Compounds and methods for skipping exon 50 in duchenne muscular dystrophy

Abstract

This article describes EEV-cargo conjugates and compositions thereof comprising (a) an endosome escape mediator (EEV) containing a cyclic cell-penetrating peptide (cCPP); and (b) cargo comprising an endosome-cargo conjugate containing a cyclic cell-penetrating peptide (cCPP). DMD Oligonucleotides complementary to the target sequence of a gene's precursor mRNA transcript, wherein the target sequence comprises at least a portion of the 5' flanking intron of exon 50, at least a portion of exon 50, at least a portion of the 3' flanking intron of exon 50, or a combination thereof. This document also describes methods for treating DMD using the compounds described herein.

Description

Cross-references to related applications

[0001] This application claims priority to the filing dates of U.S. Provisional Application Serial No. 63 / 585,311, filed September 26, 2023; U.S. Provisional Application Serial No. 63 / 588,115, filed October 5, 2023; U.S. Provisional Application Serial No. 63 / 557,125, filed February 23, 2024; and U.S. Provisional Application Serial No. 63 / 640,506, filed April 30, 2024, the disclosure of each of which is incorporated herein by reference in its entirety. Background Technology

[0002] Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to alterations in dystrophin proteins. The gene encoding dystrophin (i.e.,...) DMD Genetic modifications in genes can lead to DMD. These genetic modifications cause… DMD Gene reading frame shifts, resulting in nonfunctional truncated DMD proteins. One approach to treating DMD patients is to deliver reversible gene sequences to subjects with DMD. DMD Compounds within gene reading frames. Antisense oligonucleotides can skip readings and... DMD To restore gene reading frame shift-related internal exons DMD The gene reading frame is skipped, thus avoiding the production of non-functional truncated DMD proteins. Dystrophic proteins produced by exon skipping retain the functions lost in disease states.

[0003] DMD is caused by mutations in one or more exons, such as mutations in the exon 44-55 region. In 2016, the U.S. Food and Drug Administration (FDA) granted accelerated approval to Exondys 51 (also known as eteplirsen), the first morpholinodiaminophosphate (PMO)-based drug developed for exon 51 skipping in DMD. Approximately 13% of DMD patients have mutations suitable for exon 51 skipping (Aoki, et al., Proc Natl Acad Sci USA. 109(34):13763-13768). However, there are currently no approved therapies for DMD patients with mutations suitable for exon 50 skipping. Summary of the Invention

[0004] This article discloses an EEV-cargo conjugate for skipping exon 50 in subjects with Duchenne muscular dystrophy (DMD).

[0005] This disclosure relates to an EEV-cargo conjugate comprising: (a) Endosome escape mediators (EEVs) containing cyclic cell-penetrating peptides (“cCPP”); and (b) Goods, which include... DMD Oligonucleotides complementary to the target sequence of a gene's precursor mRNA transcript, wherein the target sequence comprises at least a portion of the 5' flanking intron of exon 50, at least a portion of exon 50, at least a portion of the 3' flanking intron of exon 50, or a combination thereof.

[0006] In the implementation scheme, the EEV-cargo conjugate may include: (a) Cyclic cell-penetrating peptide (cCPP), wherein cCPP has the structure of formula (2): Equation (2): (2), or its protonated form, in: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are independently aryl or heteroaryl side chains of amino acids; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; q is 1, 2, 3, or 4; and m′ and m′′ are each an independent integer from 0 to 3; (b) Linear exocyclic peptides (EPs), wherein the EPs contain 2 to 10 amino acid residues; (c) Connector of type (A′): Formula (A) ′): (A′) in: ** is the attachment site for linear exocyclic peptides (EP); * is the attachment site for cyclic cell-penetrating peptide (cCPP); L 1 and L 2 Independently designed as a connector arm; ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; and M is a reaction handle containing a functional group that reacts with the corresponding functional group on the cargo to form a bonding group (M'); and (d) Goods containing the human dystrophin gene ( DMD Oligonucleotides that hybridize to the 50th exon in the precursor mRNA transcript to cause exon skipping, wherein the oligonucleotides hybridize to or contain the nucleic acid sequences shown in Tables 11A-11D, 12A-12D or 13.

[0007] In the implementation scheme, the EEV-cargo conjugate may include: (a) Cyclic cell-penetrating peptide (cCPP), wherein cCPP has the structure of formula (2): Equation (2): (2), or its protonated form, in: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are independently aryl or heteroaryl side chains of amino acids; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; q is 1, 2, 3, or 4; and m′ and m′′ are each an independent integer from 0 to 3; (b) Linear exocyclic peptides (EPs), wherein the EPs contain 2 to 10 amino acid residues; (c) A joint having the structure of formula (B′): Equation (B′): (B′) in: ** is the attachment site for linear exocyclic peptides (EP); * is the attachment site for cyclic cell-penetrating peptide (cCPP); ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; M is a reaction handle, which contains a functional group that reacts with the corresponding functional group on the cargo to form a bonding group (M'); x′ is an integer from 1 to 12; z′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; j′′ is 0, 1, or 2, where j′′ is 0 when z′ is 0; and (d) Goods containing the human dystrophin gene ( DMD Oligonucleotides that hybridize to the 50th exon in the precursor mRNA transcript to cause exon skipping, wherein the oligonucleotides hybridize to or contain the nucleic acid sequences shown in Tables 11A-11D, 12A-12D or 13.

