modified nucleosides are typically used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for a 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.

In general, a nucleobase is any group containing one or more atoms or groups of atoms capable of hydrogen bonding with the base of another nucleic acid. In addition to "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (a) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are also suitable for use in the compounds described herein. The terms modified nucleobase and nucleobase mimetic may overlap, but typically modified nucleobases refer to nucleobases that are quite similar in structure to the parent nucleobase, such as, for example, 7-deazapurine, 5-methylcytosine, or G-clamp, whereas nucleobase mimetics will include more complex structures such as, for example, tricyclic phenones oxazine nucleobase mimics. Methods for preparing the modified nucleobases described above are well known to those skilled in the art.

AC may include one or more nucleosides with modified sugar moieties. The furanosyl sugar ring of a natural nucleoside can be modified in a variety of ways including, but not limited to, adding substituents, bridging two non-geminal ring atoms to form a Bicyclic Nucleic Acid (BNA), and substituting the epoxy at the 4' -position 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 antisense compounds for their targets and/or increase nuclease resistance. Representative lists of modified sugars include, but are not limited to, non-bicyclic substituted sugars, particularly non-bicyclic 2 '-substituted sugars having a 2' -F, 2'-OCH3, or 2' -O (CH 2) 2-OCH3 substituent; and 4' -thio modified sugars. The sugar may also be replaced with a sugar mimetic group or the like. Methods for preparing modified sugars are well known to those skilled in the art. Representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. patents ：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;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; WO 2005/121371.

Nucleosides can include bicyclic modified sugars (BNA), including LNA (4 '- (CH 2) -O-2' bridge), 2 '-thio-LNA (4' - (CH 2) -S-2 'bridge), 2' -amino-LNA (4 '- (CH 2) -NR-2' bridge), ENA (4 '- (CH 2) 2-O-2' bridge), 4'- (CH 2) 3-2' bridged BNA, 4'- (CH 2CH (CH 3)) -2' bridged BNA "cEt (4 '- (CH (CH 3) -O-2' bridge) and cMOE BNA (4 '- (CH (CH 2OCH 3) -O-2' bridge). Certain such BNs have been prepared and disclosed in the patent literature (see, 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, fluid et al Chembiochem 2005,6,1104-1109, singh et al chem.Commun.,1998,4,455-456, koshkin et al tetrahedron,1998,54,3607-3630, wahlestedt et al, proc.Natl.Acad.Sci.U.S. A.,2000,97,5633-5638, kumar et al, bioorg.Med.chem.Lett.,1998,8,2219-2222, WO 94/14226, WO 2005/021570, singh et al J.org.chem.,1998,63,10035-10039, WO 2007/090071; examples of published U.S. patents and published applications disclosing BNA include, for example, U.S. Pat. Nos. 7053207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, and 6,525,191, and U.S. pre-grant publication Nos. 2004-0171570, 2004-0219565, 2004-0014959, 2003-0207841, 2004-0143114, and 20030082807.

Also provided herein is a "locked nucleic acid" (LNA) wherein the 2 '-hydroxyl group of the ribosyl sugar ring is attached 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, volume 2: pages 558-561; braasch et al, chem. Biol.,2001, volume 8: pages 1-7; and Orum et al, curr. Opinion mol. Ther.,2001, volume 3: pages 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage may be a methylene (-CH 2-) group bridging the 2 'oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic moiety; in the case where the position is an ethylene group, the term ENA is used ^TM (Singh et al, chem. Commun.,1998,4,455-456; ENA) ^TM : morita et al, bioorganic MEDICINAL CHEMISTRY,2003, volume 11: pages 2211-2226). LNAs and other bicyclic sugar analogs exhibit very high duplex thermal stability (tm= +3 to +10 ℃) to complementary DNA and RNA, stability to 3' -exonucleolytic degradation, and good solubility. Effective and nontoxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al, proc. Natl. Acad. Sci. U.S.A.,2000, vol.97, pages 5633-5638).

The LNA isomer that has also been studied is alpha-L-LNA, which has been shown to have excellent stability to 3' -exonucleases. alpha-L-LNA was incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al, nucleic ACIDS RESEARCH,2003,21,6365-6372).

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

Analogs of LNA, phosphorothioate-LNA and 2' -thio-LNA (Kumar et al, biorg. Med. Chem. Lett.,1998,8,2219-2222) were also prepared. The preparation of locked nucleoside analogues containing oligodeoxyribonucleotide duplex as substrates for nucleic acid polymerase has also been described (Wengel et al, WO 99/14226). Furthermore, the synthesis of a novel conformationally constrained high affinity oligonucleotide analogue, 2' -amino-LNA, has been described in the art (Singh et al, j.org.chem.,1998,63,10035-10039). In addition, 2 '-amino-LNAs and 2' -methylamino-LNAs have been prepared and their thermal stability with duplex of complementary RNA and DNA strands has been previously reported.

Internucleoside linkage

Described herein are internucleoside linking groups that link together nucleosides or otherwise modified monomer units to form antisense compounds. Two main classes of internucleoside linkages are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate and phosphorothioate. Representative phosphorus-free internucleoside linkages include, but are not limited to, methyleneimino (-CH 2-N (CH 3) -O-CH 2-): thiodiester (-O-C (O) -S-), thiocarbamate (-O-C (O) (NH) -S-); siloxane (-O-Si (H) 2-O-); and N, N' -dimethylhydrazine (-CH 2-N (CH 3) -N (CH 3) -). Antisense compounds having non-phosphorus internucleoside linkages are referred to as oligonucleotides. Modified internucleoside linkages can be used to alter (typically increase) nuclease resistance of antisense compounds compared to native phosphodiester linkages. Internucleoside linkages having chiral atoms can be prepared as racemic, chiral or as mixtures. Representative chiral internucleoside linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free linkages are well known to those skilled in the art.

The phosphate group may be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming the oligonucleotide, the phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. Within an oligonucleotide, phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3 'to 5' phosphodiester linkage.

Conjugation group

The cargo may be modified by covalent attachment of one or more conjugate groups. Typically, the conjugate group modifies one or more properties of the cargo, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance. The conjugate groups are conventionally used in the chemical arts and are attached to the parent compound either directly or via an optional linking moiety or linking group. Conjugation groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterols, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. The conjugate group may comprise polyethylene glycol (PEG). PEG may be conjugated to cargo or cCPP. The cargo may comprise a peptide, an oligonucleotide or a small molecule.

The conjugate group may include a lipid moiety, such as a cholesterol moiety (Letsinger et al, proc.Natl. Acad.Sci. USA,1989,86,6553); cholic acid (Manoharan et al, biorg. Med. Chem. Lett.,1994,4,1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al, ann.N. Y. Acad. Sci.,1992,660,306; manoharan et al, biorg. Med. Chem. Let.,1993,3,2765); sulphur cholesterol (Oberhauser et al, nucleic acids res.,1992, volume 20: page 533); aliphatic chains such as 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 hexacosanol or triethylammonium-1, 2-di-O-hexadecyl-racemic glycerin-3-H-phosphonate (Manoharan et al, tetrahedron Lett.,1995,36,3651; shea et al, nucleic acids Res.,1990,18,3777); polyamine or polyethylene glycol chains (Manoharan et al, circulations & circulations, 1995,14,969); adamantaneacetic acid (Manoharan et al, tetrahedron lett.,1995,36,3651); palm-based moieties (Mishra et al, biochim. Biophys. Acta,1995,1264,229); or octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al, j. Pharmacol. Exp. Ter., 1996,277,923).

Linking groups or difunctional linking moieties such as those known in the art are suitable for use in the compounds provided herein. The linking groups can be used to attach chemical functional groups, conjugation groups, reporter groups, and other groups to selective sites of parent compounds (such as, for example, AC). Typically, the difunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to the parent molecule or compound of interest, and the other is selected to bind to essentially any selected group, such as a chemical functional group or a conjugate group. Any of the linkers described herein may be used. The linker may comprise a chain structure or oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups conventionally used in difunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. Difunctional linking moieties may include amino groups, hydroxyl groups, carboxylic acids, thiols, unsaturations (e.g., double or triple bonds), and the like. Some non-limiting examples of difunctional linking moieties include 8-amino-3, 6-dioxaoctanoic Acid (ADO), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), and 6-aminocaproic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C ₂-C₁₀ Alkenyl or substituted or unsubstituted C ₂-C₁₀ A non-limiting list of alkynyl groups wherein the substituents include hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

The length of AC may be about 5 to about 50 nucleotides. The length of AC may be about 5 to about 10 nucleotides. The length of AC may be about 10 to about 15 nucleotides. The length of AC may be about 15 to about 20 nucleotides. The length of AC may be about 20 to about 25 nucleotides. The length of AC may be about 25 to about 30 nucleotides. The length of AC may be about 30 to about 35 nucleotides. The length of AC may be about 35 to about 40 nucleotides. The length of AC may be about 40 to about 45 nucleotides. The length of AC may be about 45 to about 50 nucleotides.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing mechanisms these compounds may include one or more cCPP (or cCPP) conjugated to the 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 a genome. Non-limiting examples of gene editing mechanisms include gRNA, nucleases, nuclease inhibitors, combinations and complexes thereof. The following patent documents describe CRISPR gene editing mechanisms: U.S. Pat. No. 8,697,359, U.S. Pat. No. 8,771,945, U.S. Pat. No. 8,795,965, U.S. Pat. No. 8,865,406, U.S. Pat. No. 8,871,445, U.S. Pat. No. 8,889,356, U.S. Pat. No. 8,895,308, U.S. Pat. No. 8,906,616, U.S. Pat. No. 8,932,814, U.S. Pat. No. 8,945,839, U.S. Pat. No. 8,993,233, U.S. Pat. No. 8,999,641, U.S. patent application Ser. No. 14/704,551, and U.S. patent application Ser. No. 13/842,859. The above patent documents are each incorporated by reference in their entirety.

The linker may conjugate cCPP to the CRISPR gene editing mechanism. Any of the linkers described in this disclosure or known to those skilled in the art may be utilized.

gRNA

The compound may include cCPP conjugated to a gRNA. gRNA targets genomic loci in prokaryotic or eukaryotic cells.

The gRNA may be a single molecule guide RNA (sgRNA). The sgrnas include spacer sequences and scaffold sequences. The spacer sequence is a short nucleic acid sequence for targeting a nuclease (e.g., cas9 nuclease) to a particular nucleotide region of interest (e.g., genomic DNA sequence to be cleaved). The spacer may be about 17-24 base pairs in length, such as about 20 base pairs in length. The length of 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. The length of the spacer can 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. The length of 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. The spacer sequence may have a GC content of between about 40% to about 80%.

The spacer may target a site immediately preceding the 5' Protospacer Adjacent Motif (PAM). PAM sequences may be selected based on the desired nuclease. For example, the PAM sequence may be any one of the PAM sequences shown in the following table, wherein 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 or C or G.

TABLE 8

The spacer may target the sequence of a mammalian gene, such as a human gene. The spacer region may target the mutant gene. The spacer may target the coding sequence.

The scaffold sequence is the sequence within the sgRNA responsible for nuclease (e.g., cas 9) binding. The scaffold sequence does not include a spacer/targeting sequence. In embodiments, the length of the scaffold may 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. The length of the stent 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 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 50, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 63, about about 64, about 65, about 66, about 67, about 68, 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. The length of the scaffold may 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.

The gRNA may be a bimolecular guide RNA, such as crRNA and tracrRNA. The gRNA may also include a poly a tail.

The compound includes cCPP conjugated to a nucleic acid comprising a gRNA. The nucleic acid can 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. gRNA can recognize the same target. gRNA can recognize different targets. The nucleic acid comprising the gRNA comprises a sequence encoding a promoter, wherein the promoter drives expression of the gRNA.

Nuclease (nuclease)

These compounds may include a cyclic cell penetrating peptide conjugated to a nuclease (cCPP). The nuclease may be Sup>A type II, type V-A, type V-B, type VC, type V-U 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 (Cpf 1), cas12B, cas12C, tnp-B like, cas13a (C2), cas13B or Cas14 nuclease. The nuclease may be a Cas9 nuclease or a Cpf1 nuclease.

The nuclease may be a modified form or variant of a Cas9, cas12a (Cpf 1), cas12B, cas12C, tnp-B like, cas13a (C2), cas13B or Cas14 nuclease. The nuclease may be a modified form or variant of TAL nuclease, meganuclease or zinc finger nuclease. A "modified" or "variant" nuclease is, for example, a truncated, fused to another protein (such as another nuclease), catalytically inactivated, or the like nuclease. The nuclease may 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 (Cpf 1), cas12B, cas12C, tnp-B like, cas13a (C2), cas13B, cas14 nuclease or TALEN, meganuclease, or zinc finger nuclease. The nuclease may be a Cas9 nuclease (SpCas 9) derived from streptococcus pyogenes(s). 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 a Cas9 nuclease derived from streptococcus pyogenes (SpCas 9). The nuclease may be Cas9 (SaCas 9) derived from staphylococcus aureus (s.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 (SaCas 9) derived from staphylococcus aureus. Cpf1 may be a Cpf1 enzyme from the genus amino acid coccus (Acidaminococcus) (species BV3L6, uniProt accession number U2 UMQ). 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 with the Cpf1 enzyme from the genus amino acid coccus (species BV3L6, uniProt accession No. U2 UMQ).

Cpf1 may be a Cpf1 enzyme from the family Trichosporoceae (Lachnospiraceae) (species ND2006, uniProt accession A0A182DWE 3). 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 with a Cpf1 enzyme from the family chaetoviridae. The nucleic acid enzyme-encoding sequence may be codon optimized for expression in mammalian cells. The nucleic acid enzyme-encoding sequence may be codon optimized for expression in human cells or mouse cells.

The compound may include cCPP conjugated to a nuclease. The nuclease may be a soluble protein.

The compound may comprise cCPP conjugated to a nucleic acid encoding a nuclease. The nucleic acid encoding a nuclease may comprise a sequence encoding a promoter, wherein the promoter drives expression of the nuclease.

GRNA and nuclease combinations

The compound may include one or more cCPP conjugated to a gRNA and a nuclease. One or more cCPP may be conjugated to a nucleic acid encoding a gRNA and/or a nuclease. Nucleic acids encoding nucleases and grnas may include sequences encoding promoters that drive expression of the nucleases and grnas. The nucleic acid encoding the nuclease and the gRNA may include two promoters, wherein a first promoter controls expression of the nuclease and a second promoter controls expression of the gRNA. The nucleic acid encoding the gRNA and nuclease may encode about 1 to about 20 grnas, or 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, and up to about 20 grnas. gRNA can recognize different targets. gRNA can recognize the same target.

These compounds may include a cyclic cell penetrating peptide (or cCPP) conjugated to a Ribonucleoprotein (RNP) comprising gRNA and nucleases.

A composition may be delivered to a cell, the composition comprising: (a) cCPP conjugated to a gRNA and (b) a nuclease. A composition may be delivered to a cell, the composition comprising: (a) cCPP conjugated to a nuclease and (b) a gRNA.

A composition may be delivered to a cell, the composition comprising: (a) First cCPP conjugated to gRNA and (b) second cCPP conjugated to nuclease. The first cCPP and second cCPP may be identical. The first cCPP and second cCPP may be different.

Genetic element of interest

The compound may include a cyclic cell penetrating peptide conjugated to a genetic element of interest (cCPP). The genetic element of interest may replace genomic DNA sequences that are cleaved by nucleases. Non-limiting examples of genetic elements of interest include genes, single nucleotide polymorphisms, promoters or terminators.

Nuclease inhibitors

The compound can include a cyclic cell penetrating peptide (cCPP) conjugated to a nuclease inhibitor (e.g., a Cas9 inhibitor). The limitation of gene editing is potential off-target editing. Delivery of nuclease inhibitors can limit off-target editing. The nuclease inhibitor may be a polypeptide, polynucleotide or small molecule. Nuclease inhibitors are described in U.S. publication No. 2020/087354, international publication No. 2018/085288, U.S. publication No. 2018/0382741, international publication No. 2019/089761, international publication No. 2020/068304, international publication No. 2020/04384, and international publication No. 2019/076651, each of which is incorporated herein by reference in its entirety.

Therapeutic polypeptides

Antibodies to

The therapeutic moiety may comprise an antibody or antigen binding fragment. Antibodies and antigen binding fragments may be derived from any suitable source, including humans, mice, camelids (e.g., camels, alpacas, llamas), rats, ungulates, or non-human primates (e.g., monkeys, rhesus monkeys).

The term "antibody" refers to an immunoglobulin (Ig) molecule capable of binding to a particular target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term includes whole polyclonal or monoclonal antibodies and antigen binding fragments thereof. Natural immunoglobulin molecules typically comprise two heavy chain polypeptides and two light chain polypeptides. Each of the heavy chain polypeptides associates with the light chain polypeptide by means of an interchain disulfide bond between the heavy chain polypeptide and the light chain polypeptide to form two heterodimeric proteins or polypeptides (i.e., proteins consisting of two heterologous polypeptide chains). These two heterodimeric proteins are then associated by means of additional interchain disulfide bonds between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.

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 and/or light chain that binds at least one epitope of an antigen of interest. An antigen binding fragment may comprise 1,2 or 3 CDRs from a variable heavy chain (VH) sequence of an antibody that specifically binds a target molecule. An antigen binding fragment may comprise 1,2, or 3 CDRs from a variable light chain (VL) sequence of an antibody that specifically binds a target molecule. An antigen binding fragment may comprise 1,2,3, 4,5, or all 6 CDRs from the Variable Heavy (VH) and Variable Light (VL) sequences of an antibody that specifically binds a target molecule. Antigen binding fragments include proteins comprising a portion of a full length antibody (typically an antigen binding or variable region thereof), such as 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 comprise an antigen binding site or fragment of a desired specificity.

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

The term "F (ab') 2" refers to a protein fragment of IgG produced by proteolytic cleavage of pepsin. Each F (ab ') 2 fragment may comprise two F (ab') fragments and thus may be a bivalent antigen-binding fragment.

"Fv fragment" refers to a non-covalent VH:VL heterodimer that includes an antigen-binding site that retains most of the antigen recognition and binding capacity of the 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) Biochem19:4091-4096.

Bispecific antibodies (bsabs) are antibodies capable of binding two separate and distinct antigens (or different epitopes of the same antigen) simultaneously. The therapeutic moiety may comprise a bispecific antibody capable of binding two different targets of interest simultaneously. BsAb can redirect cytotoxic immune effector cells to enhance killing of tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and other cytotoxic mechanisms mediated by effector cells.

Recombinant antibody engineering has allowed the production of recombinant bispecific antibody fragments comprising Variable Heavy (VH) and 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-locked trivalent Fab), sdAb (single domain antibody), and BssdAb (bispecific single domain antibody).

Bsabs having Fc regions can be used to perform Fc-mediated effector functions such as ADCC and CDC. They have the half-life of normal IgG. On the other hand, bsabs (bispecific fragments) without Fc regions rely solely on their antigen binding capacity to achieve therapeutic activity. Because of their smaller size, these fragments have better penetration of solid tumors. BsAb fragments do not require glycosylation and they can be produced in bacterial cells. The BsAb size, valence, flexibility and half-life are suitable for use.

Using recombinant DNA technology, bispecific IgG antibodies can be assembled from two different heavy and light chains that are expressed in the same cell line. Random assembly of the different chains results in the formation of nonfunctional molecules and undesired HC homodimers. To address this problem, 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, resulting in a tetravalent BsAb that contains two binding sites for each antigen. Other approaches to address LC-HC mismatch and HC homodimerization are as follows.

Pestle-mortar BsAb IgG. The H chain heterodimerization is forced by introducing different mutations into the two CH3 domains, thereby generating an asymmetric antibody. Specifically, a "pestle" mutation is generated in one HC and a "mortar" mutation is generated in the other HC to promote heterodimerization.

Ig-scFv fusion. The addition of a new antigen binding moiety directly to full length IgG results in a fusion protein with tetravalent properties. Examples include IgG C-terminal scFv fusions and IgG N-terminal scFv fusions.

Diabody-Fc fusion. This involves replacing the Fab fragment of IgG with a bispecific diabody (a derivative of scFv).

Double variable domain IgG (DVD-IgG). VL and VH domains of IgG with one specificity are fused via linker sequences to the N-terminus of VL and VH of IgG with a different specificity, respectively, to form DVD-IgG.

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 two domains on the same chain, forcing the domains to pair with complementary domains of the other 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 designed to bind two different antigens and are bispecific antigen binding constructs.

The term "nanobody" or "single domain antibody" refers to an antigen-binding fragment of a single monomeric variable antibody domain comprising one variable domain (VH) of a heavy chain antibody. They have several advantages over traditional monoclonal antibodies (mAbs), including 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. Roll 57: page 2314 ;Siontorou,(2013)Nanobodies as novel agents for disease diagnosis and therapy.International Journal of Nanomedicine,8,4215-27). These features allow nanobodies to be modified for genetic and chemical modification (Schumacher et al ,(2018)Nanobodies:Chemical Functionalization Strategies and Intracellular Applications.Angew.Chem.Int.Ed.57,2314), Thereby facilitating their use as research tools and therapeutics (Bannas et al ,(2017)Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.Frontiers in Immunology,8,1603). nanobodies have been used for protein immobilization in the last decade (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)、 Detection of protein-protein interactions (Herce et al ,(2013)Visualization and targeted disruption of protein interactions in living cells.Nat.Commun,4,2660;MassaEt al ,(2014)Site-Specific Labeling of Cysteine-Tagged Camelid Single-Domain Antibody-Fragments for Use in Molecular Imaging.Bioconjugate Chem,25,979-988)、 as macromolecular inhibitors (Truttmann et al ,(2015)HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation.J.Biol.Chem.290,9087–9100).