[0008] In the implementation scheme, the EEV-cargo conjugate may include: (a) Cyclic cell-penetrating peptide (cCPP), wherein cCPP has the structure of formula (2): Equation (2): (2), or its protonated form, in: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are independently aryl or heteroaryl side chains of amino acids; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; q is 1, 2, 3, or 4; and m′ and m′′ are each an independent integer from 0 to 3; (b) Linear exocyclic peptides (EPs) containing 2 to 10 amino acid residues; (c) A joint having the structure of formula (C′): Equation (C′): (C′) in: ** is the attachment site for linear exocyclic peptides (EP); * is the attachment site for cyclic cell-penetrating peptide (cCPP); ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; M is a reaction handle, which contains a functional group that reacts with the corresponding functional group on the cargo to form a bonding group (M'); X o ′ is a hydrophobic component; x′ is an integer from 1 to 12; z′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; j′′ is 0, 1, or 2, where j′′ is 0 when z′ is 0; and (d) Goods containing the human dystrophin gene ( DMD Oligonucleotides that hybridize to the 50th exon in the precursor mRNA transcript to cause exon skipping, wherein the oligonucleotides hybridize to or contain the nucleic acid sequences shown in Tables 11A-11D, 12A-12D or 13.

[0009] In the implementation scheme, the EEV-cargo conjugate may include: (a) Cyclic cell-penetrating peptide (cCPP), wherein cCPP has the structure of formula (2): Mode (2)： (2), or its protonated form, in: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are independently aryl or heteroaryl side chains of amino acids; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; q is 1, 2, 3, or 4; and m′ and m′′ are each an independent integer from 0 to 3; (b) Linear exocyclic peptides (EPs) containing 2 to 10 amino acid residues; (c) A joint having the structure of formula (D′): Equation (D′): (D′) in: ** is the attachment site for linear exocyclic peptides (EP); * is the attachment site for cyclic cell-penetrating peptide (cCPP); ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; M is a reaction handle, which contains a functional group that reacts with the corresponding functional group on the cargo to form a bonding group (M'); X o ′ is a hydrophobic component; K # It consists of D-lysine or L-lysine residues; x′ is an integer from 1 to 12; z′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; j′′ is 0, 1, or 2, where j′′ is 0 when z′ is 0; and (d) Goods containing the human dystrophin gene ( DMD Oligonucleotides that hybridize to the 50th exon in the precursor mRNA transcript to cause exon skipping, wherein the oligonucleotides hybridize to or contain the nucleic acid sequences shown in Tables 11A-11D, 12A-12D or 13.

[0010] In the embodiments, the goods are oligonucleotides. In the embodiments, the oligonucleotides are therapeutic oligonucleotides. In the embodiments, the goods are antisense oligonucleotides. In the embodiments, the goods are oligonucleotides comprising at least one modified nucleotide or nucleic acid. In the embodiments, the modified nucleotide or nucleic acid is a phosphate-thioester (PS) nucleotide, a diaminophosphate morpholino oligonucleotide (PMO), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a nucleotide comprising a 2'-O-methyl (2'-OMe) modified backbone, a 2'-O-methoxy-ethyl (2'-MOE) nucleotide, a 2',4' restricted ethyl (cEt) nucleotide, or a 2'-deoxy-2'-fluoro-β-D-arabinose nucleic acid (2'F-ANA). In the embodiments, the oligonucleotide comprises at least one diaminophosphate morpholino oligonucleotide (PMO). In the embodiments, each nucleotide in the oligonucleotide is a diaminophosphate morpholino oligonucleotide (PMO).

[0011] In one embodiment, the oligonucleotide hybridizes with the nucleic acid sequence of exon 50 shown in Tables 11A-11D, 12A-12D, or 13. In another embodiment, the oligonucleotide comprises the reverse complementary sequence of a nucleic acid sequence in Tables 11A-11D, 12A-12D, or 13. In yet another embodiment, the oligonucleotide comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with the nucleic acid sequence or its reverse complementary sequence in Tables 11A-11D, 12A-12D, or 13.

[0012] In one embodiment, the oligonucleotide is complementary to a portion of exon 50. In another embodiment, the oligonucleotide is complementary to a portion of the 5' flanking intron of exon 50 and a portion of exon 50. In yet another embodiment, the oligonucleotide is complementary to a portion of the 3' flanking intron of exon 50 and a portion of exon 50.

[0013] In the implementation scheme, the first nucleotide of the oligonucleotide can be combined with... DMD SEQ ID in exon 50 of the gene NO:1 -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +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, +50, +51, +52, +53, + Nucleotide hybridization at positions 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, and 109.

[0014] This disclosure relates to a pharmaceutical composition comprising the EEV-cargo conjugate described herein.

[0015] This disclosure relates to a cell comprising the EEV-cargo conjugate described herein.

[0016] This disclosure relates to a method of treating DMD, comprising administering the EEV-cargo conjugate described herein to a patient in need. Attached Figure Description

[0017] Figure 1A and 1B This demonstrates the conjugation chemistry of conjugating oligonucleotides to endosomal escape mediators (EEVs).

[0018] Figure 2A-2B The conjugation chemistry of linking EEVs (including CPPs and linkers) to antisense oligonucleotides is shown, where the antisense oligonucleotides are shown as having no ( Figure 2A ) and containing ( Figure 2B ) Components containing PEG2.

[0019] Figures 3A-3B The percentage of exon 50 skip reads in CRL-2061™ cells treated with 5 μM 21-dimer PMO is shown.

[0020] Figures 4A-4B The percentage of exon 50 skip reads in CRL-2061™ cells treated with 5 μM 22-dimer PMO is shown.

[0021] Figures 5A-5B The percentage of exon 50 skip reads in CRL-2061™ cells treated with 5 μM 23-dimer PMO is shown.

[0022] Figures 6A-6B The percentage of exon 50 skip reads in CRL-2061™ cells treated with 5 μM 24-mer PMO is shown.

[0023] Figures 7A-7D The percentage of exon 50 skip reads in CRL-2061™ cells treated with 1 μM and 3 μM MPMO is shown. Figure 7A Exon 50 skip reads are shown in CRL-2061™ cells treated with 1 μM and 3 μM of 21-mer PMO listed in Table 14A. Figure 7B Exon 50 skip reads are shown in CRL-2061™ cells treated with 1 μM and 3 μM 22-mer PMO as listed in Table 14B. Figure 7C Exon skipping reads are shown in CRL-2061™ cells treated with 1 μM and 3 μM 23-mer PMO listed in Table 14C. Figure 7DExon skipping reads are shown in CRL-2061™ cells treated with 1 μM and 3 μM 24-mer PMO as listed in Table 14D.

[0024] Figures 8A-8C The gastrocnemius muscle (“gastroc”) of hDMD mice treated with IV injection of 15 mpk of various EEV-PMOs is shown. Figure 8A ), diaphragm ( Figure 8B ) and heart tissue ( Figure 8C The percentage of exon 50 skips in the data.

[0025] Figures 9A-9C The gastrocnemius muscle (“gastroc”) of hDMD mice treated with IV injection of 30 mpk of various EEV-PMOs is shown. Figure 9A ), diaphragm ( Figure 9B ) and heart tissue ( Figure 9C The percentage of exon 50 skips in the data.

[0026] Figure 10 EEV-PMO for displaying primary and mature RNA computer A table for off-target analysis. mm = mismatch.

[0027] Figure 11A-11E The gastrocnemius muscle of hDMD mice treated with IV injections of 15 mpk, 30 mpk, and 60 mpk EEV-PMO24 polymer-14 is shown. Figure 11A ), Triceps brachii ( Figure 11B ), Tibialis anterior muscle ( Figure 11C ),heart( Figure 11D ) and diaphragmatic tissue ( Figure 11E The percentage of exon 50 skips in the data.

[0028] Figures 12A-12E The image shows the gastrocnemius muscle of hDMD mice treated with IV injection of EEV-PMO24 polymer-14 one week after injection. Figure 12A ), Triceps brachii ( Figure 12B ), Tibialis anterior muscle ( Figure 12C ),heart( Figure 12D ) and diaphragm ( Figure 12E The percentage of exon 50 skips in the data.

[0029] Figures 13A-13E The study showed the gastrocnemius muscle of hDMD mice treated with IV injection of EEV-PMO24 polymer-14 six weeks post-injection. Figure 13A ), Triceps brachii ( Figure 13B ), Tibialis anterior muscle ( Figure 13C ),heart( Figure 13D ) and diaphragm ( Figure 13EThe percentage of exon 50 skips in the data.

[0030] Figures 14A-14E This study showed the triceps brachii muscle size in hDMD mice treated with IV EEV-PMO24 polymer-14 at 1, 2, 4, 6, 8, and 12 weeks post-injection in a single-dose, duration-of-action study. Figure 14A ), Tibialis anterior muscle ( Figure 14B ), gastrocnemius muscle ( Figure 14C ), diaphragm ( Figure 14D ) and heart ( Figure 14E The percentage of exon 50 skips in the data.

[0031] Figures 15A-15E The image shows the gastrocnemius muscle of hDMD mice treated with IV EEV-PMO24 polymer-14 at 4, 8, 16, 24, 48, 96, and 168 hours post-injection in a single-dose, early time-point study. Figure 15A ), Triceps brachii ( Figure 15B ), Tibialis anterior muscle ( Figure 15C ), diaphragm ( Figure 15D ) and heart ( Figure 15E The percentage of exon 50 skips in the data.

[0032] Figure 16 The study investigated the effects of ddPCR on iPSC-derived cardiomyocytes (CMs) from DMDΔ51 patients treated with 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM EEV-PMO24-mer-14. DMD A graph showing the percentage of exon 50 skip reads.

[0033] Figure 17 As reported by Simple Western Jess analysis, in iPSC-derived cardiomyocytes (CMs) of DMDΔ51 patients treated with 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM EEV-PMO 24-mer-14... DMD A graph showing relative expression, where dystrophin is normalized to total protein and expressed as a percentage relative to untreated normal cells.