The therapeutic moiety may be an antigen binding fragment that binds to a target of interest. An antigen-binding fragment that binds to a target of interest may include 1,2, or 3 CDRs from a variable heavy chain (VH) sequence of an antibody that specifically binds to the target of interest. An antigen binding fragment that binds to a target of interest may include 1,2, or 3 CDRs from a variable light chain (VL) sequence of an antibody that specifically binds to the target of interest. An antigen binding fragment that binds to a target of interest may include 1,2, 3, 4,5, or all 6 CDRs from a variable heavy chain (VH) and/or variable light chain (VL) sequence of an antibody that specifically binds to the target of interest. The antigen binding fragment that binds to the target may be part 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 configuration of an immunoglobulin molecule that comprises an antigen binding site or a desired specific fragment.

The therapeutic moiety may comprise a bispecific antibody. Bispecific antibodies (bsabs) are antibodies capable of binding two separate and distinct antigens (or different epitopes of the same antigen) simultaneously.

The therapeutic moiety may comprise a "diabody".

The therapeutic moiety may comprise a nanobody or a single domain antibody (which may also be referred to herein as an sdAb or VHH).

The therapeutic moiety may include a "minibody". Minibodies (Mb) include CH3 domains fused or linked to antigen binding fragments (e.g., CH3 domains fused or linked to scFv, domain antibodies, etc.). The term "Mb" may represent a CH3 single domain. The CH3 domain may represent a minibody. (S.Hu et al, cancer Res.,56,3055-3061,1996). See, e.g., 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); PCT/US92/09965; WO94/13804; holliger et al, proc.Natl. Acad. Sci. USA 90 6444-6448,1993; reiter et al, nature biotech 14,1239-1245,1996; hu et al, cancer Res.,56,3055-3061,1996.

The therapeutic moiety may include a "monomer". The term "monomer" refers to a synthetic binding protein constructed using fibronectin type III domain (FN 3) as a molecular scaffold.

The therapeutic moiety may be an antibody mimetic. An antibody mimetic is a compound that, like an antibody, can specifically bind to an antigen but is not structurally related to an antibody. They are generally artificial peptides or proteins having a molecular weight of about 3kD to 20kD (compared to antibodies having a molecular weight of about 150 kDa). Examples of antibody mimics include affibody molecules (constructed on scaffolds of the domain of protein a, see Nygren (month 6 2008). FEBS j.275 (11): 2668-76), affiln (constructed on scaffolds of gamma-B crystals or ubiquitin, see Ebersbach H et al (month 9 of 2007) J.mol.biol.372 (1): 172-85), affimer (constructed on cystatin scaffolds, see Johnson a et al, (8/7/2012.) anal. Chem.84 (15): 6553-60), affitin (constructed on Sac7d from the support of the species sulfolobus acidocaldarius (S.acidocaldarius), see Krehenbrink M et al, (2008, 11), J.mol. Biol.383 (5): 1058-68), alpha (constructed on the support of the triple helix coiled coil, see Desmet, J et al, (2014, 2, 5) Nature communications.5:5237), ANTICALINS (constructed on the support of the lipocalin, see Skerra A (2008, 6), FEBS J.275 (11): 2677-83), affinity multimers (Avimer) (constructed on the support of various membrane receptors, see Nat.Biotechnol.23 (12): 1556-61), pin (constructed on the support of the ankyrin repeat, see Stpp et al, (2008, 8), drug dispersion.695-6916), FIG. 35 (35, 35-35), and so on the support of the membrane receptor (35, 35-35) Kunitz domain peptides (constructed on scaffolds of kunitz domains of various protease inhibitors, see nixon, (3. 2006) Curr Opin Drug Discov dev.9 (2): 261-8) and monoclonal antibodies (constructed on scaffolds of type III domains of fibronectin, see koide et al (2007) methods mol. Biol. 352:95-109).

The therapeutic moiety may include a "engineered ankyrin repeat" or "DARPin". DARPin is derived from a natural ankyrin, which consists of at least three repeat motif proteins, typically consisting of four or five repeats.

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

The therapeutic moiety may comprise a F (ab) fragment.

The therapeutic moiety may comprise a F (ab') 2 fragment.

The therapeutic moiety may comprise an Fv fragment.

An antigen binding fragment may comprise a "single chain variable fragment" or "scFv". scFv refers to a fusion protein of the heavy (VH) and light (VL) variable regions of an immunoglobulin linked to a short linker peptide of ten to about 25 amino acids. Huston et al (1988), proc.Nat.Acad.Sci.USA 85 (16): 5879-5883. The linker may connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. Many methods have been described to identify the chemical structure used to convert naturally aggregated but chemically separated light and heavy chain polypeptide chains from the antibody V region into scFv molecules that will fold into a three-dimensional structure substantially similar to the structure of the antigen binding site. See, for example, U.S. Pat. nos. 5,091,513 and 5,132,405 to humin et al; U.S. Pat. No. 4,946,778 to Ladner et al.

The antigen binding construct may comprise two or more antigen binding portions. The antigen binding construct may bind two separate and distinct antigens or different epitopes of the same antigen. Pestle-mortar BsAb IgG. The H chain heterodimerization is forced by introducing different mutations into the two CH3 domains, thereby generating an asymmetric antibody. Specifically, a "pestle" mutation is generated in one HC and a "mortar" mutation is generated in the other HC to promote heterodimerization.

Peptide inhibitors

The therapeutic moiety may comprise a peptide. The peptides act as agonists, thereby increasing the activity of the target protein. The peptides act as antagonists, thereby reducing the activity of the target protein. The peptide may be configured to inhibit protein-protein interactions (PPI). Protein-protein interactions (PPI) are important in many biochemical processes, including transcription of nucleotides and various post-translational modifications of the translated protein. PPIs can be experimentally determined by biophysical techniques such as X-ray crystallography, NMR spectroscopy, surface Plasmon Resonance (SPR), biological Layer Interferometry (BLI), isothermal Titration Calorimetry (ITC), radioligand binding, spectrophotometry, and fluorescence spectroscopy. Peptides that inhibit protein-protein interactions may be referred to as peptide inhibitors.

The therapeutic moiety may include a peptide inhibitor. The peptide inhibitor may comprise from about 5 to about 100 amino acids, from about 5 to about 50 amino acids; about 15 to about 30 amino acids; or about 20 to about 40 amino acids. Peptide inhibitors may comprise one or more chemical modifications, for example to reduce proteolytic degradation and/or improve in vivo half-life. Peptide inhibitors may comprise one or more synthetic amino acids and/or backbone modifications. Peptide inhibitors may have an alpha-helical structure.

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

Small molecules

The therapeutic moiety may comprise a small molecule. The therapeutic moiety may include a small molecule kinase inhibitor. The therapeutic moiety may include a small molecule that inhibits a kinase that phosphorylates a target of interest. Inhibition of phosphorylation of a target of interest may block nuclear translocation of the target of interest. The therapeutic moiety may comprise a small molecule inhibitor of MyD 88.

Composition and method for producing the same

Compositions comprising the compounds described herein are provided.

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 according to the particular substituents found on the compound. It may be appropriate to administer the compounds as salts under conditions wherein the compounds disclosed herein are sufficiently basic or acidic to form stable, non-toxic acid or base 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, alpha-ketoglutaric acid, alpha-sugar phosphoric acid, maleic acid, toluenesulfonic acid, methanesulfonic acid, and the like. Thus, disclosed herein are hydrochlorides, nitrates, phosphates, carbonates, bicarbonates, sulfates, acetates, propionates, benzoates, succinates, fumarates, mandelates, oxalates, citrates, tartrates, malonates, ascorbates, alpha-ketoglutarates, alpha-sugar phosphates, maleates, tosylates and methanesulfonates. Pharmaceutically acceptable salts of the compounds may be obtained using standard methods well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid that provides a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids may also be prepared.

Mechanism of oligonucleotide therapeutics and target molecules

Many types of oligonucleotides are capable of modulating gene transcription, translation and/or protein function in a cell. Non-limiting examples of such oligonucleotides include, for example, small interfering rnas (siRNA), micrornas (miRNA), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense, antagomir, antimir, microrna mimics, supermir, ul adaptors, and aptamers. Additional examples include DNA-targeted triplex-forming oligonucleotides, strand invasion oligonucleotides, and synthetic guide strands of CRISPR/Cas, which function via a variety of mechanisms. See Smith and Zain, annu Rev Pharmacol Toxicol.2019,59:605-630, incorporated herein by reference.

Splice switching antisense oligonucleotides are short, synthetic, antisense, modified nucleic acids that base pair with pre-mRNA and disrupt the normal splice pool of transcripts by blocking RNA-RNA base pairing or protein-RNA binding interactions between components of the splice machinery and the pre-mRNA. Splicing of pre-mRNAs is necessary for proper expression of most protein-encoding genes, and thus, targeting this process provides a means of manipulating protein production from genes. Splice modulation is particularly valuable in the case of diseases caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of gene transcripts may be therapeutic. Such antisense oligonucleotides provide an effective and specific way to target and alter splicing in a therapeutic manner. See Havens and Hastings, nucleic Acids res.2016, 8, 19; 44 (14) 6549-6563, incorporated herein by reference.

In the case of siRNA or miRNA, these nucleic acids can down-regulate intracellular levels of a particular protein through a process known as RNA interference (RNAi). After introduction of siRNA or miRNA into the cytoplasm, these double stranded RNA constructs can bind to proteins known as RISC. The sense strand of the siRNA or miRNA is displaced from the RISC complex, thereby providing a template within the RISC that recognizes and binds mRNA having a sequence complementary to the sequence of the bound siRNA or miRNA. Upon binding to complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strand. RNAi can provide down-regulation of a particular protein by targeting the corresponding mRNA that specifically disrupts the synthesis of the encoded protein.

RNAi has a very wide range of therapeutic applications, as siRNA and miRNA constructs can be synthesized using any nucleotide sequence directed against a target protein. To date, siRNA constructs have been shown to specifically down-regulate target proteins in vitro and in vivo models as well as in clinical studies.

Antisense oligonucleotides and ribozymes also inhibit translation of mRNA into protein. In the case of antisense constructs, these single stranded deoxynucleic acids have a sequence complementary to the target protein mRNA and can bind to the mRNA by watson-crick base pairing. This binding prevents translation of the target mRNA and/or triggers rnase H degradation of the mRNA transcript, thus antisense oligonucleotides have great potential for specificity of action (i.e., down-regulation of a particular disease-related protein). To date, these compounds have shown promise in several in vitro and in vivo models, including 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 to chromosomal DNA.

Immunostimulatory nucleic acids include deoxyribonucleic acid and ribonucleic acid. In the case of deoxyribonucleic acids, certain 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 acid are believed to be specifically recognized by the endosomal receptor toll-like receptor 9 (TLR-9), which then triggers the innate and acquired immunostimulatory pathways. Certain immunostimulatory ribonucleic acid sequences have also been reported. These RNA sequences are believed to trigger immune activation by binding to toll-like receptors 6 and 7 (TLR-6 and TLR-7). In addition, double stranded RNA is reported to be immunostimulatory as well and is believed to be activated by binding to TLR-3.

Non-limiting examples of mechanisms and targets by which antisense oligonucleotides (ASOs) modulate gene transcription, translation, and/or protein function are shown in tables 9A and 9B.

TABLE 9 mechanism of ASO Regulation and target molecules

TABLE 9 mechanism of ASO Regulation and target molecules

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and related Cas proteins constitute a CRISPR-Cas system. CRISPR-Cas is a gene editing mechanism. RNA-guided (e.g., gRNA) Cas9 endonucleases specifically target and cleave DNA in a sequence-dependent manner. The Cas9 endonuclease may be substituted with any nuclease of the present disclosure. The gRNA targets a nuclease (e.g., cas9 nuclease) to a particular nucleotide region of interest (e.g., genomic DNA sequence to be cleaved) and cleaves genomic DNA. Genomic DNA can then be replaced with the genetic element of interest.

Method for regulating tissue distribution and/or retention

Provided herein are compounds and methods for modulating tissue distribution and/or retention of a therapeutic agent in a subject. Tissue distribution involves, for example: the concentration of the therapeutic agent in a particular region of tissue is increased relative to the unconjugated therapeutic agent, e.g., the concentration of the therapeutic agent in a brain region such as the cerebellum, cortex, hippocampus, or olfactory bulb is increased. Compounds that modulate the tissue distribution of therapeutic agents may include cyclic cell penetrating peptides (cCCP) and Exocyclic Peptides (EP). Methods for modulating tissue distribution may 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 the body. The tissue may be a different tissue of the same biological system, such as a different type of muscle tissue or a different tissue within the central nervous system. The tissue may be muscle tissue and there is modulation of the distribution or retention of the compound in the myocardial tissue as 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 may be CNS tissue and there is modulation of the distribution or retention of the compound in at least one CNS tissue compared to at least one other type of CNS tissue.

Any of the EPs described herein is suitable for inclusion in a compound for use in the method. The EP may be PKKKRKV. EP may 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 can be selected from KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、KKKKK、KKKRK、KBKBK、KKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV And PKKKRKG.

EP's may comprise PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry. The EP may be PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR or HBRBH, where B is β -alanine. The amino acids in EP may have D or L stereochemistry.

An EP may comprise an amino acid sequence identified in the art as a Nuclear Localization Sequence (NLS). An 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. EP may consist of NLS comprising the amino acid sequence PKKKRKV. The EP may comprise a polypeptide selected from NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK And RKCLQAGMNLEARKTKK amino acid sequence NLS. EP may be prepared from a composition selected from NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK amino acid sequence.

The amount, expression, function or activity of the compound in at least one tissue may be increased as compared to a second tissue 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%.

The amount, expression, function or activity of the compound in at least one tissue may be reduced as compared to a second tissue 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%.

The amount or expression of a compound can be assessed in different tissue types by methods known in the art, including but not limited to the methods described in the examples. The tissue may be prepared by standard methods. The amount or expression of a compound in different tissues can be measured by art-recognized techniques, such as by LC-MS/MS, western blot analysis, or ELISA. The function or activity of a compound in different tissues can be measured by techniques established for assessing the relevant function or activity, such as using RT-PCR to assess the activity of oligonucleotide-based therapeutic moieties. For example, for Antisense Compounds (ACs) used as Therapeutic Moieties (TM) to induce exon skipping in target mRNA of interest, RT-PCR can be used to quantify the level of exon skipping in different tissues.

Compounds comprising a cyclic cell penetrating peptide (cCPP) and an Exocyclic Peptide (EP) may be used to modulate tissue distribution and/or retention of a therapeutic agent in a Central Nervous System (CNS) tissue. The compound may be administered intrathecally to the subject, and the compound may modulate tissue distribution and/or retention of the therapeutic agent in a Central Nervous System (CNS) tissue. Non-limiting examples of CNS tissues include cerebellum, cortex, hippocampus, olfactory bulb, spinal cord, dorsal Root Ganglion (DRG), and cerebrospinal fluid (CSF). The compound comprising cCPP and EP may be administered intrathecally and the expression level, activity or function of the therapeutic agent may be higher in at least one CNS tissue compared to another CNS tissue. The compound comprising cCPP and EP may be administered intrathecally and the expression level, activity or function of the therapeutic agent may be lower in at least one CNS tissue compared to another CNS tissue. The therapeutic agent may include a CD 33-targeting therapeutic agent (e.g., a CD 33-targeting antisense compound), wherein the compound is administered intrathecally. The compound comprising cCPP and EP may be administered intrathecally at a dose of at least 1mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg or 50 mg/kg.

Methods of modulating tissue distribution or retention of a therapeutic agent in the Central Nervous System (CNS) of a subject can comprise: administering intrathecally to the subject a compound comprising:

(a) A cyclic cell penetrating peptide (cCPP);

(b) A Therapeutic Moiety (TM) comprising a 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 a therapeutic agent is modulated in at least one tissue of the CNS of a subject by at least 10% as compared to a second tissue of the CNS of the subject.

The amount, expression, function or activity of the therapeutic agent in at least one tissue of the CNS of the subject can be modulated at least as compared to a second tissue of the CNS of the subject 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%.

Any of the therapeutic agents described herein for CNS-related diseases or disorders are suitable for inclusion in the compounds used in the method. The therapeutic agent may include a CD 33-targeting therapeutic agent, such as any of the CD 33-targeting antisense compounds described herein.

Any of the CPPs described herein are suitable for inclusion in a compound for use in the method. The CPP may be a cyclic CPP (cCPP).

The compounds are useful for treating a subject suffering from a central nervous system disease or disorder, or a neuroinflammatory disease or disorder. In embodiments, the subject has alzheimer's disease or parkinson's disease.

Tissue distribution and/or retention of the therapeutic agent in different types of muscle tissue may be modulated. Non-limiting examples of muscle tissue include diaphragm, heart (heart) muscle, tibialis anterior, triceps, other skeletal muscles, and smooth muscles. The compound comprising cpp, EP and the therapeutic agent may be administered and the expression level, activity or function of the therapeutic agent may be higher in at least one muscle tissue compared to another muscle tissue. The compound comprising cpp, EP and the therapeutic agent may be administered and the expression level, activity or function of the therapeutic agent may be lower in at least one muscle tissue compared to another muscle tissue. The therapeutic agent may be a therapeutic agent that targets dystrophin (e.g., an antisense compound that targets DMD). The compound may be administered at a dose of at least 1mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg or 50 mg/kg.

A method of modulating tissue distribution or retention of a therapeutic agent in the muscular system of a subject comprising: administering to a subject a compound comprising:

(a) A cyclic cell penetrating peptide (cCPP);

(b) A Therapeutic Moiety (TM) comprising a therapeutic agent; and

(C) A cyclic Exopeptide (EP) comprising at least one positively charged amino acid residue, wherein the amount, expression, function or activity of a therapeutic agent is modulated in at least one tissue of the subject's musculature by at least 10% compared to a second tissue of the subject's musculature.

The amount, expression, function or activity of the therapeutic agent in at least one tissue of the subject's musculature may be modulated at least as compared to a second tissue of the subject's musculature 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%.

Any of the therapeutic agents described herein for a muscle system-related disease or disorder are suitable for inclusion in the compounds used in the method. The therapeutic agent may be a DMD-targeting therapeutic agent, such as a DMD-targeting antisense compound.

Any of the CPPs described herein are suitable for inclusion in a compound for use in the method. In embodiments, the CPP is a cyclic CPP (CPP).

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

Diseases associated with aberrant splicing and exemplary target genes

The human genome comprises over 40,000 genes, approximately half of which correspond to protein-encoding genes. However, the number of human protein species is expected to be several orders of magnitude higher due to single amino acid polymorphisms, post-translational modifications and important alternative splicing. RNA splicing, which typically occurs in the nucleus, is the process of converting precursor messenger RNA (pre-mRNA) into mature messenger RNA (mRNA) by removing non-coding regions (introns) and ligating the remaining coding regions (exons) together. The resulting mRNA can then be exported from the nucleus and translated into protein. Alternative splicing or differential splicing is a regulatory process during gene expression that produces a single gene encoding multiple proteins. In this process, specific exons of a gene may be included in or excluded from the final, processed mRNA produced by the gene. Although alternative splicing is a normal phenomenon in eukaryotes and contributes to the biodiversity of proteins encoded by the genome, abnormal variations in splicing are severely involved in disease. Most human genetic diseases are caused by splice variants; aberrant splice variants contribute to the development of cancer; and splicing factor genes are often mutated in different types of cancer.

About 10% of the about 80,000 mutations reported in the Human Gene Mutation Database (HGMD) affect splice sites. In HGMD, 3390 pathogenic mutations occurred at the +1 donor splice site. These mutations affected 2754 exons in 901 genes. The prevalence of neuromuscular disorders (NMD) is even higher due to the abnormally large size and multiple exon structure of the genes encoding the muscle structural proteins, further highlighting the importance of these mutations in NMD.

Previously, attempts to correct point mutations, e.g., splice site mutations, via the Homology Directed Repair (HDR) pathway have been extremely inefficient in postmitotic tissues such as skeletal muscle, preventing its therapeutic utility in NMD. Furthermore, standard gene therapy methods for reintroducing corrected coding regions into the genome are hampered by the large size of the genes encoding, for example, muscle structural proteins. Furthermore, many existing therapies rely on the inefficient introduction of therapeutic compounds into disease cells, making in vivo treatment impractical and experiencing higher toxicity.

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

The target gene may be a gene comprising a mutation that results in aberrant splicing. The target gene may be a gene comprising one or more mutations. The target gene may be a gene comprising one or more mutations such that transcription and translation of the target gene does not produce a functional protein. The target gene may be a gene comprising one or more mutations such that transcription and translation of the target gene results in a target protein that is less active or less functional than the wild-type target protein.

The target gene may be a potential gene for 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 neuromuscular disorder (NMD). The target gene may be a gene involved in the pathogenesis of musculoskeletal disorder (NMD). The neuromuscular disease may be pompe disease and the target gene may be GYS1.

Antisense compounds can be used to target genes that contain mutations that lead to potentially aberrant splicing by genetic diseases in order to redirect splicing to produce the desired splice product (Kole, acta Biochimica Polonica,1997,44,231-238).

CRISPR gene editing mechanisms can be used to target abnormal genes to remove or regulate gene transcription and translation.

The disease may include beta-thalassemia (Dominski and Kole, proc. Natl. Acad. Sci. USA,1993, volume 90: pages 8673-8677; sierakowska et al, nucleoside & Nucleosides, 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).

The disease may include Kobe's dystrophin disease (TAKESHIMA et al, J.Clin.invest.,1995,95,515-520).