[0034] Figure 18 The graph shows the mean fluorescence signal intensity of dystrophin in iPSC-derived cardiomyocytes (CMs) of DMDΔ51 patients treated with 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM and 10 μM EEV-PMO24-mer-14, as measured by immunofluorescence.

[0035] Figure 19This is a graph showing the area of ​​dystrophin-positive cardiomyocytes in iPSC-derived cardiomyocytes (CMs) of DMDΔ51 patients treated with 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM EEV-PMO24-mer-14, as measured by immunofluorescence.

[0036] Figure 20 As reported by ddPCR, DMDΔ51-55 inducible directly reprogrammable myotubes (iDRMs) treated with 0 μM, 1 μM, 3 μM, 10 μM, and 30 μM EEV-PMO24-mer-14 were... DMD A graph showing the percentage of exon 50 skip reads.

[0037] Figure 21 For DMDΔ51-55 inducible directly reprogrammable myotubes (iDRMs) treated with 0 μM, 1 μM, 3 μM, 10 μM, and 30 μM EEV-PMO24 polymer-14, DMD The graph shows the relative expression, which was analyzed by ddPCR and normalized to HPRT1 expression and then normalized to the control group.

[0038] Figure 22 This is a graph showing the relative expression of dystrophin in DMDΔ51-55 inducible direct reprogrammable myotubes (iDRMs) treated with 0 μM, 1 μM, 3 μM, 10 μM, and 30 μM EEV-PMO24-mer-14, as measured by Simple Western Jess analysis.

[0039] Figure 23 This is a graph showing the mean fluorescence signal intensity of dystrophin in DMDΔ51-55 inducible direct reprogrammable myotubes (iDRMs) treated with 0 μM, 1 μM, 3 μM, 10 μM, and 30 μM EEV-PMO24-mer-14, as measured by immunofluorescence.

[0040] Figure 24 This is a graph showing the area of ​​dystrophin-positive myotubes in DMDΔ51-55 inducible direct reprogrammable myotubes (iDRMs) treated with 0 μM, 1 μM, 3 μM, 10 μM, and 30 μM EEV-PMO24-mer-14, as measured by immunofluorescence.

[0041] Figure 25 The percentage of exon 50 skip reads in patient-derived DMDΔ51 skeletal myotubes treated with 0 μM, 1 μM, 3 μM, 10 μM, and 20 μM EEV-PMO24-mer-14 is shown.

[0042] Figure 26The proportion of dystrophin in total protein in AdMyoD-induced patient-derived DMDΔ51 skeletal myotubes treated with 0 μM, 1 μM, 3 μM, 10 μM, and 20 μM EEV-PMO24-mer-14 is shown.

[0043] Figure 27 The structure of the EEV-cargo conjugate is formed through amide chemistry. This conjugate is formed by passing the sequence Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG. 12 It is formed by the EEV of -OH conjugated to the 3′ base of diaminophosphate morpholino oligonucleotide (PMO). The # symbol represents the rest of the PMO.

[0044] Figure 28 The structure of the EEV-cargo conjugate is formed through amide chemistry. This conjugate is formed by passing the sequence Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG. 12 It is formed by the EEV of -K(Ac-Bip)-OH conjugated to the 3′ base of diaminophosphate morpholino oligonucleotide (PMO). The # symbol represents the rest of the PMO.

[0045] Figure 29 The structure of the EEV-cargo conjugate is formed through amide chemistry. This conjugate is formed by passing the sequence Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG. 12 It is formed by the EEV of -Nal-OH conjugated to the 3′ base of diaminophosphate morpholino oligonucleotide (PMO). The # symbol represents the rest of the PMO. Detailed Implementation

[0046] This article discloses a compound for treating patients with Duchenne muscular dystrophy (DMD), whose mutations can be treated by exon skipping. In the embodiment, DMD is caused by... DMD The DMD is caused by mutations in the exon 44-55 region of the gene. In one embodiment, the DMD is caused by a mutation in exon 50. In another embodiment, the compound comprises an EEV designed for delivering oligonucleotide cargo into cells, wherein the oligonucleotide cargo is... DMD The target sequence of the gene's precursor mRNA transcript is complementary, wherein the target sequence comprises at least a portion of the 5' flanking intron of exon 50, at least a portion of exon 50, at least a portion of the 3' flanking intron of exon 50, or a combination thereof. In an embodiment, the EEV-cargo conjugate is delivered intracellularly to a subject in need. In an embodiment, EEV delivery is associated with a conjugate containing... DMDOligonucleotide cargo complementary to the target sequence at the intron-exon junction of gene exon 50. In the embodiment, EEV delivery is coupled with an oligonucleotide cargo containing... DMD An oligonucleotide cargo complementary to the target sequence of the intron nucleotide sequence upstream (or 5′) of exon 50 of a gene. In an embodiment, EEV delivery is coupled with an oligonucleotide cargo containing... DMD Oligonucleotide cargoes that are complementary to the target sequence of the intron nucleotide sequence downstream (or 3′) of gene exon 50.

[0047] In the implementation, the oligonucleotide alters the splicing pattern of the target precursor mRNA with which it hybridizes, thereby forming a respliced ​​target protein. In the implementation, the respliced ​​target protein has enhanced function compared to the target protein produced by splicing the target precursor mRNA in the absence of the oligonucleotide. In the implementation, the respliced ​​target protein exhibits a functional improvement of at least 1%, at least 5%, 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 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, or more, including all values ​​between these values ​​and ranges. In the implementation scheme, the respliced ​​target protein restores its function to at least 1%, at least 5%, 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 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, and up to 100%, including all values ​​between these values ​​and ranges.

[0048] In embodiments, the EEV-cargo conjugate comprises an endosome escape mediator (EEV) and cargo. In embodiments, the cargo is an oligonucleotide. In embodiments, the oligonucleotide is a therapeutic oligonucleotide. In embodiments, the oligonucleotide is an antisense oligonucleotide. In embodiments, the EEV comprises a cyclic cell-penetrating peptide (cCPP). In embodiments, the EEV-cargo conjugate is capable of crossing the cell membrane and binding to a target precursor mRNA in vivo. In embodiments, an EEV-cargo conjugate is provided comprising: a) at least one endosome escape mediator (EEV) comprising a cyclic cell-penetrating peptide (cCPP); and b) at least one cargo. As used herein, “conjugation” can refer to a covalent or non-covalent association between an EEV and a cargo, including chemical conjugation of an EEV to a cargo. Non-limiting examples of conjugation chemistry used for covalent linking between an EEV and a cargo may include, but are not limited to, conjugations using click chemistry or amide-forming chemistry. A non-limiting example of non-covalent linking is via streptavidin / biotin interaction, wherein the EEV is conjugated to the cargo via non-covalent association between biotin and streptavidin.

[0049] In this embodiment, the cargo comprises an oligonucleotide. The conjugation of the oligonucleotide cargo to the EEV can occur at any suitable site on the oligonucleotide. In this embodiment, the EEV can be conjugated to the 5' end, 3' end, or backbone of the oligonucleotide cargo.

[0050] Escape medium (EEV) This document provides an endosomal escape medium (EEV) for transporting cargo molecules across the cell membrane, e.g., delivering cargo to the cytoplasm or nucleus of a cell. The cargo may be a therapeutic or diagnostic molecule. Therapeutic molecules may be oligonucleotides, peptides, small molecules, or combinations thereof. Peptides may be proteins, antibodies, or enzymes. The cargo may be an oligonucleotide. The oligonucleotide may be an antisense oligonucleotide. The oligonucleotide may be a diaminophosphate morpholino oligonucleotide (PMO). The EEV may contain a cell-penetrating peptide (CPP), e.g., a cyclic cell-penetrating peptide (cCPP) conjugated to a linear exocyclic peptide (EP). The EEV may contain one or more linkers. The EEV may contain a hydrophobic component (X). One or more linker arms of the EEV or cCPP may contain a hydrophobic component (X). In embodiments, when the linker arms of the EEV contain more than one hydrophobic component (X), (e.g., X) can be used. o ′、X o The EEV may contain a hydrocarbon component, a polyethylene glycol (PEG) component, a hydrophobic component (X), an amino acid component (AA) containing one or more amino acid residues, or a combination thereof. In one embodiment, an EEV is provided in which the amino acid stereochemistry within the EEV peptide sequence has been modified.

[0051] In the implementation scheme, the EEV is coupled to the cargo. The cargo may be coupled to the cCPP. The cargo may be coupled to the EP. The cargo may be coupled to a connector. The EP may be coupled to the cCPP. The EP may be coupled to both the cargo and the cCPP. The coupling between the EP, cargo, cCPP, or combinations thereof may be non-covalent or covalent. The EP, cargo, cCPP, or combinations thereof may be coupled via one or more connectors.

[0052] Cell-penetrating peptide (CPP) This document provides an endosome escape mediator (EEV) comprising at least one cell-penetrating peptide (CPP). The cell-penetrating peptide may be a cyclic cell-penetrating peptide (cCPP). In an embodiment, the cCPP can penetrate the cell membrane. In an embodiment, the cCPP can deliver cargo to the cytoplasm of the cell. The cCPP can deliver cargo to the cellular location of a target gene, target transcript, and / or target protein. To conjugate the cCPP with the cargo, EP, and / or linker, at least one bond or lone pair electron on the cCPP may be replaced.

[0053] In one embodiment, a hydrophobic component (X) is attached to one or more amino acid residues of the cCPP. In another embodiment, the hydrophobic component (X) is attached to the side chain of a lysine amino acid residue of the cCPP.

[0054] X can be a D-amino acid residue or an L-amino acid residue with a hydrophobic side chain. X can be a naturally occurring or non-naturally occurring amino acid residue with a hydrophobic side chain. X can be an amino acid residue with an aromatic side chain. X can be an amino acid residue with a heteroaromatic side chain. X can be selected from phenylalanine, 3-(4',4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or combinations thereof. X can be 2-naphthylalanine. X can be Na1. X can be d-N1 (nal). X can be 3-(4',4-biphenyl)-L-alanine. X can be Bip. X can be D-Bip (bip). X can be a C4-C8 alkyl hydrocarbon. X can be a C6 alkyl hydrocarbon.