The disease may include Duchenne muscular dystrophies (Dunckley et al Nucleoside & Nucleosides, 1997,16,1665-1668; dunckley et al Human mol. Genetics,1998,5,1083-90). The target gene may be the DMD gene encoding dystrophin. The protein consists of an N-terminal domain that binds to actin microfilaments, a central rod domain and a C-terminal cysteine-rich domain that binds to dystrophin-glycoprotein complexes (Hoffman et al 1987; koenig et al 1988; yoshida and Ozawa 1990). Mutations in the DMD gene that interrupt the reading frame lead to complete loss of dystrophin function, which leads to severe Duchenne Muscular Dystrophy (DMD) [ MIM 310200 ]). On the other hand, milder Becker muscular dystrophy (BMD [ MIM 300376 ]) is the result of non-frameshift processes in the same gene and results in internal deletions but partial functional dystrophin retaining its N-and C-termini (Koenig et al 1989; di Blasi et al 1996). More than two-thirds of DMD and BMD patients have a deletion of >1 exon (den Dun-nen et al 1989). Notably, patients exhibiting very mild BMD and lacking 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, despite the large deletion, a portion of the functional dystrophin may be produced, provided that the deletion brings the transcript in-frame. These observations led to the idea of using AC to alter splicing, restoring the open reading frame and converting the severe DMD phenotype to a milder BMD phenotype. Several studies have shown therapeutic AC-induced single exon skipping in cells derived from mdx mouse models (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 may be used to skip one or more exons selected from exons 2, 8, 11, 17, 19, 23, 29, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 55, and 59 of DMD. See Arartsma-Rus et al 2002, incorporated herein by reference. AC may be used to skip one or more exons selected from DMD exons 8, 11, 43, 44, 45, 50, 51, 53 and 55. In all patients with DMD, about 75% would benefit from skipping of these exons. Skipping the exons flanking the out-of-frame deletion or the out-of-frame exons containing nonsense mutations can restore the reading frame and induce synthesis of BMD-like dystrophin in the treated cells. (van Deutekom et al 2001; aartsma-Rus et al 2003). AC hybridized to its target sequence within the target DMD pre-mRNA may induce skipping of one or more exons. AC may induce expression of a re-spliced target protein comprising an active fragment of dystrophin. A non-limiting example of an AC for exon 52 is described in U.S. publication No. 2019/0365918, which is incorporated by reference in its entirety for all purposes. The compounds may comprise EP, cpp and cargo targeting the DMD gene.

Cyclic cell penetrating peptides conjugated to cargo moieties (cCPP)

The cyclic cell penetrating peptide (cCPP) may be conjugated to a cargo moiety.

The cargo moiety may be conjugated to cCPP via a linker. The cargo portion comprises a therapeutic portion. The therapeutic moiety may comprise an oligonucleotide, a peptide or a small molecule. The oligonucleotide may comprise an antisense oligonucleotide. The cargo moiety may be conjugated to a linker at the terminal carbonyl group to provide the following structure:

Wherein:

EP is a cyclic exopeptide, and M, AA _SC Goods, x ', y and z' are as defined above, are with AA _SC Is provided. x' may be 1.y may be 4.z' may be 11.- (OCH) ₂CH-₂)_x' -and/or- (OCH) ₂CH-₂)_z' independently substituted with one or more amino acids including, for example, glycine, β -alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or combinations thereof.

An Endosomal Escape Vector (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an Exocyclic Peptide (EP), and a linker, and may be conjugated to cargo to form an EEV-conjugate comprising a structure of formula (C):

Or a protonated form thereof, wherein:

R ₁、R₂And R is ₃ Amino acid residues which may each independently be H or have a side chain comprising an aromatic group;

R ₄ is H or an amino acid side chain;

EP is an exocyclic peptide as defined herein;

Cargo is part 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; and

Z' is an integer from 2 to 20.

R ₁、R₂、R₃,R₄ EP, cargo, m, n, x ', y, q and z' are as defined herein.

EEV may be conjugated to cargo, and EEV-conjugates may comprise a structure of formula (C-a) or (C-b):

Or a protonated form thereof, wherein EP, m and z are as defined above in formula (C).

EEV may be conjugated to cargo, and EEV-conjugates may comprise a structure of formula (C-C):

Or a protonated form thereof, wherein EP, R ¹、R²、R³、R⁴ And m is as defined above in formula (III); AA may be an amino acid as defined herein; n may be an integer from 0 to 2; x may be an integer from 1 to 10; y may be an integer from 1 to 5; z may be an integer from 1 to 10.

EEV may be conjugated to an oligonucleotide cargo, and EEV-oligonucleotide conjugates may comprise a structure of formula (C-1), (C-2), (C-3), or (C-4):

/>

/>

EEV can be conjugated to an oligonucleotide cargo, and EEV-conjugates can comprise the following structure:

Cytoplasmic delivery efficiency

Modification of the cyclic cell penetrating peptide (cCPP) may increase cytoplasmic delivery efficiency. By comparing the cytoplasmic delivery efficiency of cCPP with the modified sequence to a control sequence, improved cytoplasmic uptake efficiency can be measured. Regulatory sequences do not include specific replacement amino acid residues in the modified sequence (including, but not limited to, arginine, phenylalanine, and/or glycine), but are otherwise identical.

As used herein, cytoplasmic delivery efficiency refers to the ability of cCPP to cross the cell membrane and enter the cytosol of the cell. cCPP are not necessarily dependent on the receptor or cell type. Cytoplasmic delivery efficiency may refer to absolute cytoplasmic delivery efficiency or relative cytoplasmic delivery efficiency.

Absolute cytosolic 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 cytosol delivery efficiency refers to the concentration of cCPP in the cytosol compared to the concentration of control cCPP in the cytosol. Quantification may be achieved by fluorescent labeling cCPP (e.g., with FITC dye) and measuring the fluorescence intensity using techniques well known in the art.

The relative cytoplasmic delivery efficiency is determined by comparing the amount of the invention cCPP that is internalized by a cell type (e.g., a HeLa cell) to the amount of the control cCPP that is internalized by the same cell type. To measure relative cytoplasmic delivery efficiency, the cell types can be incubated in the presence of cCPP for a specified period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), after which the amount of cCPP internalized by the cell can be quantified using methods known in the art, such as fluorescence microscopy. Separately, the same concentration of control cCPP was incubated in the presence of this cell type for the same period of time and the amount of control cCPP internalized by the cells was quantified.

Relative cytoplasmic delivery efficiency can be measured by measuring the IC of cCPP with modified sequence to intracellular targets ₅₀ And comparing cCPP with the modified sequence to the control sequence ₅₀ a comparison is made (as described herein) to determine.

The relative cytoplasmic transfer efficiency of cCPP can be in the range of about 50% to about 450%, such as 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 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, or about 590%, including all ranges and values therebetween, as compared to ring (Ff RrRrQ). The relative cytoplasmic delivery efficiency of cCPP can be improved by greater than about 600% as compared to a cyclic peptide comprising a ring (Ff Φ RrRrQ).

The absolute cytoplasmic delivery potency is about 40% to about 100%, for example 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.

The cCPP of the present disclosure can increase cytoplasmic delivery efficiency by a factor of about 1.1 to about 30 times, e.g., about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10, about 10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.5, about 19.0, about 19.5, about 21.0, about 20.5, about 25.0, about 25.5, about 21.5, about 22.0, about 25.5, about 25.0, about 22.5, about 23.5, about 25.0, about 26.5, about 20.0, about 26.0, about 26.5, about 25.0, about 26.0, about 25.5, about 26.0, about 0, about 26.5, about 0, about 25.5, about 0, about 26.0, about 0, about 26.0, about 26.5, about 10.5, about 10.0, and the equivalent therebetween.

Preparation method

The compounds described herein may be prepared in a number of ways known to those skilled in the art of organic synthesis or in variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. The optimal reaction conditions may vary with the particular reactants or solvents used, but such conditions may be determined by one skilled in the art.

Variations of the compounds described herein include addition, subtraction, or movement of the various components as described for each compound. Similarly, the chirality of a molecule may change when one or more chiral centers are present in the molecule. In addition, compound synthesis may involve protection and deprotection of various chemical groups. The use of protection and deprotection and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemical nature of the protecting groups can be found, for example, in wuts and greene, protective Groups in Organic Synthesis, 4 th edition, wiley & sons,2006, which is incorporated herein by reference in its entirety.

Starting materials and reagents for preparing the disclosed compounds and compositions are commercially available from commercial suppliers such as Aldrich Chemical Co.(Milwaukee,WI)、Acros Organics(Morris Plains,NJ)、Fisher Scientific(Pittsburgh,PA)、Sigma(St.Louis,MO)、Pfizer(New York,NY)、GlaxoSmithKline(Raleigh,NC)、Merck(Whitehouse Station,NJ)、Johnson&Johnson(New Brunswick,NJ)、Aventis(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 by methods known to those skilled in the art following procedures described in references such as 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 supplements (ELSEVIER SCIENCE publishers, 1989); organic Reactions, volumes 1-40 (John Wiley and Sons, 1991); march' S ADVANCED Organic Chemistry, (John Wiley and Sons, 4 th edition); larock's Comprehensive Organic Transformations (VCH publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, are available from commercial sources.

The reaction to prepare the compounds described herein may be carried out in a solvent, which may be selected by one skilled in the art of organic synthesis. The solvent may be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) under which the reaction is carried out. The reaction may be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation may be monitored according to any suitable method known in the art. For example, product formation may be achieved by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³ C) Infrared spectrometry, spectrophotometry (e.g., UV-visible) or mass spectrometry, or by chromatography such as High Performance Liquid Chromatography (HPLC) or thin layer chromatography.

The disclosed compounds can be prepared by solid phase peptide synthesis in which the amino acid α -N-terminus is protected by an acid or base protecting group. Such protecting groups should have properties that are stable to the conditions under which the peptide linkage is formed, while being readily removable without disrupting the growing peptide chain or racemizing any chiral centers contained therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropoxycarbonyl, t-pentyloxycarbonyl, isobornyloxycarbonyl, α -dimethyl-3, 5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-t-butoxycarbonyl and the like. A9-fluorenylmethoxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. For side chain amino groups such as lysine and arginine, other preferred side chain protecting groups are 2,5,7, 8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, cbz, boc and adamantoxycarbonyl; for tyrosine are benzyl, o-bromobenzyloxy-carbonyl, 2, 6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl and acetyl (Ac); for serine are tert-butyl, benzyl and tetrahydropyranyl; for histidine are trityl, benzyl, cbz, p-toluenesulfonyl and 2, 4-dinitrophenyl; for tryptophan is formyl; benzyl and tert-butyl for aspartic acid and glutamic acid, and triphenylmethyl (trityl) for cysteine.

In the solid phase peptide synthesis method, the α -C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful in the above synthesis are those materials which are inert to the reagents and reaction conditions of the progressive condensation-deprotection reaction and insoluble in the medium used. The solid support used for the synthesis of the α -C-terminal carboxy peptide is a 4-hydroxymethylphenoxymethyl-co (styrene-1% divinylbenzene) or 4- (2 ',4' -dimethoxyphenyl-fmoc-aminomethyl) phenoxyacetamido ethyl resin available from Applied Biosystems (foster city, calif.). alpha-C-terminal amino acids are prepared by reacting 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) Hexafluorophosphate (BOP) or bis (2-oxo-3-/>) Oxazolidinyl) phosphine chloride (BOPCl) is coupled to the resin at a temperature of 10 ℃ to 50 ℃ for about 1 to about 24 hours in a solvent such as dichloromethane or DMF. 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) prior to coupling with the α -C-terminal amino acid as described above. One method for coupling with the deprotected 4- (2 ',4' -dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (HBTU, 1 eq.) and 1-hydroxybenzotriazole (HOBT, 1 eq.) in DMF. The coupling of the consecutive protected amino acids can be performed in an automated polypeptide synthesizer. In one example, fmoc is used to protect the alpha-N-terminus in the amino acid of the growing peptide chain. Removal of the Fmoc protecting group from the alpha-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about a 3-fold molar excess and preferably coupled in DMF. The coupling agent may be O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (HBTU, 1 eq.) and 1-hydroxybenzotriazole (HOBT, 1 eq.). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either continuously 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 comprising anisole, water, ethylene dithiol and trifluoroacetic acid. In the case where the α -C-terminus of the polypeptide is an alkylamide, the resin is cleaved by ammonolysis with the alkylamine. Alternatively, the peptide may be removed by transesterification (e.g., with methanol), followed by ammonolysis, or by direct transamidation. The protected peptide may be purified at this point or used directly in the next step. The removal of the side chain protecting groups can be accomplished using the cleavage mixtures described above. The fully deprotected peptide may be purified by a series of chromatographic steps using any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography (e.g., on Sephadex G-25, LH-20) or countercurrent distribution; high Performance Liquid Chromatography (HPLC), particularly reverse phase HPLC on octyl-octadecylsilyl-silica bonded phase column packing.

Methods of synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited by the method of synthesizing the AC. In embodiments, provided herein are compounds having reactive phosphorus groups that can be used to form internucleoside linkages (including, for example, phosphodiester and phosphorothioate internucleoside linkages). The methods of preparation and/or purification of the precursor or antisense compound are not limitations of the compositions or methods provided herein. Methods for synthesizing and purifying DNA, RNA, and antisense compounds are well known to those skilled in the art.

Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, agrawal, inc. (1993), humana Press) and/or RNA (SCARLINGE, methods (2001), vol.23:206-217 p. Gait et al, applications of Chemically synthesized RNA in RNA: protein Interactions, smith, inc. (1998), pages 1-36:Gallo et al, tetrahedron (2001), vol.57:5707-5713).

Antisense compounds provided herein can be conveniently and routinely prepared by well known solid phase synthesis techniques. The equipment used for this synthesis is sold by several suppliers including, for example Applied Biosystems (Foster City, calif.). Any other means known in the art for such synthesis may additionally or alternatively be employed. The preparation of oligonucleotides such as phosphorothioates and alkylated derivatives using similar techniques is well known. The invention is not limited by the synthetic method of antisense compounds.

Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analytical methods include Capillary Electrophoresis (CE) and electrospray mass spectrometry. Such synthetic and analytical methods can be performed in multiwell plates. The method of the present invention is not limited by the method of oligomer purification.

Application method

Also provided herein are methods of using the compounds or compositions described herein. Also provided herein are methods for treating a disease or condition in a subject in need thereof, the methods comprising administering to the subject an effective amount of any of the compounds or compositions described herein. The compounds of the compositions are useful for treating any disease or disorder that can be treated with the therapeutic moieties disclosed herein.

Also provided herein are methods of treating cancer in a subject. The method comprises administering to the subject an effective amount of one or more compounds or compositions described herein or a pharmaceutically acceptable salt thereof. The compounds and compositions described herein, or pharmaceutically acceptable salts thereof, are useful for treating cancer in humans (e.g., children and the elderly) and animals (e.g., veterinary applications). The disclosed methods may optionally include identifying patients in need or likely to be in need of treatment for cancer. Examples of types of cancers that can be treated 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 ducts, bones, bone marrow, intestines (including colon and rectum), eyes, gall bladder, kidneys, mouth, throat, esophagus, stomach, testes, 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 that can be treated by the compounds and compositions described herein include carcinomas, kaposi's sarcoma, melanomas, mesothelioma, soft tissue sarcomas, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid and others) and lymphomas (hodgkin and non-hodgkin's) and multiple myeloma.

The methods of treating or preventing cancer described herein may also include treatment with one or more additional agents (e.g., anticancer agents or ionizing radiation). The one or more additional agents as described herein, as well as the compounds and compositions or pharmaceutically acceptable salts thereof, may be administered in any order, including simultaneous administration, as well as sequential administration at intervals of up to several days. The method may also include more than a single administration of one or more additional agents and/or compounds and compositions as described herein or pharmaceutically acceptable salts thereof. Administration of one or more additional agents as described herein, as well as compounds and compositions or pharmaceutically acceptable salts thereof, may be by the same or different routes. When treated with one or more additional agents, the compounds and compositions as described herein, or pharmaceutically acceptable salts thereof, can be combined into a pharmaceutical composition comprising one or more additional agents.

For example, A compound or composition as described herein, or A pharmaceutically acceptable salt thereof, may be combined with an additional anticancer agent to form A pharmaceutical composition, such as 13-cis retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, ackutane, actinomycin-D, adriamycin, adrucil, agrylin, ala-Cort, aldesleukin, alemtuzumab, alisretin, alkaban-AQ, alkeran, all-trans retinoic acid, interferon alpha, altretamine, methotrexate, amifostine, aminoglutethimide, anagrelide, anandron, anastrozole, cytarabine, aranesp, aredia, arimidex, aromascin, arsenic trioxide, asparaginase, ATRA, avastin, BCG, BCNU, bevacizumab, bexarotene, bicalutamide, biCNU, blenoxane, bleomycin, bortezomib, busulfan, C225, calcium sulfite, folic acid Campath, camptosar, camptothecin-11, capecitabine, carac, carboplatin, carmustine tablet, casodex, CCNU, CDDP, ceeNU, cerubidine, cetuximab, chlorambucil, cisplatin, aureofactor, cladribine, cortisone, cosmegen, CPT-11, cyclophosphamide, cytadren, cytarabine liposome, cytosar-U, cytoxan, dacarbazine, dactinomycin Alfadapatine, daunorubicin hydrochloride, daunorubicin liposome, daunoXome, decadron, delta-Cortef, deltasone, dimesleukin, depoCyt, dexamethasone acetate, dexamethasone sodium phosphate, dexasone, dexrazoxane, DHAD, DIC, diodex, docetaxel, doxil, doxorubicin liposome, droxia, DTIC, dexrazoxane, and pharmaceutical compositions, DTIC-Dome, duralone, efudex, eligard, ellence, eloxatin, elspar, emcyt, epirubicin, alfazoparylene, erbitux, erwinia L-asparaginase, estramustine, ethyol, etopophos, etoposide phosphate, eulexin, evista, exemestane Fareston, faslodex, femara, febuxostat, fluorouridine, fludarabine, fluoroplex, fluorouracil (cream), fluoxymesterone, flutamide, folinic acid, FUDR fulvestrant, G-CSF, gefitinib, gemcitabine, gemtuzumab ozantine, gemzar, gleevec, lupron, lupron depot, matulane, maxidex, nitrogen mustard hydrochloride, medralone, medrol, megace, megestrol acetate, melphalan, mercaptopurine, mesna, mesnex, methotrexate, sodium methotrexate, methylprednisolone, mylocel, letrozole, neosar, neulasta, neumega, neupogen, nilandron, nilutamide nitrogen mustard, novaldex, novantrone, octreotide acetate, oncospar, oncovin, ontak, onxal, oprevelkin, orapred, orasone, oxaliplatin, paclitaxel, pamidronate, panretin, paraplatin, pediapred, PEG interferon, pegapase, pefemagistin, PEG-INTRON, PEG-L-asparaginase, phenylalanine nitrogen mustard, platinol, platinol-AQ, prednisolone, prednisone, prelone, procarbazine, PROCRIT, proleukin, prolifeprospan containing A carmustine implant, purinethol, raloxifene, rheumatrex, rituxan, rituximab, roveron-A (interferon alpha-2A), rubex, erythromycins hydrochloride, sandostatin LAR, sargramostim, solu-Cortef, solu-Medrol, STI-571, streptozotocin, tamoxifen, TARGRETIN, TAXOL, TAXOTERE, TEMODAR, temozolomide, teniposide, teSPA, thalidomide, thalomid, theraCys, thioguanine Tabloid Thiophosphamide, thioplex, thiotepa, TICE, toposar, topotecan, toremifene, trastuzumab, retinoic acid, trexall, trisenox, TSPA, VCR, velban, velcade, vePesid, vesanoid, viadur, vinblastine sulfate, VINCASAR PFS, vincristine, vinorelbine tartrate, VLB, VP-16, vumon, xeloda, zanosar, zevalin, zinecard, zoladex, zoledronic acid, zometa, GLIADEL WAFER, glivc, GM-CSF, goserelin, granulocyte colony stimulating factor, halotestin, herceptin, hexadrol, hexalen, altretamine, HMM, hycamtin, hydrea, hydrocortisone acetate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone, hydroxyurea Titamoxifen, idamycin, idarubicin, ifex, IFN-alpha, ifosfamide, IL 2, IL-11, imatinib mesylate, imidazole carboxamide, interferon alpha-2 b (PEG conjugate), interleukin 2, interleukin 11, intron A (interferon alpha-2 b), leucovorin, leukeran, leukine, leuprolide, leurocristine, leustatin, liposomal Ara-C, liquid pred, lomustine, L-PAM, L-Sarcolysin, meticorten, mitomycin-C, mitoxantrone, M-Prednisol, MTC, MTX, mustargen, mustine, mutamycin, myleran, iressa, irinotecan, isotretinoin, kidrolase, lanacort, L-asparaginase, and LCR. Additional anticancer agents may also include biological agents, such as antibodies.

Many tumors and cancers have viral genomes present in tumor or cancer cells. For example, epstein-barr virus (EBV) is associated with many mammalian malignancies. The compounds disclosed herein may also be used alone or in combination with an anticancer or antiviral agent such as ganciclovir, azidothymidine (AZT), lamivudine (3 TC), and the like, to treat patients infected with viruses that may cause cell transformation and/or to treat patients suffering from tumors or cancers that are associated with the presence of viral genomes in cells. The compounds disclosed herein may also be used in combination with virus-based oncological disease therapies.