[0055] The total number of amino acid residues in a cCPP is 6 to 20, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, including all ranges and subranges therein. A cCPP may contain 6 to 13 amino acid residues. A cCPP may contain 6 to 10 amino acids. By way of example, a cCPP containing 6-10 amino acid residues may have a structure according to any one of formulas IA to IE: Formula IA to 1-E: , , , or Among them, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9 and AA 10 It consists of amino acid residues.

[0056] cCPP can contain 6 to 8 amino acids. cCPP can contain 8 amino acids.

[0057] Each amino acid in cCPP can be either natural or non-natural. Some abbreviations for natural and non-natural amino acids used in this paper are shown in Table 1.

[0058] As used herein, the term "amino acid" refers to a compound having both an amino group and a carboxylic acid group. Most amino acids (except glycine) also have side chains. As used herein, "amino acid side chain" or "side chain" refers to a characterizing substituent bound to the α-carbon of an amino acid.

[0059] An "α-amino acid" is an amino acid in which the amino group is attached to the first (α) carbon adjacent to the carboxylic acid group, such that the carbon atom of the carbonyl group is separated from the nitrogen atom of the amino group by one carbon atom. A "β-amino acid" (also called "beta-amino acid" or "β-amino acid") is an analogue of an α-amino acid in which the amino group is attached to the second (β) carbon, instead of the α-carbon, such that the carbonyl group is separated from the nitrogen atom of the amino group by two carbon atoms. Examples of β-amino acids include, but are not limited to, β-alanine and β-homophenylalanine.

[0060] "Uncharged" amino acids are those that do not have a charge at physiological pH (e.g., 6.5 to 8.0 or 6.8 to 7.6). It is worth noting that histidine can exist in a neutral or positively charged form at physiological pH.

[0061] Side chains that do not contain aryl or heteroaryl groups are referred to herein as “non-aryl” side chains. In embodiments, side chains that do not contain aryl or heteroaryl groups may be uncharged and are referred to herein as uncharged non-aryl side chains. Amino acids having uncharged non-aryl amino side chains include, but are not limited to, histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, 3-(4-thienyl)-alanine, and their β-amino acid derivatives.

[0062] The term "non-natural amino acid" refers to an organic compound that is a homolog of a natural amino acid because it has a similar structure to the natural amino acid, thus mimicking its structure and reactivity. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of the 20 commonly found naturally occurring amino acids, nor are they the rare natural amino acids selenocysteine ​​or pyrrolidone. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives, or combinations thereof.

[0063] Table 1: Amino Acid Abbreviations

[0064] In one embodiment, cCPP may comprise two adjacent amino acids having hydrophobic side chains. In another embodiment, cCPP may comprise three adjacent amino acids having hydrophobic side chains. In another embodiment, the amino acids having hydrophobic side chains are selected from phenylalanine and naphthylalanine. In another embodiment, the adjacent amino acids having hydrophobic side chains may have D-stereochemistry or L-stereochemistry. In another embodiment, the adjacent amino acids having hydrophobic side chains may have the same stereochemistry. In another embodiment, the adjacent amino acids having hydrophobic side chains may have alternating stereochemistry. In another embodiment, the adjacent amino acids having hydrophobic side chains may have all L-stereochemistry. In another embodiment, the adjacent amino acids having hydrophobic side chains may have all D-stereochemistry.

[0065] In the embodiments, one or two amino acids in cCPP may not have side chains. In the embodiments, all amino acids in cCPP have side chains. As used herein, when no side chain is present, the amino acid has two hydrogen atoms (e.g., -CH2) on one or more carbon atoms of the amine and carboxylic acid that link the amino acid residues. An amino acid without a side chain may be glycine. An amino acid without a side chain may be β-alanine.

[0066] In one embodiment, cCPP may comprise two adjacent amino acids having hydrophobic side chains, not having side chains, or combinations thereof. In another embodiment, cCPP may comprise three adjacent amino acids having hydrophobic side chains, not having side chains, or combinations thereof. In another embodiment, the adjacent amino acids having hydrophobic side chains, not having side chains, or combinations thereof may have alternating stereochemistry. In another embodiment, the adjacent amino acids having hydrophobic side chains, not having side chains, or combinations thereof may have all L-stereochemistry. In another embodiment, the adjacent amino acids having hydrophobic side chains, not having side chains, or combinations thereof may have all D-stereochemistry.

[0067] cCPP may contain 6 to 20, 6 to 10, or 6 to 8 amino acid residues, wherein: (i) at least two amino acids may independently be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid; (ii) at least two amino acids may have side chains containing aryl or heteroaryl groups; and (iii) at least two amino acids may independently have side chains containing guanidine or its protonated form. In an embodiment, (i) the two amino acids may independently be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid; (ii) two or three amino acids may have side chains containing aryl or heteroaryl groups; and (iii) the two amino acids may independently have side chains containing guanidine or its protonated form.

[0068] In the embodiments, one amino acid of cCPP may be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid. In the embodiments, two amino acids may independently be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid. In the embodiments, three amino acids may be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid.

[0069] In an embodiment, one amino acid of cCPP may have a side chain containing an aryl or heteroaryl group. In an embodiment, two amino acids of cCPP may have side chains containing an aryl or heteroaryl group. In an embodiment, three amino acids of cCPP may have side chains containing an aryl or heteroaryl group. In an embodiment, none of the amino acids having side chains containing an aryl or heteroaryl group are adjacent. In an embodiment, two amino acids having side chains containing an aryl or heteroaryl group may be adjacent. In an embodiment, two adjacent amino acids may have opposite stereochemistry. In an embodiment, two adjacent amino acids may have the same stereochemistry. In an embodiment, three amino acids having side chains containing an aryl or heteroaryl group may be adjacent. In an embodiment, three adjacent amino acids may have the same stereochemistry. In an embodiment, three adjacent amino acids may have alternating stereochemistry. The amino acid residues having side chains containing aryl or heteroaryl groups may each independently be phenylalanine, naphthylalanine, or β-homophenylalanine residues, each optionally substituted with one or more substituents. At least one amino acid residue having a side chain containing an aryl or heteroaryl group may be a phenylalanine residue. At least two amino acid residues having side chains containing aryl or heteroaryl groups may be phenylalanine residues. Each amino acid residue having a side chain containing an aryl or heteroaryl group may be a phenylalanine residue. At least one amino acid residue having a side chain containing an aryl or heteroaryl group may be a naphthylalanine residue. At least one amino acid residue having a side chain containing an aryl or heteroaryl group may be a naphthylalanine residue. At least one amino acid residue having a side chain containing an aryl or heteroaryl group may be a β-homophenylalanine residue. An amino acid residue having a side chain containing an aryl or heteroaryl group may be a β-homophenylalanine residue.

[0070] In an embodiment, one amino acid of cCPP may have a side chain that does not contain an aryl or heteroaryl group, referred to herein as a "non-aryl" side chain. In an embodiment, the side chain that does not contain an aryl or heteroaryl group may be uncharged, and referred herein as an uncharged non-aryl side chain. In an embodiment, two amino acids of cCPP may have uncharged non-aryl side chains. In an embodiment, three amino acids of cCPP may have uncharged non-aryl side chains. Amino acids having uncharged non-aryl amino side chains include, but are not limited to, histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine.

[0071] In one embodiment, one amino acid of cCPP has a side chain containing a guanidinium group or its protonated form. In another embodiment, two amino acids of cCPP may have side chains containing a guanidinium group or its protonated form. In another embodiment, three amino acids of cCPP may have side chains containing a guanidinium group or its protonated form. In another embodiment, four amino acids of cCPP may have side chains containing a guanidinium group or its protonated form. In another embodiment, the amino acids of cCPP having side chains containing a guanidinium group or its protonated form are adjacent. In another embodiment, the amino acids of cCPP having side chains containing a guanidinium group or its protonated form are not adjacent. In another embodiment, the amino acid containing a guanidinium group or its protonated form is arginine. In another embodiment, the amino acids of cCPP having side chains containing a guanidinium group or its protonated form have D-stereochemistry or L-stereochemistry. In another embodiment, the amino acids of cCPP having side chains containing a guanidinium group or its protonated form have alternating D-stereochemistry and L-stereochemistry. In another embodiment, the amino acids of cCPP having side chains containing a guanidinium group or its protonated form have the same stereochemistry. In one embodiment, the amino acid having a cCPP containing a side chain comprising a guanidinium group or its protonated form has D-stereochemistry. In another embodiment, the amino acid having a cCPP containing a guanidinium group or its protonated form has L-stereochemistry.

[0072] In the embodiments, cCPP may contain 6 to 10 amino acid residues, wherein: (i) at least two amino acids may be independently glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least two amino acids may have side chains containing aryl or heteroaryl groups; and (iii) at least two amino acids may independently have side chains containing guanidine or its protonated form.

[0073] In the implementation, cCPP may contain 6 to 10 amino acid residues, wherein: (i) two amino acids may be independently glycine, β-alanine or 4-aminobutyric acid residues; (ii) two or three amino acids may have side chains containing aryl or heteroaryl groups; and (iii) two amino acids may independently have side chains containing guanidine or its protonated form.

[0074] In the implementation scheme, cCPP may contain 6 to 10 amino acid residues, wherein: (i) at least two amino acids may be independently glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least three amino acids may have side chains containing aryl or heteroaryl groups; and (iii) at least two amino acids contain arginine.

[0075] glycine and related amino acid residues In some embodiments, cCPP may contain 1, 2, 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. In some embodiments, cCPP may contain 2 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. In some embodiments, cCPP may contain 3 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. In some embodiments, cCPP may contain 4 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. In some embodiments, cCPP may contain 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. In some embodiments, cCPP may contain 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. In some embodiments, cCPP may contain 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. In the embodiments, cCPP may contain 3, 4, or 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof.