Also described herein are methods of killing tumor cells in a subject. The method comprises contacting the tumor cells with an effective amount of a compound or composition as described herein, and optionally comprising the step of irradiating the tumor cells with an effective amount of ionizing radiation. In addition, provided herein are methods of tumor radiotherapy. The method comprises contacting the tumor cells with an effective amount of a compound or composition as described herein and irradiating the tumor with an effective amount of ionizing radiation. As used herein, the term ionizing radiation refers to radiation comprising particles or photons having sufficient energy or that can be generated via nuclear interactions to produce ionization. One example of ionizing radiation is x-radiation. An effective amount of ionizing radiation refers to a dose of ionizing radiation that produces increased cell damage or death when administered in combination with a compound described herein. The ionizing radiation may be delivered according to methods known in the art, including administration of radiolabeled antibodies and radioisotopes.

The methods and compounds described herein are useful for prophylactic and therapeutic treatment. As used herein, the term treatment (treatment) or treatment (treatment) includes prophylaxis; delaying onset; reduction, eradication, or delay of post-onset sign or symptom exacerbation; and can be used for preventing recurrence. For prophylactic use, a therapeutically effective amount of a compound and compositions as described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject prior to the onset of cancer (e.g., prior to a significant sign of cancer), during early onset (e.g., after the initial sign and symptom of cancer), or after the progression is determined. Prophylactic administration can occur days to years before symptoms of infection develop. Prophylactic administration can be used, for example, in chemo-prophylactic treatment of subjects presenting with pre-cancerous lesions, subjects diagnosed with early stage malignancy, and subgroups (e.g., family, race, and/or occupation) susceptible to a particular cancer. Therapeutic treatment involves administering to a subject a therapeutically effective amount of a compound and composition as described herein, or a pharmaceutically acceptable salt thereof, after diagnosis of cancer.

In some examples of therapeutic methods of treating cancer or tumor in a subject, a compound or composition administered to a subject can comprise a therapeutic moiety that can comprise a targeting moiety that can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, pin1, grb2 SH2, or a combination thereof.

The disclosed subject matter also relates to methods for treating a subject suffering from a metabolic disorder or condition. An effective amount of one or more compounds or compositions disclosed herein may be administered to a subject suffering from a metabolic disorder and in need of treatment thereof. In some examples, the metabolic disorder may include type II diabetes. In some examples of therapeutic methods of treating metabolic disorders in a subject, a compound or composition administered to a subject may comprise a therapeutic moiety that may comprise a targeting moiety that may act as an inhibitor against PTP 1B. In one specific example of the method, the subject is obese, and the method may comprise treating the subject for obesity by administering a composition as disclosed herein.

The disclosed subject matter also relates to methods for treating a subject suffering from an immune disorder or condition. An effective amount of one or more compounds or compositions disclosed herein is administered to a subject suffering from an immune disorder and in need of treatment thereof. In some examples of therapeutic methods of treating an immune disorder in a subject, a compound or composition administered to a subject may comprise a therapeutic moiety that may comprise a targeting moiety that may act as an inhibitor against Pin 1.

The disclosed subject matter also relates to methods for treating a subject suffering from an inflammatory disorder or condition. An effective amount of one or more compounds or compositions disclosed herein may be administered to a subject suffering from an inflammatory disorder and in need of treatment thereof.

The disclosed subject matter also relates to methods for treating a subject suffering from cystic fibrosis. An effective amount of one or more compounds or compositions disclosed herein may be administered to a subject suffering from cystic fibrosis and in need thereof. In some examples of methods of treating cystic fibrosis in a subject, a compound or composition administered to a subject can comprise a therapeutic moiety that can comprise a targeting moiety that can act as an inhibitor against CAL PDZ.

The compounds disclosed herein are useful for detecting or diagnosing a disease or disorder in a subject. For example, cCPP can comprise a targeting moiety and/or a detectable moiety that can interact with a target (e.g., a tumor).

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

In some embodiments, the disease is a CNS disorder. In some embodiments, the disease is Alzheimer's Disease (AD) (Zhao et al, gerontology, 2019; vol.65: pages 323-331). In some embodiments, the target gene is the CD33 gene. The CD33 gene is located on human chromosome 19q13.33, encoding a 67kDa transmembrane glycoprotein. Human CD33 preferentially binds α -2, 6-linked sialic acid. CD33 is expressed only on immune cells. CD33 is an inhibitory receptor through which an immunoreceptor tyrosine-based inhibitory motif (ITIM) recruits inhibitory proteins such as SHP phosphatase. CD33 is also involved in the process of adhesion in immune or malignant cells, inhibition of cytokine release by monocytes, immune cell growth and survival by inhibition of proliferation, and induction of apoptosis. Polymorphism of CD33 is associated with modulation of AD susceptibility. rs3865444C is an allele associated with increased risk of AD in european, chinese and north american populations due to increased CD33 expression. Skipping of CD33 exon 2 results in reduced expression of CD33 and increased expression of the CD33 isoform D2-CD33 that does not contain a ligand binding domain. Expression of D2-CD33 is associated with reduced risk of developing AD. In some embodiments, the AC of the present disclosure is used to skip an exon selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7a, and exon 7b of CD 33. In some embodiments, the exon is exon 2. In some embodiments, AC hybridized to its target sequence within the target CD33 pre-mRNA induces skipping of one or more exons. In some embodiments, AC induces expression of a re-spliced target protein comprising a CD33 inactive fragment.

In some embodiments, the disease is cancer (Laszlo et al Oncotarget, month 7, 12 of 2016; volume 7, 28: pages 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, renal 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 cancers described above expresses CD33. In some embodiments, the AC of the present disclosure is used to skip the exon of CD33. In some embodiments, the exon is 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 target gene is Myc, STAT3, MDM4, ERRB4, BCL2L1, GLDC, PKM2, MCL1, MDM2, BRCA2, IL5R, FGFR1, MSTR1, USP5, or CD33.

In some embodiments, provided herein are compounds comprising AC and CPPs targeting the CD33 gene. Non-limiting examples of the above compounds are shown below, which may be further modified to conjugate a cyclic Exopeptide (EP) as described herein. Antisense oligonucleotides are underlined.

ENTR-0036：

ENTR-0081：

ENTR-0087：

ENTR-0085：

ENTR-0179：

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

In some embodiments, the disease is osteogenesis imperfecta (Wang and Marini, J.Clin invest.,1996, volume 97: pages 448-454).

In some embodiments, the disease is cystic fibrosis (Friedman et al, J.biol. Chem.,1999, volume 274: pages 36193-36199).

In some embodiments, the disease is Merosin-deficient congenital muscular dystrophy type 1A (MDC 1A). MDC1A is an autosomal recessive neuromuscular disease characterized by muscle weakness, hypotonia, demyelinating neuropathy, and mild brain abnormalities in newborns. Splice site mutations were estimated to affect approximately 40% of MDC1A patient populations. Pathogenic mutations are located in the LAMA2 gene, which encodes the a2 chain (LAMA 2) of the heterotrimeric protein complex of laminin-211 (or merosin) expressed in the basement membrane of muscle cells and schwann cells. In MDC1A, laminin-211 loses its proper interaction with receptors such as integrin α7β1 and dystrophin, leading to apoptosis and degeneration of muscle cells and schwann cells, which leads to fibrosis and loss of muscle function. In some embodiments, AC hybridizes to LAMA2 target pre-mRNA. To date, the development of therapeutic strategies for MDC1A has focused mainly on the prevention of fibrosis and apoptosis. The extent of LAMA2 deficiency is highly correlated with clinical severity in patient and mouse models. The lack of functional Lama2 results in mice suffering from severe muscle atrophy and hind limb paralysis. Thus, restoration of LAMA2 expression has great potential for the treatment of MDC 1A. It has been previously demonstrated that muscle-specific overexpression of laminin-211 in merosin-deficient mice improves myology, but does not improve the associated paralysis, suggesting that correction of peripheral neuropathy requires recovery of Lama2 beyond skeletal muscle. In some embodiments, AC restores correct splicing of the gene.

In some embodiments, antisense compounds can be used to alter the ratio of long and short forms of bcl-x pre-mRNA. See U.S. patent No. 6,172,216;6,214,986; taylor et al, nat. Biotechnol, 1999, volume 17: pages 1097-1100, each of which is incorporated herein by reference. More and more genes and gene products are involved in apoptosis. One of these genes and gene products is bcl-2, an intracellular membrane protein that is shown to block or delay apoptosis. Overexpression of bcl-2 has been shown to be associated with proliferation, autoimmunity and resistance to apoptosis, including chemotherapy-induced overexpression (Fang et al, J.Immunol.1994, vol.153: pp.4388-4398). The bcl-2 related gene family has been described. All bcl-2 family members share two highly conserved domains: BH1 and BH2. Such family members include, but are not limited to, A-1, mcl-1, bax, and bcl-x. Due to the sequence homology of Bcl-x with Bcl-2, bcl-x was isolated at low stringency using Bcl-2cDNA probes. Bcl-x has been found to act as a regulator of apoptosis independent of Bcl-2 (Boise et al, cell,1993, volume 74: pages 597-608). Two isoforms of bcl-x have been reported in humans. Bcl-xl (long form) contains highly conserved BH1 and BH2 domains. When transfected into IL-3 dependent cell lines, bcl-xl inhibits apoptosis during withdrawal of growth factors in a manner similar to bcl-2. In contrast, bcl-x short isoforms of the 63 amino acid region containing BH1 and BH2 domains, produced by alternative splicing and lacking exon 1, antagonize the anti-apoptotic effects of bcl-2 or bcl-xl. As Boise et al, cell,1993, volume 74: numbered on pages 597-608, bcl-x transcripts can be categorized as follows into regions described by those skilled in the art: nucleotides 1-134,5 'untranslated region (5' -UTR); nucleotides 135-137, translation initiation codon (AUG); nucleotides 135-836, coding region, wherein 135-509 is the shorter exon 1 of bcl-xs transcript and 135-698 is the longer exon 1 of bcl-xl transcript; nucleotides 699-836, exon 2; nucleotides 834-836, a stop codon; nucleotides 837-926,3 'untranslated region (3' -UTR). Between exons 1 and 2 (between nucleotides 698 and 699), introns are spliced out of pre-mRNA when mature bcl-xl (long form) mRNA transcripts are produced. Alternative splicing from position 509 to position 699 produces bcl-xs (short form) mRNA transcripts that are 189 nucleotides shorter than long transcripts, 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 a "5' splice site", position 509 is sometimes referred to as a "recessive 5' splice site", and nucleotide 699 is sometimes referred to as a "3' splice site". In some embodiments, AC hybridizes to a sequence comprising a recessive 5' splice site of bcl-x pre-mRNA, thereby inhibiting the production of short isoforms and increasing the ratio of bcl-xl to bcl-xs isoforms.

In some embodiments, AC facilitates the skipping of a specific exon containing a premature stop codon. See Wilton et al, neurokul. Discord, 1999, volume 9: pages 330-338, which are incorporated herein by reference.

In some embodiments, AC counteracts or corrects aberrant splicing in the target pre-mRNA. See U.S. patent No. 5,627,274 and WO 94/26887, each of which is incorporated herein by reference, and which discloses compositions and methods for combating aberrant splicing in mutant-containing pre-mRNA molecules using antisense oligonucleotides that do not activate rnase H.

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 deletion of two copies of motor neuron survival gene 1 (SMN 1), protein SMN1 being part of a multiprotein complex thought to be involved in snRNP biogenesis and recycling. Almost the same gene SMN2 exists in the repeat region on chromosome 5q 13. Although SMN1 and SMN2 have the potential to encode the same protein, SMN2 contains a translational silencing mutation at position +6 of exon 7, which results in inefficient inclusion of exon 7 in the SMN2 transcript. Thus, the major form of SMN2 is a truncated version lacking exon 7, which is unstable and inactive (Cartegni and Drainer, nat. Genet.,2002, volume 30: pages 377-384). In some embodiments, the AC targets intron 6, exon 7, or intron 7 of SMN 2. In some embodiments, AC modulates splicing of SMN2 pre-mRNA. In some embodiments, modulation of splicing results in an increase in exon 7 inclusion.

In some embodiments, the target gene is a beta globulin gene. See Sierakowska et al, 1996, incorporated herein by reference. In some embodiments, the target gene is a cystic fibrosis membrane conductance regulator. See Friedman et al, 1999, which is incorporated herein by reference. In some embodiments, the target gene is the BRCA1 gene. In some embodiments, the target gene is an 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 associated with neuromuscular disorder (NMD). In some embodiments, the target gene is a gene associated with cancer.

In some embodiments, the target gene is a gene that is subject to alternative splicing. In some embodiments, the compounds and methods of the invention can be used to preferentially increase the proportion of a protein isoform by preferentially increasing splicing of a target pre-mRNA to produce an mRNA encoding that isoform.

In some embodiments, the disease is a disease caused by repeated amplification of nucleotide repeat sequences (e.g., trinucleotide repeat amplification, tetranucleotide repeat amplification, pentanucleotide repeat amplification, or hexanucleotide repeat amplification). In some embodiments, the disease is huntington's disease, huntington's disease-like 2 (HDL 2), myotonic dystrophy, spinocerebellar ataxia, spinal and Bulbar Muscular Atrophy (SBMA), dentate nucleus pallidum atrophy (DRPLA), amyotrophic lateral sclerosis, frontotemporal dementia, fragile X syndrome, fragile X mental retardation 1 (FMR 1), fragile X mental retardation 2 (FMR 2), fragile XE mental retardation (FRAXE), friedreich's ataxia (FRDA), fragile X-related tremor/ataxia syndrome (FXTAS), myoclonus epilepsy, ocular Pharyngeal Muscular Dystrophy (OPMD), or syndrome or non-syndrome X-linked mental retardation. In some embodiments, the disease is huntington's disease. In some embodiments, the disease is amyotrophic lateral sclerosis. In some embodiments, the disease is a form of spinocerebellar ataxia (e.g., SCA1, SCA2, SAC3/MJD, SCA6, SCA7, SCA8, SCA10, SCA12, or SCA 17).

In some embodiments, the disease is friedreich's ataxia. In some embodiments, the target gene is FXN, which encodes frataxin. In some embodiments, a compound provided herein comprises an antisense oligonucleotide that targets FXN. Exemplary oligonucleotides targeting FXN are provided in table 9.

TABLE 9 exemplary oligonucleotides targeting FXN

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In some embodiments, the disease is a form of myotonic dystrophy (e.g., type 1 myotonic dystrophy or type 2 myotonic dystrophy). In some embodiments, the target gene is a DMPK gene encoding myotonic protein kinase. In some embodiments, the compounds provided herein comprise antisense oligonucleotides targeted to DMPK. Exemplary oligonucleotides targeting DMPK are provided in table 10.

TABLE 10 exemplary oligonucleotides targeting DMPK

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In some embodiments, the disease is Dravet syndrome. Dravet syndrome is a severe progressive genetic epilepsy. Dravet syndrome is an autosomal dominant disorder caused by more than 1250 nascent mutations in SCN1A, resulting in 50% nav1.1 protein expression. In 85% of patients, dravet syndrome is caused by pathogenic mutations or deletions of the SCN1A gene. Existing antiepileptic drugs only address the occurrence of seizures, and more than 90% of patients with Dravet syndrome still report incomplete seizure control. In some embodiments, the antisense oligonucleotide targets SCN1A. In some embodiments, the antisense oligonucleotide targeting SCN1A has a sequence of 5'-CCATAATAAAGGGCTCAG-3'. In some embodiments, the efficacy of antisense compounds to target SCN1A is assessed in a mouse model. Non-limiting examples of mouse models include those with SCN1A exon 1 (SCN 1A ^tm1Kea ) And exon 26 (Scn 1 a) ^tm1Wac ) A mouse model with targeted deletions, a mouse model with specific point mutations knocked in such as SCN1a R1407X, scn1a R1648H and SCN1a E1099X, and a transgenic mouse model expressing bacterial artificial chromosomes (bacs) with human SCN1A R1648H mutations. In some embodiments, the efficacy of antisense compounds to target SCN1A is assessed in an in vitro model, e.g., in wild-type fibroblasts.

In some embodiments, the disease is Fragile X Syndrome (FXS). FXS is the most common form of genetic intellectual and developmental diseases. FXS is caused by the silent expression of fragile X mental retardation protein (FMRP) by the presence of >200 CGG trinucleotide repeats in FMR1 encoding FMRP. FMRP is encoded by FMR1. In some embodiments, antisense compounds of the present disclosure target FMR1. In some embodiments, the efficacy of antisense compounds to target FMR1 (e.g., those described in Dahlhaus et al), which is incorporated herein by reference in its entirety, is evaluated in a mouse model: dahlhaus, r. (2018). In male mice: brittle syndrome X was modeled. Frontiers in molecular neuroscience, volume 11: page 41.

In some embodiments, the disease is Fragile X Tremor Ataxia Syndrome (FXTAS). FXTAS is a late onset, progressive neurodegenerative disorder characterized by cerebellar ataxia and intention tremor. FXTAS is caused by a pre-FMR 1 mutation, which is defined as having 55 to 200 CGG repeats in the 5' untranslated region of FMR1. In some embodiments, antisense compounds of the present disclosure target FMR1.

In some embodiments, the disease is Huntington's Disease (HD). HD is an autosomal dominant disease characterized by reduced cognitive ability, psychosis and chorea. HD is often fatal. HD is caused by the CAG triplet repeat sequence amplified in the HTT gene, which amplification results in the production of mutant huntingtin (mHTT). Accumulation of mHTT results in progressive loss of neurons in the brain. In some embodiments, the target gene is HTT. In some embodiments, antisense compounds of the present disclosure target HTT. In some embodiments, the efficacy of antisense compounds and/or oligonucleotides is assessed in an in vivo model. An exemplary model is described in Pouladi et al, which is incorporated herein by reference in its entirety: pouladi, mahmoud a et al, "Choosing an animal model for the study of Huntington's disease", nature Reviews Neuroscience, volume 14, phase 10 (2013): pages 708-721. In some embodiments, the antisense oligonucleotide is non-allele selective. In some embodiments, the non-allele selective antisense oligonucleotide is HTTRx spacer oligonucleotide (Ionis) or a bivalent siRNA (UMass). In some embodiments, the antisense oligonucleotide is allele-selective. In some embodiments, the allele-selective antisense oligonucleotide is a sterically pure spacer oligonucleotide targeting a single nucleotide polymorphism in HTT. In some embodiments, the antisense oligonucleotide targets exon 1, exon 30, exon 36, exon 50, or exon 67 of the HTT. The following references describe exemplary antisense oligonucleotides and are incorporated herein by reference in their entirety: yu, dongbo et al, cell, volume 150, stage 5 (2012): pages 895-908; alterman, julia f. Et al, nature biotechnology, volume 37, 8 (2019): pages 884-894. Tabrizi, sarah J. Et al, NEW ENGLAND Journal of Medicine, volume 380, 24 (2019): pages 2307-2316; kordasiewicz, holly b. Et al, neuron, volume 74, phase 6 (2012): pages 1031-1044.

In some embodiments, the disease is Wilson's Disease (WD). WD is a recessive, deadly copper homeostasis disorder, usually diagnosed in patients between 5 and 35 years of age, resulting in liver and nervous system symptoms due to free copper accumulation. WD is caused by a loss-of-function mutation in the ATP7B gene. ATP7B encodes copper-transporting atpase 2, an transmembrane copper transporter and is responsible for transporting copper from the liver to other parts of the body. In some embodiments, provided herein are antisense oligonucleotides or compounds thereof that target ATP 7B. In some embodiments, the antisense oligonucleotide or compound thereof targets a T1934G (or Met-645-Arg) mutation in ATP 7B. The above ATP7B variant is described in Merico et al, which is incorporated herein by reference in its entirety: merico, daniele et al, NPJ Genomic Medicine, volume 5, phase 1 (2020): pages 1-7. In some embodiments, the antisense oligonucleotide has a sequence of 5'-CAGCTGGAGTTTATCTTTTG-3'.

In some embodiments of this aspect, the sequences of the corresponding genes responsible for such diseases readily form RNA clusters comprising tandem nucleotide repeats (e.g., multiple nucleotide repeats comprising at least 10, 15, 20, 25, 30, 40, 50, 60, 70 or more contiguous repeat nucleotide sequences). In some embodiments, the tandem nucleotide repeat is a trinucleotide repeat. The trinucleotide repeat sequence may be a CAG repeat sequence, a CGG repeat sequence, a GCC repeat sequence, a GAA repeat sequence or a CUG repeat sequence. In some embodiments, the trinucleotide repeat sequence is a CAG repeat sequence. In some embodiments, the RNA sequence comprises at least 10 trinucleotide repeats (e.g., CAG, CGG, GCC, GAA or CUG repeats), such as at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, or at least 70 trinucleotide repeats. In some embodiments, the target gene is selected from FMR1, AFF2, FXN, DMPK, SCA, PPP2R2B, ATN1, DRPLA, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP. See U.S. patent application publication number 2016/0355796 and U.S. patent application publication number 2018/0344817, each of which is incorporated herein by reference, and which discloses diseases and corresponding genes that readily form and/or amplify tandem nucleotide repeats.

In some embodiments, the AC of the present disclosure is administered to treat any of the diseases described by the present disclosure, such as Huntington's disease, huntington's disease-like 2 (HDL 2), myotonic dystrophy, spinocerebellar ataxia, spinal and Bulbar Muscular Atrophy (SBMA), dentate nucleus pallidum atrophy (DRPLA), amyotrophic lateral sclerosis, frontotemporal dementia, fragile X syndrome, fragile X mental retardation 1 (FMR 1), fragile X mental retardation 2 (FMR 2), fragile XE mental retardation (FRAXE), friedel-crafts ataxia (FRDA), fragile X-related tremor/ataxia syndrome (FXTAS), myoclonus epilepsy, ocular Pharyngeal Muscular Dystrophy (OPMD) syndrome or non-syndrome X-linked mental retardation, cystic fibrosis, proximal spinal muscular atrophy, duchenne muscular dystrophy, spinal muscular atrophy, steinert myotonic dystrophy, merosin-deficient congenital muscular dystrophy type 1A, osteogenic insufficiency, cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, or Alzheimer's disease.

In some embodiments, the AC of the present disclosure is a spacer oligonucleotide as disclosed in U.S. patent No. 9,550,988, the disclosure of which is incorporated herein by reference.

In some embodiments, the disclosed ACs comprise the sequence and/or structure of any of the SMN 2-targeted ACs disclosed in U.S. patent No. 8,361,977, the disclosure of which is incorporated herein by reference.

In some embodiments, the ACs of the present disclosure comprise the sequence and/or structure of any of the DMD, SMN2, or DMPK-targeted ACs disclosed in U.S. patent No. 2017/0260524, the disclosure of which is incorporated herein by reference.

In some embodiments, the AC of the present disclosure comprises the disclosure of U.S. patent US20030235845A1、US20060099616A1、US 2013/0072671 A1、US 2014/0275212 A1、US 2009/0312532 A1、US20100125099A1、US 2010/0125099 A1、US 2009/0269755 A1、US 2011/0294753 A1、US 2012/0022134 A1、US 2011/0263682 A1、US 2014/0128592 A1、US 2015/0073037 A1 And the sequence and/or structure of any of the AC or oligonucleotides disclosed in US20120059042A1, the contents of each of which are incorporated herein in their entirety for all purposes.

Compositions, formulations and methods of administration

In vivo application of the disclosed compounds and compositions containing them may be accomplished by any suitable method and technique currently or contemplated to be known to those skilled in the art. For example, the disclosed compounds may be formulated in a physiologically or pharmaceutically acceptable form and administered by any suitable route known in the art, including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal and intrasternal administration, such as by injection. The administration of the disclosed compounds or compositions may be a single administration, or at successive or different intervals, as readily determinable by one of skill in the art.

The compounds disclosed herein and compositions comprising them may also be administered using liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide uniform doses over an extended period of time. The compounds may also be administered in the form of their salt derivatives or in crystalline form.

The compounds disclosed herein may be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in many sources well known and readily available to those skilled in the art. For example, remington's Pharmaceutical Science, e.w. martin (1995) describes formulations that can be used in conjunction with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound. The composition used may also be in various forms. Such dosage forms include, for example, solid, semi-solid, and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The composition also preferably comprises conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethylsulfoxide, glycerol, alumina, starch, saline and equivalent carriers and diluents. To provide for administration of such doses for the desired therapeutic treatment, the compositions disclosed herein may advantageously comprise between about 0.1% and 100% by weight of one or more of the subject compounds, in total, based on the weight of the total composition comprising the carrier or diluent.

Formulations suitable for administration include, for example, sterile injectable aqueous solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only a sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets and the like. It should be understood that the compositions disclosed herein may contain other conventional agents in the art regarding the type of formulation in question, in addition to the ingredients specifically mentioned above.

The compounds disclosed herein and compositions comprising them may be delivered to cells by direct contact with the cells or via carrier means. Carrier means for delivering the compounds and compositions to cells are known in the art and include, for example, encapsulation of the compositions in a liposomal fraction. Another means for delivering the compounds and compositions disclosed herein to a cell may include attaching the compounds to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S. application publication nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow translocation of the composition across a biological membrane. U.S. application publication number 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. The compounds may also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymers for intracranial tumors; poly [ bis (p-carboxyphenoxy) propane: sebacic acid ] (as used in GLIADEL) in a molar ratio of 20:80; chondroitin; chitin; and chitosan.

For the treatment of oncological disorders, the compounds disclosed herein may be administered to a patient in need of treatment in combination with other anti-tumor or anti-cancer substances and/or with radiation and/or photodynamic therapy and/or with surgical treatment to remove tumors. These other substances or treatments may be administered simultaneously or at different times with the compounds disclosed herein. For example, the compounds disclosed herein may be used in combination with mitotic inhibitors such as paclitaxel or vinca alkaloids, alkylating agents such as cyclophosphamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as doxorubicin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecins, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and/or other anticancer drugs or antibodies such as, for example GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (genentech, inc.) or immunotherapeutic agents such as ipilimumab and bortezomib, respectively.

In certain examples, the compounds and compositions disclosed herein may be topically applied at one or more anatomical sites, e.g., sites of undesired cell growth (such as tumor sites or benign skin growth, e.g., injection or topical application to tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. The compounds and compositions disclosed herein may be administered systemically, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent or an edible carrier that can be assimilated for oral delivery. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be mixed directly with the food of the patient's diet. For oral therapeutic administration, the active compounds may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays and the like.

The disclosed compositions are bioavailable and may be delivered orally. The oral composition may be a tablet, lozenge, pill, capsule, or the like, and may further comprise the following: binders, such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid and the like; lubricants such as magnesium stearate; and may be added with a sweetener such as sucrose, fructose, lactose or aspartame, or a flavoring such as peppermint, oil of wintergreen, or cherry flavoring. When the unit dosage form is a capsule, it may contain, in addition to materials of the type described above, a liquid carrier such as a vegetable oil or polyethylene glycol. Various other materials may be present as coatings or otherwise alter the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used to prepare any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compounds can be incorporated into sustained release formulations and devices.

The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, may be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or salt thereof may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these formulations may contain preservatives to prevent microbial growth.

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

Sterile injectable solutions are prepared by incorporating the compounds and/or agents disclosed herein in the required amounts with various other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solution thereof.

For topical administration, the compounds and agents disclosed herein may be applied as liquid or solid forms. However, it is generally desirable to apply them topically to the skin as a composition in combination with a dermatologically acceptable carrier, which may be solid or liquid. The compounds and agents and compositions disclosed herein may be topically applied to the skin of a subject to reduce the size of malignant or benign growths (and may include complete removal), or to treat an infection site. The compounds and agents disclosed herein may be applied directly to the locus of growth or infection. Preferably, the compounds and agents are applied to the growth or infection site in a formulation such as an ointment, cream, lotion, solution, tincture, or the like.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol mixtures in which the compounds can be dissolved or dispersed at an effective level, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents may be added to optimize the characteristics of a given use. The resulting liquid composition may be applied from an absorbent pad for impregnating bandages and other dressings, or sprayed onto the affected area using, for example, a pump or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials may also be used with the liquid carrier to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the skin of a user.

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

The dosage range in which the composition is administered is a dosage range large enough to produce the desired effect affecting the symptom or disorder. The dosage should not be so large as to cause adverse side effects such as undesired cross-reactions, allergic reactions, etc. Generally, the dosage will vary with the age, condition, sex and degree of disease of the patient and can be determined by one skilled in the art. In the case of any contraindications, the dosage can be adjusted by the individual physician. The dosage may vary, and may be administered in one or more doses per day for one or more days.

Also disclosed are pharmaceutical compositions comprising a compound disclosed herein in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions suitable for oral, topical or parenteral administration comprising an amount of a compound constitute a preferred aspect. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient within a reasonable time frame without lethal toxicity, and preferably cause no more than an acceptable level of side effects or morbidity. Those skilled in the art will recognize that the dosage will depend on a variety of factors including the condition (health) of the subject, the weight of the subject, the type of concurrent therapy (if any), the frequency of treatment, the rate of treatment, and the severity and stage of the pathological condition.

Kits comprising the compounds disclosed herein in one or more containers are also disclosed. The disclosed kits may optionally include a pharmaceutically acceptable carrier and/or diluent. The kit may include one or more other components, adjuvants or adjuvants as described herein. The kit includes one or more anti-cancer agents, such as those described herein. The kit may include instructions or packaging materials describing how to administer the compounds or compositions of the kit. The container of the kit may be of any suitable material, such as glass, plastic, metal, etc., and may be of any suitable size, shape or configuration. The compounds and/or agents disclosed herein may be provided in a kit as a solid (such as in tablet, pill, or powder form). The compounds and/or agents disclosed herein may be provided in a kit as a liquid or solution. Kits may include ampoules or syringes containing compounds and/or medicaments disclosed herein in liquid or solution form.

Certain definitions

As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a mixture of two or more such compositions, reference to "an agent" includes a mixture of two or more such agents, reference to "the component" includes a mixture of two or more such components, and so forth.

The term "about" when immediately preceding a numerical value means a range (e.g., plus or minus 10% of the value). For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc. Unless the context of the present disclosure indicates otherwise or is inconsistent with such interpretation. For example, in a list of values such as "about 49, about 50, about 55, …," about 50 "means a range extending to less than half the interval between the front and back values, e.g., greater than 49.5 to less than 52.5. Furthermore, the phrase "less than about" value or "greater than about" value should be understood in accordance with the definition of the term "about" provided herein. Similarly, the term "about" when preceding a series of values or ranges of values (e.g., "about 10, 20, 30" or "about 10-30"), refers to all values in the series or endpoints of the range, respectively.

As used herein, the term "cyclic cell penetrating peptide" or "cCPP" refers to a peptide that facilitates delivery of cargo (e.g., a therapeutic moiety) into a cell.

As used herein, the term "endosomal escape vector" (EEV) refers to cCPP conjugated to a linker and/or an Exocyclic Peptide (EP) by chemical ligation (i.e., covalent or non-covalent interactions). The EEV may be an EEV of formula (B).

As used herein, the term "EEV-conjugate" refers to an endosomal escape carrier as defined herein that is conjugated to cargo by chemical attachment (i.e., covalent bond or non-covalent interaction). The cargo may be a therapeutic moiety (e.g., an oligonucleotide, peptide, or small molecule) that can be delivered into the cell by EEV. The EEV-conjugate may be an EEV-conjugate of formula (C).

As used herein, the terms "exocyclic peptide" (EP) and "modulator peptide" (MP) are used interchangeably to refer to two or more amino acid residues joined by peptide bonds that can be conjugated to a cyclic cell penetrating peptide (cCPP) as disclosed herein. When conjugated to the cyclic peptides disclosed herein, EP may alter the tissue distribution and/or retention of the compound. Typically, an EP comprises at least one positively charged amino acid residue, e.g. at least one lysine residue and/or at least one arginine residue. Non-limiting examples of EPs are described herein. An EP may be a peptide identified in the art as a "nuclear localization sequence" (NLS). Non-limiting examples of nuclear localization sequences include the nuclear localization sequence of the SV40 viral large T antigen, whose smallest functional units are the seven amino acid sequence PKKKRKV, the double-typed nucleoplasmin NLS with sequence NLSKRPAAIKKAGQAKKKK, the c-myc nuclear localization sequence with amino acid sequence PAAKRVKLD or RQRRNELKRSF, the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of the input protein-alpha, the sequences VSRKRPRP and PPKKARED of the myoma T protein, the sequence PQPKKKPL of human p53, the sequence SALIKKKKKMAP of mouse c-abl IV, the sequences DRLRR and PKQKKRK of influenza virus NS1, the sequence RKLKKKIKKL of hepatitis virus delta antigen and the sequence REKKKFLKRR of mouse Mxl protein, the sequence KRKGDEVDGVDEVAKKKSKK of human poly (ADP-ribose) polymerase and the sequence RKCLQAGMNLEARKTKK of steroid hormone receptor (human) glucocorticoid. Additional examples of NLS are described in International publication No. 2001/038547 and incorporated herein by reference in its entirety.

As used herein, "linker" or "L" refers to a moiety that covalently binds one or more moieties (e.g., exocyclic Peptides (EPs) and cargo, such as oligonucleotides, peptides, or small molecules) to a cyclic cell penetrating peptide (cCPP). The linker may comprise a natural or unnatural amino acid or polypeptide. The linker may be a synthetic compound containing two or more suitable functional groups suitable for binding cCPP to the cargo moiety to form the compounds disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. cCPP may be covalently bound to the cargo via a linker.

As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides of the oligonucleotide may be modified. The oligonucleotides may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides may consist of natural and/or modified nucleobases, sugars, and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.

The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked to an alpha amino group of one amino acid through a carboxyl group of another amino acid. Two or more amino acid residues may be linked to an alpha amino group through the carboxyl group of one amino acid. Two or more amino acids of a polypeptide may be joined by peptide bonds. A polypeptide may include peptide backbone modifications in which two or more amino acids are covalently linked by a bond other than a peptide bond. The polypeptide may include one or more unnatural amino acids, amino acid analogs, or other synthetic molecules that are capable of being integrated into the polypeptide. The term polypeptide includes naturally occurring and artificially occurring amino acids. The term polypeptide includes, for example, peptides comprising about 2 to about 100 amino acid residues as well as proteins comprising more than about 100 amino acid residues or more than about 1000 amino acid residues, including but not limited to therapeutic proteins such as antibodies, enzymes, receptors, soluble proteins, and the like.

The term "therapeutic polypeptide" refers to a polypeptide that has therapeutic, prophylactic or other biological activity. The therapeutic polypeptide may be produced in any suitable manner. For example, the therapeutic polypeptide may be isolated or purified from a naturally occurring environment, may be chemically synthesized, may be recombinantly produced, or a combination thereof.

The term "small molecule" refers to an organic compound that is pharmacologically active and has a molecular weight of less than about 2000 daltons, or less than about 1000 daltons, or less than about 500 daltons. Small molecule therapeutics are typically manufactured by chemical synthesis.

As used herein, the term "contiguous" refers to two amino acids joined by a covalent bond. For example, in a representative cyclic cell penetrating peptide (cCPP) such as AA₁/AA₂、AA₂/AA₃、AA₃/AA₄ And AA (alpha) ₅/AA₁ Adjacent amino acid pairs are exemplified in the context of (a).

As used herein, a residue of a chemical refers to a derivative of a chemical that is present in a particular product. To form a product, at least one atom of the substance is substituted with a bond to another moiety such that the product contains a derivative or residue of the chemical substance. For example, the cyclic cell penetrating peptides (cCPP) described herein have amino acids (e.g., arginine) incorporated therein by formation of one or more peptide bonds. The amino acid incorporated into cCPP may be referred to as a residue, or simply as an amino acid. Thus, arginine or arginine residues refer to

The term "protonated form thereof" refers to a protonated form of an amino acid. For example, the guanidine group on the arginine side chain can be protonated to form guanidine A group. The structure of the protonated form of arginine is/>

As used herein, the term "chiral" refers to a molecule having more than one stereoisomer that differs in the three-dimensional arrangement of atoms, wherein one stereoisomer is a non-superimposable mirror image of the other stereoisomer. In addition to glycine, amino acids have a chiral carbon atom adjacent to a carboxyl group. The term "enantiomer" refers to a chiral stereoisomer. Chiral molecules may be amino acid residues having the "D" and "L" enantiomers. Molecules without chiral centers, such as glycine, may be referred to as "achiral". .

As used herein, the term "hydrophobic" refers to a moiety that is insoluble or has minimal solubility in water. Typically, the neutral and/or non-polar moiety, or predominantly neutral and/or non-polar moiety, is hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein below.

As used herein, "aromatic" refers to an unsaturated ring molecule having 4n+2 pi electrons, wherein n is any integer. The term "non-aromatic" refers to any unsaturated ring molecule that does not fall within the definition of aromatic.

"Alkyl", "alkyl chain" or "alkyl group" refers to a fully saturated, straight or branched hydrocarbon chain group having one to forty carbon atoms and linked to the remainder of the molecule by a single bond. Including any number of alkyl groups containing 1 to 40 carbon atoms. Alkyl containing up to 40 carbon atoms is C ₁-C₄₀ Alkyl, alkyl containing up to 10 carbon atoms is C ₁-C₁₀ Alkyl, alkyl containing up to 6 carbon atoms is C ₁-C₆ Alkyl, and alkyl containing up to 5 carbon atoms is C ₁-C₅ An alkyl group. C ₁-C₅ Alkyl includes C ₅ alkyl, C ₄ alkyl, C ₃ alkyl, C ₂ Alkyl and C ₁ Alkyl (i.e., methyl). C ₁-C₆ Alkyl includes the above for C ₁-C₅ All moieties described for alkyl, but also C ₆ An alkyl group. C ₁-C₁₀ Alkyl includes the above for C ₁-C₅ Alkyl and C ₁-C₆ All moieties described for alkyl, but also C ₇、C₈、C₉And C ₁₀ An alkyl group. Similarly, C ₁-C₁₂ Alkyl includes all of the foregoing moieties, but also includes C ₁₁And C ₁₂ An alkyl group. C ₁-C₁₂ Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless specifically stated otherwise in the specification, an alkyl group may be optionally substituted.

"Alkylene", "alkylene chain" or "alkylene group" refers to a fully saturated straight or branched divalent hydrocarbon chain group having one to forty carbon atoms. C ₂-C₄₀ non-limiting examples of alkylene groups include ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. Unless specifically stated otherwise in the specification, the alkylene chain may be optionally substituted.

"Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain group having two to forty carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the remainder of the molecule by a single bond. Including any number of alkenyl groups containing 2 to 40 carbon atoms. Alkenyl containing up to 40 carbon atoms is C ₂-C₄₀ Alkenyl, alkenyl containing up to 10 carbon atoms is C ₂-C₁₀ Alkenyl, alkenyl containing up to 6 carbon atoms is C ₂-C₆ Alkenyl, and alkenyl containing up to 5 carbon atoms is C ₂-C₅ Alkenyl groups. C ₂-C₅ Alkenyl groups include C ₅ Alkenyl, C ₄ Alkenyl, C ₃ Alkenyl and C ₂ Alkenyl groups. C ₂-C₆ Alkenyl groups include those described above with respect to C ₂-C₅ all moieties described for alkenyl groups, but also C ₆ Alkenyl groups. C ₂-C₁₀ alkenyl groups include those described above for C ₂-C₅ Alkenyl and C ₂-C₆ all moieties described for alkenyl groups, but also C ₇、C₈、C₉And C ₁₀ Alkenyl groups. Similarly, C ₂-C₁₂ Alkenyl includes all of the foregoing moieties, but also includes C ₁₁And C ₁₂ Alkenyl groups. C ₂-C₁₂ Non-limiting examples of alkenyl groups include vinyl (ethyl/vinyl)), 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-hexenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless specifically stated otherwise in the specification, an alkyl group may be optionally substituted.

"Alkenylene", "alkenylene" or "alkenylene group" refers to a straight or branched divalent hydrocarbon chain radical having two to forty carbon atoms and having one or more carbon-carbon double bonds. C ₂-C₄₀ non-limiting examples of alkenylenes include ethylene, propylene, butene, and the like. Unless specifically stated otherwise in the specification, alkenylene chains may be optional.

"Alkoxy" OR "alkoxy group" refers to the group-OR, wherein R is alkyl, alkenyl, alkynyl, cycloalkyl, OR heterocyclyl as defined herein. Unless specifically stated otherwise in the specification, an alkoxy group may be optionally substituted.

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

"Alkylcarbamoyl" or "alkylcarbamoyl radical" means the radical-O-C (O) -NR _aR_b wherein R is _aAnd R is _b Identical or different and independently are alkyl, alkenyl, alkynyl, aryl, heteroaryl, or R as defined herein _aR_b May together form a cycloalkyl group or a heterocyclyl group as defined herein. Unless specifically stated otherwise in the specification, alkylcarbamoyl groups may be optionally substituted.

"Alkylcarboxamide" or "alkylcarboxamide group" means the group-C (O) -NR _aR_b wherein R is _aAnd R is _b identical or different and independently are alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl or heterocyclyl groups, or R as defined herein _aR_b May together form a cycloalkyl group as defined herein. Unless specifically stated otherwise in the specification, the alkylcarboxamido groups may be optionally substituted.

"Aryl" refers to a hydrocarbon ring system group comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from acetate (ACEANTHRYLENE), acenaphthylene (ACENAPHTHYLENE), acetenaphthalene (acetenaphthalene), anthracene, azulene (azulene), benzene, chrysene (chrysene), fluoranthene (fluoranthene), fluorene, asymmetric indacene (as-indacene), symmetric indacene (s-indacene), indane, indene, naphthalene, phenalene, phenanthrene, obsidiene (pleiadiene), pyrene, and benzophenanthrene. Unless specifically stated otherwise in the specification, the term "aryl" is meant to include optionally substituted aryl groups.

"Heteroaryl" refers to a group of a 5 to 20 membered ring system comprising a hydrogen atom, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, heteroaryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, and benzofuranyl azolyl, benzothiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxenyl, 1, 4-benzodi/> Alkyl, benzonaphthofuranyl, and benzo Oxazolyl, benzodioxolyl, benzodioxanyl, benzopyranyl, benzopyronyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothienyl), benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, iso/> Azolyl, naphthyridinyl,/> Diazolyl, 2-oxo-azepinyl,/> Oxazolyl, oxiranyl, 1-oxypyridyl, 1-oxypyrimidinyl, 1-oxypyrazinyl, 1-oxypyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, pheno/> oxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless specifically stated otherwise in the specification, heteroaryl groups may be optionally substituted.

The term "substituted" as used herein means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamide, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom is replaced by a non-hydrogen atom such as, but not limited to: halogen atoms such as F, cl, br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, ester groups, and the like; a sulfur atom in groups such as a thiol group, a thioalkyl group, a sulfone group, a sulfonyl group, and a sulfoxide group; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in a group such as a trialkylsilyl group, a dialkylarylsilyl group, an alkyldiarylsilyl group, and a triarylsilyl group; and other heteroatoms in various other groups. "substituted" also means any of the above groups in which one or more atoms are replaced with Gao Jiejian (e.g., double or triple bonds) to heteroatoms (such as oxygen in oxo, carbonyl, carboxyl, and ester groups); and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes where one or more atoms are replaced -NR_gR_h、-NR_gC(＝O)R_h、-NR_gC(＝O)NR_gR_h、-NR_gC(＝O)OR_h、-NR_gSO₂R_h、-OC(＝O)NR_gR_h、-OR_g、-SR_g、-SOR_g、-SO₂R_g、-OSO₂R_g、-SO₂OR_g、＝NSO₂R_g and-SO ₂NR_gR_h Substituted any of the above groups. "substituted" also means that one or more hydrogen atoms are replaced -C(＝O)R_g、-C(＝O)OR_g、-C(＝O)NR_gR_h、-CH₂SO₂R_g、-CH₂SO₂NR_gR_h Substituted any of the above groups. In the above, R _gAnd R is _h The same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. "substituted" also means any of the foregoing groups in which one or more atoms is replaced with an amino, cyano, hydroxy, imino, nitro, oxo, thio, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and/or heteroarylalkyl group. "substituted" may also mean an amino acid in which one or more atoms in the side chain are replaced by alkyl, alkenyl, alkynyl, acyl, alkylcarboxamide, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl or heteroaryl groups. Furthermore, each of the foregoing substituents may also be optionally substituted with one or more of the substituents described above.

As used herein, "subject" refers to an individual. Thus, a "subject" may include domestic animals (e.g., cats, dogs, etc.), farm animals (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "subject" may also include mammals, such as primates or humans. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under treatment by a clinician (e.g., physician).

The term "inhibition" refers to a decrease in activity, response, disorder, disease or other biological parameter. This may include, but is not limited to, complete elimination of an activity, reaction, condition or disease. This may also include, for example, a 10% reduction in activity, response, disorder or disease as compared to a natural or control level. Thus, a decrease may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any amount decrease therebetween as compared to a native or control level.

"Reduction" or other forms of the word, such as "reduction" or "reduction", means a decrease in an event or feature (e.g., tumor growth). It will be appreciated that this is typically associated with some standard or expected value, in other words it is relative, but reference to a standard or relative value is not always required. For example, "reducing tumor growth" means reducing the growth rate of a tumor relative to a standard or control (e.g., untreated tumor).

The term "treatment" refers to the medical management of a patient with the aim of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term includes active therapies, i.e. therapies directed specifically to ameliorating a disease, pathological condition or disorder, and also includes causal therapies, i.e. therapies directed to eliminating the etiology of the associated disease, pathological condition or disorder. Furthermore, the term includes palliative treatment, i.e. treatment intended to alleviate symptoms rather than cure a disease, pathological condition or disorder; prophylactic treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a related disease, pathological condition, or disorder; and supportive treatment, i.e., treatment for supplementing another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder.

The term "therapeutically effective" means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of the disease or disorder. Such improvements need only be reduced or altered and need not be eliminated.

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

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

As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers may include sugars such as lactose.

Table 10

Examples

EXAMPLE 1 construction of cell penetrating peptide-antisense Compound conjugates

Oligonucleotide design. Antisense Compounds (ACs) are designed to bind to and block mRNA expression of a target protein of interest, and are constructed as Phosphorodiamidate Morpholino Oligomers (PMOs) with C6-thiol 5' modification.

Cell penetrating peptides. Cell penetrating peptides are formulated using Fmoc chemistry and conjugated to AC, for example, as described in international application No. PCT/US20/66459, filed by Entrada Therapeutics, inc. At 12, 21, 2021, the disclosure of which is hereby incorporated herein in its entirety. In an embodiment, the CPP is cCPP, which has the amino acid sequence of Ff Φ RrRr.

EXAMPLE 2 construction of cell penetrating peptide-antisense Compound conjugates

Oligonucleotide design. Antisense Compounds (ACs) are designed to bind to and block mRNA expression of a target protein of interest, and are constructed as Phosphorodiamidate Morpholino Oligomers (PMOs) consisting only of phosphorodiamidate morpholino bases.

Cell penetrating peptides. Cell penetrating peptides comprising arginine derivatives are formulated using Fmoc chemistry and conjugated to AC, for example, as described in U.S. provisional application No. 63/171,860, entitled "NOVEL CYCLIC CELL PENETRATING PEPTIDES," filed by Ziqing Qian, 4, 7, 2021, the disclosure of which is hereby incorporated herein in its entirety. In embodiments, the CPP has the sequence acetyl-Pro-Lys-Lys-Lys-Arg-Lys-Val-PEG ₂ -Lys (cyclo [ Phe-D-Phe-2-Nal-Cit-D-Arg-Cit-D-Arg-gamma-Glu) -PEG ₁₂-Lys(N₃)-NH₂.

EXAMPLE 3 construction of cell penetrating peptide-antisense Compound conjugates containing regulatory peptide sequences

The compounds of table a were prepared as follows. Compounds ENTR-0047, ENTR-0168, ENTR-0203 and ENTR-0207 each contain an Exocyclic Peptide (EP) sequence represented by "NLS", whereas compounds ENTR-0006, ENTR-0070, ENTR-0059 and ENTR-0121 do not contain NLS.

Table A. EGFP-654 targeting compounds

And (5) designing a target gene. Missense EGFP gene ("EGFP-654") was designed in which mutant intron 2 of the human beta globulin gene interrupted the EGFP coding sequence. A mutation is introduced at nucleotide 654 of intron 2 that activates an aberrant splice site and results in the retention of the intron fragment in the spliced mature mRNA, thereby preventing proper translation of EGFP.

Oligonucleotide design. Antisense Compounds (ACs) are designed to bind to and block aberrant splice sites of target genes in order to correct pre-mRNA splicing and restore EGFP expression. AC has the sequence "5'-GCTATTACCTTAACCCAG-3'" and was designed as Phosphorodiamidate Morpholino Oligomer (PMO) with C6-thiol 5' modification (ENTR-0006, table A).

Cell penetrating peptides. Cell penetrating peptides comprising cyclo (Phe-D-Phe-SNal-Arg-D-Arg-Arg-D-Arg-gamma-Glu) -b-Ala-b-Ala-Lys (maleimide) -NH2 ("CPP 12-maleimide") were formulated as TFA salts. Peptides were synthesized using standard Fmoc chemistry according to the following procedure:

a.1. At N ₂ DCM was added to a vessel containing Rink amide resin (1 mmol,1g,1.0 mmol/g) and Fmoc-Lys (Trt) -OH (1 eq.) under bubbling.

B.2. DIEA (4.0 eq.) was added dropwise and mixed for 4 hours.

C.3. MeOH (0.2 mL) was added to the resin and mixed for 30 minutes.

D.4. the solution was drained and washed 5 times with DMF.

E.5. 20% piperidine/DMF was added and reacted for 30 minutes.

F.6. the solution was drained and washed 5 times with DMF.

G.7. Fmoc-amino acid solution was added to the resin and mixed for 30 seconds, followed by the addition of the activation buffer and N ₂ Bubbling was carried out for about 1 hour.

H.8. steps 4 to 7 were repeated for the following amino acid couplings. Completion of all coupling reactions was confirmed by the negative ninhydrin test. After coupling, the resin was washed 5 times with DMF.

I.9. The allyl protecting group was removed by adding a solution of PhSiH3 (10 equivalents) and Pd (PPh 3) 4 (0.1 equivalents) in DCM, repeated twice.

J.10. The peptide was cyclized by HATU (1.0 eq.) and DIEA (2 eq.). Cyclization was monitored by ninhydrin test. The resin was washed five times with DMF.

K.11. The Trt protecting group was removed with 20% HIFP/80% DCM and the resulting primary amine was reacted with OAt activated 3-maleimidopropionic acid using HATU/DIPEA for 2 hours.

After coupling, the resin was washed 3 times with MeOH and dried under reduced pressure.

The following table shows materials and coupling reagents for solid phase peptide synthesis:

Peptides were cleaved from the solid phase peptide synthesis resin and purified according to the following procedure.

A. Cleavage buffer (95% TFA, 2.5% TIPS, 2.5% H) was added at room temperature ₂ O) was added to a flask containing the side chain protected peptide and stirred for 1 hour, and repeated once.

B. The peptide solution was filtered and collected.

C. The peptide was concentrated under reduced pressure to give a residue.

D. Dissolving the obtained solid in CH ₃ CN and H ₂ o, then lyophilized to give the crude peptide (2 g,70.1% yield) as a white solid.

CPP-AC conjugate formation. The steps of the conjugation process are shown in figure 11. Compound 1 was treated with 1M TCEP and MeCN/H ₂ O treatment to reduce the 5' end gives compound 2. To this solution of compound 2 (about 13mg, from about 10mg reduction of 2pmo 654-G) in H2O/CH3CN (3:2, 2 ml) was added CPP 12-maleimide (4.5 mg,1.2 eq, pre-dissolved in H2O/CH) in one portion ₃ CN (3:2), 98.4. Mu.L). To the mixture was then added PB buffer (ph=7, 1 ml) and stirred at 25 ℃ for 1 hour. Another batch (12 mg) was treated in parallel. LCMS showed that compound 1 had been completely consumed and the solution was directly injected and purified by C18 reverse phase column. The mixture was first conditioned with TEEA (2 mM TEAA in water, CH ₃ CN solution) and then eluting with TFA conditions (0.075% TFA in water, CH3CN solution) to give CPP 12-maleimide-PMO-654 (28 mg,96.3% purity, total yield: 56.8%).

Purification conditions:

/>

PMO synthesis scheme and linker installation at the 3' end. The following scheme was used to synthesize PMO. For each step of the following synthesis scheme, it is ensured that the volume of reagents or solvents used completely covers the resin (more if necessary). The volumes listed below are estimates expressed in milliliters per gram of resin used, which increase during synthesis as the size of the resin increases. All synthetic steps in the table were performed at room temperature. The resin was swelled in NMP for 1 hour prior to synthesis. The resin was washed twice with DCM, then 2 times with 30% TFE/DCM (15 mL/g resin). The following table describes PMO synthesis and joint installation schemes:

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CYTFA solution = 100mM 4-cyanopyridine +100mM TFA in 4:1dcm: tfe +1% ethanol solution

The following are solutions used for the synthesis:

a. neutralization solution = 3:1dcm: iproh solution of 5% dipea.

B. coupling solution = equivalent number and concentration will increase throughout the synthesis using the following guidelines:

c. residues 1-10 = 3 equivalents of morpholino monomer, 5 equivalents of NEM in DMI (0.2M morpholino monomer, 0.5M NEM), room temperature, 4.25 hours.

D. residue 1: coupling was carried out for 5 hours at room temperature.

E. Residues 11-20 = 4 equivalents of morpholino monomer, 6.5 equivalents of a DMI solution of NEM (0.3M morpholino monomer, 0.5M NEM), room temperature, 4.25 hours.

F. residues 21-25 = 5 equivalents of morpholino monomer, 8 equivalents of NEM in DMI (0.3M morpholino monomer, 0.5M NEM), room temperature, 4.25 hours.

G. guanine morpholino monomer: coupling was performed at room temperature for 4.75 hours.

Some couplings used 6 equivalents of morpholino monomer, 8 equivalents of NEM in DMI (0.4M morpholino monomer, 0.5M NEM) for 4.75 hours at 45 ℃.

The PMO synthetic resin having a linker has the following structure:

The morpholino monomers used in the coupling process have the following structure:

The following protocol was used for PMO synthesis:

Deprotection: the resin was first washed with 30% TFE/DCM solution and allowed to stand for 15 seconds before draining. Then CYTFA solution was added to the drained resin and reacted for 15 minutes. After draining the resin, a new CYTFA solution was added and reacted again for 15 minutes. The resin was drained, rinsed twice with DCM for 15 seconds, and then neutralized.

And (3) neutralization: the neutralized solution was added to the resin, stirred and allowed to stand for 5 minutes, and then drained. The fresh neutralization solution of the second wash was delivered to the resin, stirred and reacted for 5 minutes. The resin was washed once with DCM and once with DCM or anhydrous DMI prior to coupling.

Coupling: using the guidelines listed above, two coupling solutions were prepared: 1) PMO monomer dissolved in DMI, and 2) NEM dissolved in DMI. The two solutions were mixed immediately before addition to the resin. The resin was stirred and reacted for 4.25 to 5 hours. The resin was washed once with DCM.

And (3) end capping: a capping solution consisting of 0.55M benzoic anhydride and 0.55M NEM in NMP was added to the resin and reacted for 15 minutes. The resin was drained and a neutralizing solution was added to the resin to react for 5 minutes. The resin was drained again, washed once with DCM, then twice with 30% TFE/DCM solution.

Post synthesis: after the final coupling step, the resin-bound PMO can be stored until it is cleaved (note: for this purpose, the 3' -trityl protecting group must still be located on the PMO) by washing the resin eight times with iPrOH, then vacuum drying the resin at room temperature. For PMO modification at 3', the trityl protecting group was removed, the resin was neutralized, then a solution of the appropriate bifunctional linker (TFA-protected amino or cyclooctyne) PFP ester (4 eq.) in NMP and DIPEA (8 eq.) were added to the resin and reacted for 3 hours. The solution was drained and the resin was washed once with DCM and then twice with 30% TFE/DCM solution.

Cutting: the following options are available for making PMO cuts:

a. PMO was cleaved from the resin with a 1:1 solution of ammonium hydroxide (25% ammonia) and methylamine (8 mL/g) at 65℃for 15 minutes.

B. The PMO was cut from the resin with ammonium hydroxide (25% ammonia) at 65 ℃ for 16 hours.

C. PMO was cleaved from the resin (8 mL/g resin) with 7M ammonia in methanol at 65℃for 16 hours.

D. The deprotected PMO solution is then desalted prior to lyophilization and purification.

E. The resulting PMO was purified by Clarity 5 μm, C18 oligonucleotide reverse phase (250 mm. Times.30 mm), AXIA packed column and gradient 10% -30% over 40 min, flow rate 30mL/min, solvent A was water containing 0.05% TFA and solvent B was acetonitrile.

Design and preparation of CPP-PMO654 conjugate. For 3 'covalent conjugation of primary amine modified PMO, a solution of the desired peptide-TFP ester in DMF (4 eq, 5 mM) was added to a solution of PMO-3' -primary amine (1 eq, 2 mM) in PBS-10X. The reaction is carried out to completion at room temperature over a period of 4-8 hours, e.g., by LCMS (Q-TOF) using BEH C18 column [ ] 1.7 μm,2.1 mm. Times.150 mm), buffer A: water, 0.1% fa), buffer B: acetonitrile, 0.1% fa), flow rate (0.3 mL/min), starting with 2% buffer B and gradually increasing to 70%, for 11 minutes, confirming a total of 20 minutes of running. For 3 'or 5' conjugation via click reaction, a solution of peptide-azide in water without nuclease (1 mM) was added to PMO-3 '-cyclooctyne or cyclooctyne-5' -PMO solid. The mixture was vortexed to dissolve the peptide-PMO conjugate, centrifuged to settle the solution, and incubated at room temperature for 8-12 hours to complete, as confirmed by LCMS (Q-TOF). For purification, the crude mixture was diluted with DMSO, loaded onto a C18 reverse phase column (150 mm x 21.2 mm) at a flow rate of 20mL/min and purified over 20 minutes by using a suitable gradient of water and acetonitrile containing 0.05% tfa as solvents. The desired fractions were combined, the pH of the solution was adjusted to 5-6 by 1M NaOH, and the solution was subjected to a lyophilization process to give a white lyophilized powder. For in vitro and in vivo formulations, the conjugate was reconstituted to the desired concentration (2 mg/mL-10 mg/mL) in an appropriate amount of PBS or saline. The concentration of non-LSR labeled conjugates was measured by preparing 10, 20 and 50-fold dilutions in formulated buffer and reading absorbance at 260nm or 280nm using Nanodrop. Once the linear range of dilution is reached, absorbance is measured in triplicate and the average absorbance and epsilon are used ₂₆₀Or epsilon ₂₈₀ the concentration was calculated. Epsilon of conjugate ₂₈₀ Calculated by the following formula: epsilon ₂₈₀ = 100356+ (n×3550); n=number of CPPs. For LSR modified PMO, the concentration was measured at 566nm, where ε ₅₆₆＝100000LMol^-1Cm^-1 . Diluted samples were analyzed by LCMS (Q-TOF) for conjugate identity confirmation. The following table summarizes the calculated MW and experimental MW. All experimental MW reasonably matched the calculated average MW with the expected assay variation of.+ -. 6Da.

Name of the name	Calculated MW	Experimental average MW	Purity of
				ENTR 0006	6038.19
ENTR 0070	6151.27	6148.22	>99
				ENTR 0059	7411.06	7409.78	99
ENTR 0121	7938.3	7933.15	97
				ENTR 0047	9039.81	9034.81	99
ENTR 0168	10444.78	10447.3	95
				ENTR 0203	9293.79	9295.2	92
ENTR 0207	9532.05	9532.8	>99

The structure of ENTR-0203 is as follows:

the structure of ENTR-0207 is as follows:

EXAMPLE 4 use of cell penetrating peptides coupled to oligonucleotides and Nuclear localization sequences for splicing correction of exon 23 of DMD in MDX mouse model

The purpose is that. This study used an MDX mouse model (model of DMD) to study the effect of compositions comprising AC, CPP and nuclear localization sequences (NLS, or regulatory peptide EP) on dystrophin expression and myofiber injury.

Preparation and design of CPP-PMO targeting murine DMD exon 23. The design of Antisense Compounds (AC) targeting DMD exon 23 is shown in table B1 below. The design of the CPP-NLS-PMO constructs (ENTR-0164, ENTR-0165, ENTR-0201) is shown in Table B2 below.

TABLE B1 DMD-targeting Compounds

Table B2 CPP-NLS-PMO compound targeting DMD

For 3 'or 5' conjugation via click reaction (ENTR-0164, ENTR-0165, ENTR-0201), a solution of peptide-azide in water without ribozyme (1 mM) was added to PMO-3 '-cyclooctyne or cyclooctyne-5' -PMO solid. The mixture was vortexed to dissolve the peptide-PMO conjugate, centrifuged to settle the solution, and incubated at room temperature for 8-12 hours to complete, as confirmed by LCMS (Q-TOF). For purification, the crude mixture was diluted with DMSO, loaded onto a C18 reverse phase column (150 mm x 21.2 mm) at a flow rate of 20mL/min and purified by an appropriate gradient using water with 0.05% tfa and acetonitrile as solvents. The desired fractions were combined, the pH of the solution was adjusted to 5-6 by 1M NaOH, and the solution was subjected to a lyophilization process to give a white lyophilized powder. For in vitro and in vivo formulations, the conjugate was reconstituted to the desired concentration (2 mg/mL-10 mg/mL) in an appropriate amount of PBS or saline. The concentration of non-LSR labeled conjugates was measured by preparing 10, 20 and 50-fold dilutions in formulated buffer and reading absorbance at 260nm or 280nm using Nanodrop. Once the linear range of dilution is reached, absorbance is measured in triplicate and the average absorbance and epsilon are used ₂₆₀Or epsilon ₂₈₀ the concentration was calculated. Epsilon of conjugate ₂₈₀ Calculated by the following formula: epsilon ₂₈₀ = 138993+ (n×3550); n=number of CPPs. Diluted samples were analyzed by LCMS (Q-TOF) to confirm conjugate identity. The following table summarizes the calculated MW and experimental MW. All experimental MW reasonably matched the calculated average MW with the expected assay variation of.+ -. 6Da.

Name of the name	Calculated MW	Experimental average MW	Purity of
				ENTR-0013	8413.1	8410.02	99
ENTR-0017	8487	8484	95
				ENTR-0066	9001.7	9003.8	95
ENTR-0068	8751.07	8748.02	85
				ENTR-0149	8635.07	8635.8	82
ENTR-0164	11838.19	11836.3	97
				ENTR-0165	11944.31	11942.3	99
ENTR-0201	11669.5	11669.5	>99

Exemplary structures of compounds comprising antisense oligonucleotides (underlined) targeting murine DMD and CPP are shown below.

ENTR-0164：

ENTR-0165：

ENTR-0201：

Study design. Compositions comprising AC having the 5'-GGCCAAACCTCGGCTTACCTGAAAT-3' sequence, cCPP12 (amino acid sequence is Ff Φ RrRr) and the nuclear localization sequence PKKKRKV (referred to herein as "ENTR-201") were applied to MDX mice to assess the ability of the composition to skip exon 23 and thus treat DMD. Control compositions lack cCPP and nuclear localization sequences. The AC sequence of the control composition is 5'-GGC CAA ACC TCG GCT TAC CTG AAA T-3'. The ENTR-201 composition is administered Intravenously (IV) to mice at a dose of 10mg/kg once a week for four weeks or at a dose of 20mpk once a week. The control composition was administered Intravenously (IV) to mice at a dose of 20 mpk. Total RNA was extracted from tissue samples and analyzed by RT-PCR, protein was extracted from tissue samples and analyzed by Western blot to observe the efficiency of splice correction and to detect dystrophin products. The percentage of exon 23 corrected product was assessed. The dystrophin level was assessed relative to α -actin (loading control) compared to dystrophin expression in wild-type mice. Serum levels of creatine kinase increased in DMD patients due to myofiber injury were also assessed by a commercially available kit from SIGMA CHEMICALS.

Evaluation of peptide fusions with CPP12-PMO constructs. To further increase functional delivery of PMO, we have also explored various peptide fusions of CPP-PMO constructs. For example, it has been demonstrated that enhanced muscle targeting of T9 peptide (SKTFNTHPQSTP) (Y. Seow et al, peptides, volume 31 (2010): pages 1873-1877) and muscle specific peptide (MSP peptide, ASSLNIA (Gao et al, molecular Therapy (2014), volume 22, 7: pages 1333-1341) are also fused to CPP12 constructs, as in ENTR-0119 and ENTR-0163, respectively.

Notably, CPP12 with the nuclear localization sequence (PKKKRKV) is significantly superior to CPP12 alone (e.g., ENTR-164, ENTR-0165, and ENTR-0201, table B2). ENTR-164 produced 59.5% + -2.2%, 29.1% + -0.8% and 61.0% + -13.7% exon 23 skipping in quadriceps, heart and diaphragm, respectively, after a single intravenous dose of 20mpk for one week. ENTR-165 produced 38.5% + -2.5%, 30.5% + -17.0% and 30.2% + -2.8% exon 23 skipping in quadriceps, heart and diaphragm, respectively, after a single intravenous dose of 20mpk for one week. ENTR-201 produced 73.5% + -8.4%, 60.5% + -17.2% and 79.0% + -6.8% exon 23 skipping in quadriceps, heart and diaphragm, respectively, after a single intravenous dose of 20mpk for one week. The preparation of these NLS fusions is described in detail above. The structures of ENTR-164, ENTR-165 are as described above. In agreement with the exon skipping data, ENTR-0164 and ENTR-0165 also significantly corrected expression of dystrophin expression, as analyzed by Western blot. The percentage of dystrophin correction was further quantified as a percentage of wild type by comparison with the level in the corresponding tissue from wild type (C57 BL/10) and using the actin as a loading control. In addition to using a modified PMO with 3' amide bond formation, we also tested the incorporation of cyclooctyne onto a solid support by modification of morpholino amino groups with a bifunctional linker consisting of cyclooctyne moieties for click reaction with CPP-azide and PFP esters to form carbamates with PMO, and thus produced a precursor useful in the synthesis of ENTR-201. Like ENTR-165, ENTR-201 also exhibits high exon skipping activity within all muscle groups.

Activity of ENTR-201 in MDX mice. The following table summarizes injection, sample collection and bioassays to investigate the duration of action of ENTR-201 following a single IV injection.

Animals were sacrificed at 1 week, 2 weeks and 4 weeks post injection after a single dose of ENTR-201 at 20mg/kg on day 1. Vehicle only (PBS) was used as negative control. Heart, diaphragm, quadriceps and transverse abdominal muscles were collected, RT-PCR was performed to detect dystrophin exon 23 jump products, and Western blot analysis was performed to detect dystrophin expression (relative to α -actin). Samples 4 weeks after a single IV injection of 20mpk of ENTR-201 or PBS were also analyzed by immunohistochemical staining to detect the expression and distribution of dystrophin in various muscle tissues.

Treatment of mice with a single dose of 20mg/kg ENTR-0201 resulted in splicing correction of dystrophin in heart, diaphragm, quadriceps and transverse abdominal muscles (TrA). ENTR-0201 provides a significant enhancement in exon skipping efficiency up to four weeks after a single IV injection. The corresponding dystrophin levels were analyzed by Western blot. Recovered dystrophin remained for up to four weeks after a single IV injection at 20mpk in heart, diaphragm, quadriceps and transverse abdominal muscles (TrA). The level of protein correction was consistent with RNA analysis. Tissue samples from the last injection were also analyzed by immunohistochemistry, which showed that all skeletal muscle fibers were immunostained positive for dystrophin, as observed by brown staining. The intensity of dystrophin expression is significant in cardiac muscle tissue, reaching near normal levels. Extensive uniform expression of dystrophin was analyzed on multiple tissue sections within each of the muscle groups.

Activity of ENTR-201 in MDX mice after repeated doses. The following table outlines injection, sample collection and bioassays to investigate the activity of ENTR-201 after repeated doses.

Mice were treated once a week with 10mg/kg of ENTR-201 for 4 weeks. 20mg/kg of ENTR-013 (PMO only) and vehicle (PBS) only were used as control groups. All animals were sacrificed 1 week after the last injection. Heart, diaphragm, quadriceps and transverse abdominal muscles were collected, RT-PCR was performed to detect products of dystrophin exon skipping, western blot analysis and immunohistochemical staining were performed to detect dystrophin expression, and thus dystrophin expression and distribution in various tissues. Serum creatine kinase levels were quantified as muscle function biomarkers.

Treatment of MDX mice with 10mg/kg ENTR-201 once a week for four weeks also resulted in significant splice correction of dystrophin mRNA and dystrophin levels in various muscle tissues (heart, diaphragm, quadriceps and transverse abdominal muscles (TrA)). Treatment with 10mg/kg of ENTR-201 resulted in higher amounts of both splice correction and dystrophin in all four muscle tissues than treatment with 20mpk PMO. Notably, mRNA correction and dystrophin expression in the heart was only observed in MDX mice treated with 10mpk ENTR-201, but not in MDX mice treated with 20mg/kg PMO. Findings from IHC studies are also consistent with RT-PCR and WB analysis. Treatment with 10mpk ENTR-201 once a week for four weeks also normalized serum creatine kinase levels as a biomarker for muscle injury, indicating that oligonucleotide 201 treatment reduced muscle fiber injury in the DMD mouse model. In contrast, PMO alone (ENTR-0013) treatment did not significantly reduce elevated serum CK levels. Serum samples were collected from the repeat dosing study one week after the last injection. The CK level was analyzed using a commercially available CK measurement kit (Millipore SIGMA CHEMICALS, MAK 116) according to the manufacturer's instructions. Quantification of dystrophin showed that nearly 40% of cells were positive for dystrophin in heart tissue, in contrast to 5% or less cells in vehicle-treated or PMO-treated heart tissue alone.

Treatment of mice once a week with 20mg/kg oligonucleotide 201 resulted in splicing correction of dystrophin in heart and diaphragm. Treatment of mice four times a week with 10mg/kg of oligonucleotide 201 or 5mg/kg of oligonucleotide 201 also resulted in correction of splicing of dystrophin in heart and diaphragm. Treatment with 10mg/kg of oligonucleotide 201 produced a higher amount of splicing correction in the heart and diaphragm than treatment with 5 mg/kg. Treatment with 5mg/kg or 10mg/kg of oligonucleotide 201 four times per week also resulted in a decrease in creatine kinase expression compared to the control, indicating that oligonucleotide 201 treatment reduced myofiber injury in DMD patients.

Example 5 tissue Regulation in muscle of cell penetrating peptide of exon 23 for splicing correction DMD in MDX mouse model Using coupling with oligonucleotide and Nuclear localization sequence

The compounds of table C below include additional non-limiting examples of NLS containing compounds. The compounds were prepared as described in the previous examples.

Table C:

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non-limiting examples of CCP-AC chemical structures that also contain a modulatory peptide (MP; or NLS) are shown below:

EEV-PMO-MDX23-1

EEV-PMO-MDX23-2

EEV-PMO-MDX23-3

Exemplary compounds were tested in the MDX model described in example 4. Mice were treated on day 1 with a single intravenous dose of 20 mg/kg. On day 5 post injection, animals were sacrificed and specific tissues were harvested and flash frozen. RNA was extracted and exon skipping in the diaphragm (fig. 12A), heart (fig. 12B), tibialis anterior (fig. 12C) and triceps (fig. 12D) was quantified by RT-PCR as previously described. The results indicate that treatment with NLS-containing compounds (ENTR-1491093, ENTR-1491094, ENTR-1491096) resulted in lower levels of exon skipping in heart tissue than the three types of muscle tissue examined. In addition, on day 5 post-injection, dystrophin levels in the diaphragm (fig. 13A), heart (fig. 13B) and tibialis anterior (fig. 13C) were quantified by western blot analysis. The results indicate that treatment with NLS-containing compounds (EEV-PMO-ENTR-1491093, EEV-PMO-ENTR-1491096) also resulted in lower levels of dystrophin expression in heart tissue and tibialis anterior tissue compared to diaphragm tissue.

Example 6A. Use of cell penetrating peptides conjugated to oligonucleotides for CD33 knockout in human macrophages.

The compounds of table D were prepared. Exemplary experiments are described below and in examples 6B and 6C.

CD 33-targeting compounds

And (3) cells. Differentiated THP-1 cells (human monocytes) and glioblastoma cells (human neuronal cells) were used in this study.

Study design. CD33 is associated with diseases such as cancer and alzheimer's disease ("AD"). Targeting CD33 expression represents a therapeutic strategy for AD and cancer.

Targeting CD33 expression represents a therapeutic strategy for AD and cancer. Skipping of exon 2 of the gene expressing CD33 results in D2-CD33 (a CD33 isoform lacking the sialic acid binding domain). In the absence of such ligand binding domains, CD33 is unable to inhibit microglial activation and microglial phagocytosis of amyloid β protein. The result of this is the prevention of AD. This example evaluates the efficacy of the platform described in examples 1-5 for treating AD or cancer. Briefly, THP1 and glioblastoma cells were treated with AC having a nucleic acid sequence of 5'-GTAACTGTATTTGGTACTTCC-3' ("PMO-CD 33"), PMO-CD33 conjugated to CPP ("EEV-PMO-CD 33-2"), or PMO-CD33 conjugated to both CPP and NLS (EEV-PMO-CD 33-5) in the presence of 10% Fetal Bovine Serum (FBS).

PMO sequence development and optimization. Nucleic acid sequence 5'-CTGTATTTGGTACTT-3' has been previously reported to induce human CD33 exon 2 skipping in THP1 cells (Bergeijk p. Et al Molecular and Cellular biol., 2019). We first modified oligonucleotide chemistry from 2' -MOE modified RNA to Phosphorodiamidate Morpholino Oligomer (PMO), as in conjugated construct ENTR-085, with only modest success. To improve efficacy, we further developed 21nt-long PMO. PMO-CD33 of Table 5: 5'-GTAACTGTATTTGGTACTTCC-3' and its CPP conjugate (iEEV-PMO-CD 33-4) showed excellent efficacy. Thus, the PMO sequence PMO-CD33 was used in subsequent studies.

Exon 2 skipping was detected by RT-PCR and flow cytometry. Semi-quantitative PCR analysis after reverse transcription showed that treatment of THP1 cells with EEV-PMO-CD33-4 for 48 hours in the presence of 10% FBS resulted in skipping of exon 2 and production of D2-CD33 (a CD33 isoform lacking the ligand binding domain). Treatment of THP1 cells with PMO-CD33 alone resulted in a smaller number of exon skipping compared to treatment with EEV-PMO-CD 33-4. Exon 2 skipping is dependent on the dose of EEV-PMO-CD 33-4. Flow cytometry demonstrated a reduction in the production of CD33 in cells treated with EEV-PMO-CD33-4 compared to untreated (NT) cells.

Dose-dependent exon 2 skipping induced by EEV-PMO-CD33-5 in THP1 cells. CD33 mRNA from differentiated THP1 cells (human monocytes) treated with various concentrations of EEV-PMO-CD33-5, PMO-CD33 together with Endoporter (6. Mu.L/mL) transfection reagent or with PMO-CD33 alone for 48 hours in the presence of 10% FBS was analyzed by RT-PCR. The results show a dose-dependent jump of exon 2CD33 by EEV-PMO-CD33-5 treatment with a significant improvement (more than 2-fold) compared to transfection and more than 1000-fold improvement compared to unconjugated PMO-CD 33. CD33 mRNA of glioblastoma cells (human neuronal cell line, U-87 MG) treated with various concentrations of EEV-PMO-CD33-5 for 48 hours in the presence of 10% FBS was analyzed by RT-PCR. The results show a dose-dependent jump of exon 2CD33 by EEV-PMO-CD33-5 treatment.

Duration of action of EEV-PMO-CD33 in differentiated THP1 cells. Differentiated THP1 cells (human monocytes) were treated with EEV-PMO-CD33-4 for 1 day and the cells continued to be cultured with complete growth medium. Cells were harvested and analyzed for CD33 mRNA 2-8 days after incubation. The results show that ingested EEV-PMO-CD33-4 can induce CD33 exon 2 skipping for a period of time (> 8 days).

Exon 2 skipping induced by monovalent EEV-PMO-CD33 and divalent EEV-PMO-CD 33. CD33 mRNA of differentiated THP1 cells (human monocytes) treated with PMO-CD33, monovalent EEV-PMO-CD33-2 and divalent EEV-PMO-CD33-4 for 48 hours was analyzed by RT-PCR. The results show that EEV-PMO-CD33-4 is more effective in inducing CD33 exon 2 skipping than EEV-PMO-CD 33-2.

Example 6B use of cell penetrating peptides conjugated to oligonucleotides for CD33 knockout in human macrophages.

This example uses the protocol of example 6A to evaluate PMO having 5'-GTAACTGTATTTGGTACTTCC-3' ("PMO-CD 33") or conjugated to CPP in the presence of 10% Fetal Bovine Serum (FBS) ^CD33 AC of the nucleic acid sequence of ("EEV-PMO-CD 33-2"). The CPP used was cCPP, which had the amino acid sequence of FfΦ RrRr.

Exon 2 skipping was detected by RT-PCR and flow cytometry. RT-PCR analysis showed that treatment of THP1 cells with EEV-PMO-CD33-2 in the presence of 10% FBS for 48 hours resulted in skipping of exon 2 and production of D2-CD33 (a CD33 isoform lacking the ligand binding domain). Treatment of THP1 cells with PMO-CD33 alone resulted in a smaller number of exon skipping compared to treatment with EEV-PMO-CD33-2. Exon 2 skipping is dependent on the dose of EEV-PMO-CD33-2. Flow cytometry demonstrated a reduction in the production of CD33 in cells treated with EEV-PMO-CD33-2 compared to untreated (NT) cells.

Example 6C use of a cell penetrating peptide conjugated to an oligonucleotide for the production of D2-CD33 in a non-human primate.

EEV-PMO-CD33-2 and EEV-PMO-CD33-5 described in examples 6A-6C are used in animal studies, such as in rodents, monkeys, and humans. Various doses (0.5 mpk, 1mpk, 2.5mpk, 5mpk, 10mpk, 20mpk, 40 mpk) of EEV-PMO-CD33 or PMO-CD33 conjugate targeted to skipping of exon of the gene expressing CD33 (exon 2 of human CD33 or exon 5 of monkey CD 33) are administered intravenously or intrathecally to animals or humans. The results show that the oligonucleotide therapeutic induces exon skipping of the target CD33 gene, down-regulates CD33 levels and can treat AD.

NHP study design. To explore the tolerance of EEV-PMO in non-human primate (NHP), two CPP-PMO constructs, as well as PMO itself, were administered in a staggered fashion at d0 and d3 by intravenous infusion at 2mpk and 5mpk, respectively. Oligonucleotides included "PMO-CD33", PMO-CD33 conjugated to CPP (EEV-PMO-CD 33-2) or PMO-CD33 conjugated to both CPP and NLS (EEV-PMO-CD 33-5), and were formulated in saline (0.9% weight/volume sodium chloride). The CPP used was CPP12, which has the cyclic amino acid sequence of FfΦ RrRr. PBMCs (peripheral blood mononuclear cells) were isolated 1 day (d 4) and 7 days (10) after 5mpk injection to detect splice correction. Blood, serum and urine samples were collected 1 day and/or 7 days after each injection for hematology, clinical chemistry, clotting, urine testing, cytokine and histamine analysis.

Exon exclusion was detected by RT-PCR. Although humans and non-human primates share high sequence homology of the CD33 gene, the 5' -UTR and splicing patterns are different. The sequence encoding the IgV domain of CD33 is located in exon 2 of human and in exon 5 of the non-human primate CD33 gene. Thus, the skipping of exon 2 in human CD33 (D2-CD 33) resulting in the ΔIgV-CD33 protein corresponds to the skipping of exon 5 of non-human primate CD 33. Monkey PBMCs were collected at the above specific time points. Total RNA was extracted and RT-PCR was performed using forward primer 5'-CTCAGACATGCCGCTGCT-3' and reverse primer 5'-TTGAGCTGGATGGTTCTCTCCG-3' to yield 700bp full length CD33 mRNA (FL-CD 33) and 320bp exon-5 skipped CD33 mRNA (D5-CD 33). Semi-quantitative PCR analysis following reverse transcription showed that treatment of monkey PBMC cells showed that IV administration of EEV-PMO-CD33-2 and EEV-PMO-CD33-5 instead of PMO resulted in skipping of exon 5 of the CD33 gene and production of D2-CD 33. And the activity of both EEV-PMO-CD33-2 and EEV-PMO-CD33-5 persists for at least 7 days after treatment.

Example 7 tissue Regulation in the CNS Using intrathecally administered cell penetrating peptides coupled to oligonucleotides and Nuclear localization sequences

The purpose is as follows: the objective of this study was to evaluate the tolerability and CNS tissue distribution of PMO-containing test preparations, including test preparations containing regulatory peptides (MP; or NLS), administered to rats by intrathecal injection.

The general method comprises the following steps: fifteen (15) male Sprague Dawley rats with JVC were purchased from Envigo. Animals were assigned to seven (7) treatment groups plus one (1) spare animals. All groups were dosed by intrathecal injection at 50 μl/animal. Group 1 received vehicle. Groups 2-1 and 2-2 received PMO-CD33 at 10 μg/animal and 25 μg/animal, respectively. Groups 3-1 and 3-2 received EEV-PMO-CD33-2 at 10. Mu.g/animal and 25. Mu.g/animal, respectively. Groups 4-1 and 4-2 received EEV-PMO-CD33-1 at 10. Mu.g/animal and 25. Mu.g/animal, respectively. All animals were dosed once on day 0.

Temporary blood was collected 0.5 hours, 2 hours, 6 hours, 10 hours and 24 hours after dosing prior to dosing. Blood was processed into plasma and stored frozen at nominally-70 ℃. On day 1, the end-program was performed approximately 24 hours after dosing. By CO ₂ all animals were euthanized by asphyxiation, followed by thoracotomy and final blood collection via cardiac puncture. The maximum available volume of whole blood was collected into heparin lithium tubes and processed into plasma. The clinical chemistry of the plasma was analyzed by the test facility. The residual plasma was stored frozen. After euthanasia, the maximum volume of CSF available was collected and stored frozen. Brains (dissected into cerebellum, cortex, hippocampus, hypothalamus and olfactory bulb), spinal cord and DRG were removed and frozen separately. All frozen samples were stored at nominal-70 ℃.

Compound synthesis: EEV-PMO-CD33-1 was synthesized according to the following procedure (see also FIG. 14A). PMO-CD33 (5'-GTA ACT GTA TTT GGT ACT TCC-3' -primary amine) having the following sequence was reacted with cyclooctyne-PEG 4-PFP carbonate to obtain cyclooctyne modified PMO. Briefly, a solution of cyclooctyne-PEG 4-PFP in DMF (3 eq-4 eq, 500. Mu.L) was added to a solution of PMO-CD33 in 100mM NaHCO3 (1 eq, 5mM, 500. Mu.L). The reaction mixture was vortexed, centrifuged and incubated at room temperature for 2 hours. The reaction was monitored by LCMS (Q-TOF) using BEH C18 column @ 1.7 μm,2.1 mm. Times.150 mm), buffer A: water, 0.1% fa), buffer B: acetonitrile, 0.1% fa), flow rate (0.3 mL/min), starting with 2% buffer B and gradually increasing to 70%, for 11 minutes, running for a total of 20 minutes. After completion, the reaction mixture was loaded onto a pre-equilibrated PD-10 desalting column and the product was eluted using 1.5mL nuclease-free buffer. azide-EEV (1.5 eq.) was added as a solid to the PMO-PEG4COT solution. The mixture was vortexed to dissolve EEV-PMO-CD33-1, centrifuged to settle the solution, and incubated at room temperature for 8-12 hours to complete as confirmed by LCMS (Q-TOF). The crude mixture was diluted with DMSO, loaded onto a C18 reverse phase column (150 mm x 21.2 mm) at a flow rate of 20mL/min and purified over 20 minutes by using a 5% -25% gradient of water with 0.1% tfa and acetonitrile as solvents. The desired fractions were combined, the pH of the solution was adjusted to 7 by 1M NaOH, and the solution was subjected to a lyophilization process to give a white lyophilized powder. The product was subjected to salt exchange to chloride ion by reconstitution of EEV-PMO-CD33-1 in 1M aqueous NaCl solution. The solution was transferred to pre-equilibrated Amicon tube 3KD and centrifuged at 3500rpm for 20 minutes. This procedure was repeated twice, followed by three saline treatments (0.9% nacl, sterile, endotoxin free) until the conductivity of the final filtrate reached that of the reference saline solution. The solution was further diluted with saline to the desired formulation concentration and sterile filtered in a biosafety cabinet. The concentration of each formulation according to the following table was re-measured after filtration. Purity and identity of each formulation was assessed by LCMS (QTOF). The endotoxin amount, residual free peptide, TFA content and pH of the formulation were further determined.

EEV-PMO-CD33-2 was synthesized according to the following procedure (see also FIG. 14B). Using PYAOP as a coupling agent, PMO-CD33 (5'-GTA ACT GTA TTT GGT ACT TCC-3' -primary amine) having the following sequence was reacted with EEV. Briefly, EEV (1.5 eq), PYAOP (1.5 eq) and DIPEA (3 eq) were dissolved in NMP and reacted for 2 minutes. The solution was then added to a DMSO solution of PMO-CD33 (1 eq.) with a final PMO concentration of 50mg/mL. The reaction mixture was vortexed, centrifuged and incubated at room temperature for 2 hours. The reaction was monitored by LCMS (Q-TOF) using BEH C18 column @ 1.7 μm,2.1 mm. Times.150 mm), buffer A: water, 0.1% fa), buffer B: acetonitrile, 0.1% fa), flow rate (0.3 mL/min), starting with 2% buffer B and gradually increasing to 70%, for 11 minutes, running for a total of 20 minutes. After completion, the reaction mixture was loaded onto a C18 reverse phase column (150 mm x 21.2 mm) at a flow rate of 20mL/min and purified over 20 minutes by using a 5% -25% gradient of water with 0.1% tfa and acetonitrile as solvents. The desired fractions were combined, the pH of the solution was adjusted to 7 by 1M NaOH, and the solution was subjected to a lyophilization process to give a white lyophilized powder. The product was subjected to salt exchange to chloride and formulated as described above.

The chemical structures of PMO-CD33, EEV-PMO-CD33-1 and EEV-PMO-CD33-2 are shown below:

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Experiment design: animals were treated as shown in the following table:

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No=number; an=animal; IT = intrathecal

Bioassay sample analysis: tissues were thawed, weighed and homogenized (w/v, 1/5) with RIPA buffer spiked with 1x protease inhibitor cocktail. The tissue homogenate was centrifuged at 5000rpm for 5 minutes at 4 ℃. The supernatant was precipitated with a mixture of H2O, acetonitrile and MeOH and centrifuged at 15000rpm for 15 minutes at 4 ℃. The supernatant was transferred to an injection plate for LC-MS/MS analysis. The dynamic range of LC-MS/MS assay is 25ng/g tissue to 50,000ng/g tissue.

Results: individual rat body weight was measured immediately after PMO (antisense compound (AC) alone) and EEV-PMO administration and 24 hours after dose. No significant decrease in body weight was observed after EEV-PMO-CD33-1, EEV-PMO-CD33-2 and PMO-CD33 administration compared to vehicle controls. 30 minutes and 8 hours after dosing, rats were observed for clinical characteristics and body temperature was measured. No serious side effects were observed. Clinical chemistry (albumin, albumin-globulin ratio, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, calcium, cholesterol, creatine kinase, and creatinine) to measure liver and kidney toxicity was also assessed 24 hours after IT injection. No significant toxicity was detected by clinical chemical evaluation in EEV-PMO-CD33-1, EEV-PMO-CD33-2 and PMO-CD33 treated rats compared to vehicle control.

After treatment, various rat brain tissue sections (cerebellum, cortex, hippocampus, olfactory bulb) were collected and frozen 24 hours after IT injection. Rat brain tissues were pooled together and homogenized. The tissue homogenates were analyzed using LC-MS/MS to quantify the amount of PMO, EEV-PMO-CD33-2 and EEV-PMO-CD33-1 detected in various tissue sections of rat brain. Both EEV-PMO-CD33-2 and EEV-PMO-CD33-1 showed an increase in uptake in brain tissue compared to PMO-CD33 alone (FIG. 15). EEV-PMO-CD33-1 was detected at higher concentrations in rat brain compared to PMO-CD33 alone (12-fold increase in cerebellum, 4-fold increase in cortex, 15-fold increase in hippocampus, and 11-fold increase in olfactory bulb) and EEV-PMO-CD33-2 (9-fold increase in cerebellum, 1.5-fold increase in hippocampus, 6.7-fold increase in olfactory bulb). In particular, EEV-PMO-CD33-1 shows much higher expression in the cerebellum and olfactory bulb than PMO or EEV-PMO-CD33-2 alone.

Rat spinal cord, dorsal Root Ganglion (DRG) and cerebrospinal fluid (CSF) were also collected 24 hours after IT injection. The concentrations of PMO-CD33, EEV-PMO-CD33-2 and EEV-PMO-CD33-1 in rat spinal cord, DRG and CSF were measured by LC-MS/MS. At 24 hours after IT injection, a higher concentration of EEV-PMO-CD33-1 was detected in spinal cord, DRG and CSF than the same dose of PMO-CD33 and EEV-PMO-CD33-2 alone (FIGS. 16A-16C). 24 hours after injection, a higher concentration of EEV-PMO was detected in both spinal cord (fig. 16a,40 fold increase) and DRG (fig. 16b,60 fold increase) compared to PMO alone. In particular, EEV-PMO-CD33-1 containing NLS showed much higher expression in spinal cord, DRG and CSF than PMO alone or EEV-PMO lacking NLS at comparable doses.

In summary, LC-MS data demonstrated superior delivery efficiency of EEV-PMO-CD33-1 containing a modulator peptide (MP; or NLS) into the CNS compared to both PMO alone and EEV-PMO-CD33-2 without modulator peptide.

Conclusion: taken together, these data indicate that these conjugated PMOs administered to rats by intramedullary injection are well tolerated and well retained within the CNS. Notably, EEV-PMO-CD33-1 is delivered in the central nervous system (including cortex, hippocampus, olfactory bulb, cerebellum, spinal cord, and DRG). EEV-conjugation enhances PMO delivery into the CNS compared to PMO alone or EEV-conjugates lacking NLS.

Claims (99)

1. A compound, the compound comprising:

(a) Cell Penetrating Peptide (CPP);

(b) A Therapeutic Moiety (TM); and

(C) An Exocyclic Peptide (EP), wherein the tissue distribution or retention of said compound is modulated compared to a compound comprising said CPP and said TM but not comprising said EP.

2. The compound of claim 1, wherein the EP is conjugated to the CPP.

3. The compound of claim 1, wherein the EP is conjugated to the TM.

4. A compound according to any one of claims 1 to 3, wherein the CPP is a cyclic cell penetrating peptide (cCPP).

5. The compound of any one of claims 1 to 4, wherein the EP comprises at least one positively charged amino acid residue.

6. The compound of claim 5, wherein the EP comprises at least one lysine residue and/or at least one arginine residue.

7. The compound of claim 5, wherein the EP comprises at least one lysine residue.

8. The compound of claim 5, wherein the EP comprises the sequence PKKKRKV.

9. The compound of claim 5, wherein the EP comprises a polypeptide selected from the group consisting of KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、KKKKK、KKKRK、KBKBK、KKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV And PKKKRKG.

10. The compound of any one of claims 1 to 4, wherein the EP is a Nuclear Localization Signal (NLS).

11. The compound of any one of claims 1 to 10, wherein the therapeutic moiety is a protein, polypeptide, small molecule, or oligonucleotide other than an antisense compound.

12. The compound of any one of claims 1 to 10, wherein the therapeutic moiety is an Antisense Compound (AC).

13. The compound of claim 12, wherein the AC comprises at least one modified nucleotide or nucleic acid selected from Phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino nucleotides, locked nucleic acids (lnas), peptide nucleic acids (pnas), nucleotides comprising a 2' -0-methyl (2 ' -OMe) modified backbone, 2'0-methoxy-ethyl (2 ' -MOE) nucleotides, 2',4' -constrained ethyl (cet) nucleotides, and 2' -deoxy-2 ' -fluoro- β -D-arabinonucleic acid (2 ' f-ANA), and

Wherein hybridization of the AC to the target sequence reduces or prevents splicing, inhibits or modulates translation, mediates degradation, or blocks amplification of nucleotide repeat sequences.

14. The compound of claim 12, wherein the AC comprises a small interfering RNA (siRNA), microrna (miRNA), ribozyme, immunostimulatory nucleic acid, antisense, antagomir, antimir, microrna mimetic, supermir, ul adapter, aptamer, or CRISPR gene editing mechanism.

15. The compound of any one of claims 12 to 14, wherein the cyclic peptide is conjugated to the 3' end of the AC.

16. The compound of any one of claims 12 to 14, wherein the cyclic peptide is conjugated to the 5' end of the AC.

17. The compound of any one of claims 12 to 14, wherein the cyclic peptide is conjugated to the backbone of the AC.

18. The compound of any one of claims 12 to 17, comprising a linker that conjugates the cyclic peptide with the AC.

19. The compound of claim 18, wherein the linker is covalently bound to the 5' end of the AC.

20. The compound of claim 18, wherein the linker is covalently bound to the 3' end of the AC.

21. The compound of claim 18, wherein the linker is covalently bound to the backbone of the AC.

22. The compound of any one of claims 18 to 21, wherein the linker is covalently bound to a side chain of an amino acid residue on the cyclic peptide.

23. The compound of any one of claims 18 to 22, wherein the linker comprises a divalent or trivalent C ₁-C₅₀ Alkylene wherein 1 to 25 methylene groups are optionally and independently substituted with-N (H) -, -N (C) ₁-C₄ Alkyl) -, -N (cycloalkyl) -, -O-, -C (O) O-, -S (O) ₂-、-S(O)₂N(C₁-C₄ Alkyl) -, S (O) ₂ N (cycloalkyl) -, -N (H) C (O) -, -N (C) ₁-C₄ Alkyl) C (O) -, -N (cycloalkyl) C (O) -, -C (O) N (H) -, -C (O) N (C) ₁-C₄ alkyl), -C (O) N (cycloalkyl), aryl, heteroaryl, cycloalkyl or cycloalkenyl substitution.

24. The compound of any one of claims 18 to 22, wherein the linker comprises:

(i) One or more D or L amino acid residues, each of which is optionally substituted;

(ii) Optionally substituted alkylene;

(iii) Optionally substituted alkenylene;

(iv) Optionally substituted alkynylene;

(v) Optionally substituted carbocyclyl;

(vi) Optionally substituted heterocyclyl;

(vii) - (J-R) ¹ ) z-, wherein each R ¹ independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently NR ³、-NR³ C (O) -, S or O, wherein each R ³ independently is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted, and z is independently an integer from 1 to 20;

(viii) - (J-R) ² ) x-, wherein each R ² in each case independently alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, each J is independently in each case NR ³、-NR³ C (O) -, S or O, wherein each R ³ Independently is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted, and z is independently an integer from 1 to 20; or (b)

(Ix) A combination thereof.

25. The compound of claim 24, wherein the linker comprises:

(i) Residues of lysine, glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminopentanoic acid, or combinations thereof;

(ii)-(J-R¹)z；

(iii) - (J-R) ² ) x; or (b)

(Iv) A combination thereof.

26. The compound of claim 24 or 25, wherein each R ¹And R is ² Each independently is alkylene, each J is O, each x is independently an integer from 1 to 20, and each z is independently an integer from 1 to 20.

27. The compound of claim 18, wherein the linker comprises:

(i) - (OCH) ₂CH₂)_z -subunits, wherein z is an integer from 2 to 20;

(ii) Residues of glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminopentanoic acid, or combinations thereof; or (b)

(Iii) A combination of (i) and (ii).

28. The compound of any one of claims 18 to 27, wherein the linker has the structure:

Wherein:

each AA is independently a residue of glycine, β -alanine, 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminopentanoic acid;

AA (AA) _SC Is a side chain of an amino acid on the cyclic peptide;

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.

29. The compound of any one of claims 18 to 27, wherein the linker has the structure:

Wherein:

x is an integer from 1 to 20;

y is an integer from 1 to 5;

z is an integer from 2 to 20;

m is a binding moiety; and

AA (AA) _SC is a side chain of an amino acid on the cyclic peptide.

30. The compound of claim 29, wherein M is:

-C(O)-、

Wherein: r ¹ Is alkylene, cycloalkyl or wherein m is an integer from 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, and wherein each B is independently selected from nucleobases.

31. The compound of claim 30, wherein M is-C (O).

32. The compound according to any one of claims 29 to 31, wherein z is 11.

33. The compound according to any one of claims 29 to 32, wherein x is 1.

34. A compound according to any one of the preceding claims comprising an Exocyclic Peptide (EP) conjugated to a cyclic peptide according to any one of the preceding claims or to a linker according to any one of the preceding claims.

35. The compound of any one of claims 29 to 34, comprising an Exocyclic Peptide (EP) conjugated to an amino group of the linker.

36. The compound of claim 34 or 35, wherein the EP comprises 2 to 10 amino acid residues.

37. The compound of any one of claims 34 to 36, wherein the EP comprises 4 to 8 amino acid residues.

38. The compound of any one of claims 34 to 37, wherein the EP comprises 1 or 2 amino acids comprising a side chain comprising a guanidino group or protonated form thereof.

39. The compound of any one of claims 34 to 38, wherein the EP comprises 1,2, 3 or 4 lysine residues.

40. The compound of claim 39, wherein the amino group on the side chain of each lysine residue is trifluoroacetyl (-COCF) ₃ ) A group, allyloxycarbonyl (Alloc), 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl (Dde) or (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene-3) -methylbutyl (ivDde) group.

41. The compound of any one of claims 34 to 40, wherein the EP comprises at least 2 amino acid residues having a hydrophobic side chain.

42. The compound of claim 41, wherein the amino acid residue having a hydrophobic side chain is valine, proline, alanine, leucine, isoleucine or methionine.

43. The compound of any one of claims 34-42, wherein the exocyclic peptide comprises one of the following sequences ：PKKKRKV;KR;RR;KKK;KGK;KBK;KBR;KRK;KRR;RKK;RRR;KKKK;KKRK;KRKK;KRRK;RKKR;RRRR;KGKK;KKGK;KKKKK;KKKRK;KBKBK;KKKRKV;PGKKRKV;PKGKRKV;PKKGRKV;PKKKGKV;PKKKRGV; Or PKKKRKG.

44. The compound of any one of claims 34 to 43, wherein the exocyclic peptide has the structure: ac-P-K-K-K-R-K-V-.

45. A compound according to any one of the preceding claims comprising the structure:

Wherein:

x is an integer from 1 to 20;

y is an integer from 1 to 5;

z is an integer from 1 to 20;

EP is a cyclic exopeptide;

M is a binding moiety;

AC is an antisense compound; and

AA (AA) _SC Is an amino acid residue of the cyclic peptide.

46. The compound of any one of claims 1 to 45, wherein the cyclic peptide comprises 4 to 20 amino acid residues in the cyclic peptide, wherein at least two amino acid residues comprise a guanidino group or protonated form thereof, and at least two amino acid residues independently comprise a hydrophobic side chain.

47. The compound of any one of claims 1 to 46, wherein the cyclic peptide comprises 2,3 or 4 amino acid residues comprising a guanidino group or protonated form thereof.

48. The compound of claim 46 or 47, wherein the cyclic peptide comprises 2,3, or 4 amino acid residues comprising a hydrophobic side chain.

49. The compound of any one of claims 46 to 48, wherein the cyclic peptide comprises at least one amino acid comprising a peptide selected from the group consisting of seq id no Or a side chain of a protonated form thereof.

50. The compound of any one of claims 46 to 49, wherein the cyclic peptide comprises 1,2,3 or 4 amino acids comprising a polypeptide selected from the group consisting of

Or a side chain of a protonated form thereof.

51. The compound of any one of claims 46 to 50, wherein the cyclic peptide comprises at least one glycine residue.

52. The compound of any one of claims 46 to 51, wherein the cyclic peptide comprises 1,2, 3 or 4 glycine residues.

53. The compound of any one of the preceding claims, wherein the cyclic peptide has the structure of formula (I):

Or a protonated form thereof,

Wherein:

R ₁、R₂And R is ₃ Amino acid residues which may each independently be H or have a side chain comprising an aromatic group;

R ₁、R₂And R is ₃ at least one of which is an aromatic or heteroaromatic side chain of an amino acid;

R ₄And R is ₆ Independently H or an amino acid side chain;

AA (AA) _SC Is an amino acid side chain;

q is 1, 2, 3 or 4; and

Each m is independently an integer of 0, 1, 2 or 3.

54. The compound of claim 53, wherein R is ₄ Is H or an amino acid residue comprising a side chain containing an aromatic group.

55. The compound of claim 53 or 54, wherein the amino acid residue comprising a side chain comprising an aromatic group is phenylalanine.

56. The compound of any one of claims 53 to 55, wherein R ₁、R₂、R₃And R is ₄ Comprises phenylalanine.

57. The compound of any one of claims 53 to 56, wherein R ₁、R₂、R₃And R is ₄ Both of which are H.

58. The compound of any one of claims 53 to 57, wherein the cyclic peptide has the structure of formula (I-1), (I-2), (I-3), or (I-4):

Or a protonated form thereof,

Wherein:

AA (AA) _SC Is an amino acid side chain; and

Each m is independently an integer from 0 to 3.

59. The compound of any one of claims 53 to 58, wherein the cyclic peptide has the structure of formula (V-1):

Or a protonated form thereof,

Wherein:

AA (AA) _SC Is an amino acid side chain; and

Each m is independently an integer from 0 to 3.

60. The compound of any one of claims 53 to 59, wherein AA _SC a side chain comprising an asparagine residue, an aspartate residue, a glutamine residue, a glutamate residue, a homoglutamate residue or a homoglutamate residue.

61. The compound of any one of claims 53 to 59, wherein AA _SC Side chains comprising glutamine residues.

62. The compound of any one of claims 1 to 61, wherein the cyclic peptide comprises FGFGRGR.

63. The compound of any one of claims 1 to 61, wherein the cyclic peptide comprises GfFGrGr.

64. The compound of any one of claims 1 to 61, wherein the cyclic peptide comprises Ff Φ GRGR.

65. The compound of any one of claims 1 to 64 comprising a structure of formula (C):

Or a protonated form thereof,

Wherein:

R ₁、R₂And R is ₃ Amino acid residues which may each independently be H or have a side chain comprising an aromatic group;

R ₄And R is ₆ 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 2 to 20;

y is an integer from 1 to 5;

q is an integer from 1 to 4;

z' is an integer from 2 to 20;

Wherein the AC is an oligonucleotide.

66. The compound of any one of claims 1 to 64, having the structure of formula (C-1), (C-2), (C-3), or (C-4):

/>

/>

Or a protonated form thereof.

67. The compound of any one of the preceding claims, wherein the compound modulates splicing of exons 2, 8, 11, 17, 19, 23, 29, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 55, and 59 of DMD.

68. The compound of any one of the preceding claims, wherein the compound modulates splicing of exons 2, 8, 11, 23, 43, 44, 45, 50, 51, 53 and 55 of DMD.

69. The compound of any one of the preceding claims, wherein the compound modulates splicing of exon 2, 23, 44 or 51 of DMD.

70. The compound of any one of the preceding claims, wherein the compound modulates splicing of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7a, and exon 7b of CD 33.

71. The compound of any one of the preceding claims, wherein the C-terminus of the EP sequence is conjugated to the CPP.

72. The compound of claim 1 selected from the group consisting of EEV-PMO-MDX1,2,3, EEV-PMO-CD33-1 and the compounds shown in table C of example 5.

73. A pharmaceutical composition comprising a compound according to any one of claims 1 to 72.

74. A cell comprising a compound according to any one of claims 1 to 72.

75. A method of modulating tissue distribution or retention of a therapeutic agent in a subject in need thereof, the method comprising administering a compound of any one of claims 1-72.

76. The method of claim 75, wherein the compound is administered intrathecally to the subject and the compound modulates tissue distribution or retention of the therapeutic agent in the Central Nervous System (CNS) tissue.

77. The method of claim 75, wherein the compound modulates tissue distribution or retention of the therapeutic agent in muscle tissue.

78. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering a compound of any one of claims 1-72.

79. The method of claim 78, wherein 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 spatially blocks mRNA.

80. The method of claim 78, wherein administration of the compound modulates splicing of target pre-mRNA.

81. The method of any one of claims 78 to 80, wherein the disease or disorder is a central nervous disorder, a neuromuscular disorder, or a musculoskeletal disorder.

82. The method of any one of claims 78 to 80, wherein the disease or disorder is Duchenne muscular dystrophy, β -thalassemia, kobe's dystrophy, osteogenesis imperfecta, cystic fibrosis, merosin-deficient congenital muscular dystrophy type 1A, or spinal muscular atrophy.

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

84. The method of any one of claims 78 to 80, wherein the disease or disorder is fragile X, friedreich's ataxia (FRDA), huntington's Disease (HD), myotonic dystrophy type 1 (DM 1), myotonic dystrophy type 2 (DM 2), spinal and Bulbar Muscular Atrophy (SBMA), spinocerebellar ataxia type 1 (SCA 1), spinocerebellar ataxia type 2 (SCA 2), or spinocerebellar ataxia type 3 (SCA 3).

85. The method of claim 84, wherein the disease or disorder is fragile X, friedreich's ataxia (FRDA), huntington's Disease (HD), myotonic dystrophy type 1 (DM 1).

86. The method of any one of claims 78 to 80, wherein the disease or disorder comprises a neuroinflammatory disease.

87. The method of claim 86, wherein the neuroinflammatory disorder comprises alzheimer's disease or parkinson's disease.

88. A method of modulating tissue distribution or retention of a therapeutic agent in the Central Nervous System (CNS) of a subject, the method comprising:

Administering intrathecally to the subject a compound comprising:

(a) Cell Penetrating Peptide (CPP);

(b) A Therapeutic Moiety (TM) comprising a therapeutic agent; and

(C) A cyclic Exopeptide (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 CNS of the subject as compared to a second tissue of the CNS of the subject.

89. The method of claim 88, wherein the therapeutic agent is a CD 33-targeting antisense compound.

90. The method of claim 89, wherein the CPP is a cyclic CPP.

91. The method of claim 88, wherein the EP is selected from the group consisting 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 And PKKKRKG.

92. The method of claim 88, wherein the subject has a central nervous system disease or disorder, or a neuroinflammatory disease or disorder.

93. The method of claim 92, wherein the subject has alzheimer's disease or parkinson's disease.

94. A method of modulating tissue distribution or retention of a therapeutic agent in a subject's musculature, the method comprising:

Administering to the subject a compound comprising:

(a) Cell Penetrating Peptide (CPP);

(b) A Therapeutic Moiety (TM) comprising a therapeutic agent; and

(C) A cyclic Exopeptide (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 muscular system of the subject as compared to a second tissue of the muscular system of the subject.

95. The method of claim 94, wherein the therapeutic agent is an antisense compound that targets DMD.

96. The method of claim 94, wherein the CPP is a cyclic CPP.

97. The method of claim 94, wherein the EP is selected from the group consisting 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 And PKKKRKG.

98. The method of claim 94, wherein the subject has a neuromuscular disorder or a musculoskeletal disorder.

99. The method of claim 98, wherein the subject has Duchenne type muscular dystrophy.