[0076] In some embodiments, cCPP may contain 1, 2, 3, 4, 5, or 6 glycine residues. cCPP may contain 2 glycine residues. In some embodiments, cCPP may contain 3 glycine residues. In some embodiments, cCPP may contain 4 glycine residues. In some embodiments, cCPP may contain 5 glycine residues. In some embodiments, cCPP may contain 6 glycine residues. In some embodiments, cCPP may contain 3, 4, or 5 glycine residues. In some embodiments, cCPP may contain 3 or 4 glycine residues. In some embodiments, cCPP may contain 2 or 3 glycine residues. In some embodiments, cCPP may contain 1 or 2 glycine residues.

[0077] In one embodiment, cCPP may contain at least three glycine residues. In another embodiment, cCPP may contain 3, 4, 5, or 6 glycine residues. In another embodiment, cCPP may contain 3 glycine residues. In another embodiment, cCPP may contain 4 glycine residues. In another embodiment, cCPP may contain 5 glycine residues. In another embodiment, cCPP may contain 6 glycine residues. In yet another embodiment, cCPP may contain 3, 4, or 5 glycine residues. cCPP may contain 3 or 4 glycine residues.

[0078] In one embodiment, none of the glycine, β-alanine, or 4-aminobutyric acid residues in cCPP are adjacent. In another embodiment, two or three glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent. In yet another embodiment, two glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent.

[0079] In this implementation, none of the glycine residues in the cCPP are adjacent. For example, each glycine residue in the cCPP can be separated by an amino acid residue that is not glycine.

[0080] In one embodiment, two or more glycine residues in cCPP are adjacent. In another embodiment, two or three glycine residues may be adjacent. In yet another embodiment, two glycine residues are adjacent.

[0081] Amino acid side chains with aryl or heteroaryl groups In some embodiments, cCPP may contain 2, 3, 4, 5, or 6 amino acid residues that independently have a side chain containing an aryl or heteroaryl group. In some embodiments, cCPP may contain 2 amino acid residues that independently have a side chain containing an aryl or heteroaryl group. In some embodiments, cCPP may contain 3 amino acid residues that independently have a side chain containing an aryl or heteroaryl group. In some embodiments, cCPP may contain 2, 3, or 4 amino acid residues that independently have a side chain containing an aryl or heteroaryl group. In some embodiments, cCPP may contain 2 or 3 amino acid residues that independently have a side chain containing an aryl or heteroaryl group.

[0082] The aryl group can be a 6- to 14-membered aryl group. The aryl group can be phenyl, naphthyl, or anthracene, each optionally substituted. The heteroaryl group can be a 6- to 14-membered heteroaryl group having one, two, or three heteroatoms selected from N, O, and S. The heteroaryl group can be pyridyl, quinolinyl, or isoquinolinyl.

[0083] The amino acid residues having side chains containing aryl or heteroaryl groups can each independently be residues of phenylalanine, naphthylalanine, phenylglycine, β-homophenylalanine, holonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan, or tyrosine, each optionally substituted by one or more substituents. The amino acid residues having side chains containing aryl or heteroaryl groups can each independently be residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothiophene alanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, holophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 3-(9-anthrayl)-alanine. The amino acid residues having side chains containing aryl or heteroaryl groups can each independently be 3-(3-benzothiophene)-alanine, 3-(2-quinolinyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N The amino acid residues containing aryl or heteroaryl side chains may be phenylalanine, 1-naphthylalanine, 2-naphthylalanine, phenylglycine, β-homophylline, or holonaphthylalanine residues, each optionally substituted with one or more substituents. The amino acid residues containing aryl or heteroaryl side chains may be phenylalanine, 2-naphthylalanine, β-homophylline, or holonaphthylalanine residues, each optionally substituted with one or more substituents. The amino acid residues containing aryl or heteroaryl side chains may be phenylalanine or 2-naphthylalanine residues, each optionally substituted with one or more substituents. At least one amino acid residue containing an aryl or heteroaryl side chain may be a phenylalanine residue. At least two amino acid residues having side chains containing aryl or heteroaryl groups may be phenylalanine residues. Each amino acid residue having a side chain containing an aryl or heteroaryl group may be a phenylalanine residue. One amino acid residue having a side chain containing an aryl or heteroaryl group may be a 2-naphthylalanine residue. One amino acid residue having a side chain containing an aryl or heteroaryl group may be a β-homophenylalanine residue.

[0084] In one embodiment, none of the amino acids having side chains containing aryl or heteroaryl groups are adjacent. In another embodiment, two amino acids having side chains containing aryl or heteroaryl groups may be adjacent. In another embodiment, two adjacent amino acids may have opposite stereochemistry. In another embodiment, two adjacent amino acids have the same stereochemistry. In another embodiment, three amino acids having side chains containing aryl or heteroaryl groups may be adjacent. In another embodiment, three adjacent amino acids having side chains containing aryl or heteroaryl groups have the same stereochemistry. In another embodiment, three adjacent amino acids have alternating stereochemistry.

[0085] Amino acid residues containing aryl or heteroaryl groups may be L-amino acids. Amino acid residues containing aryl or heteroaryl groups may be D-amino acids. Amino acid residues containing aryl or heteroaryl groups may be a mixture of D-amino acids and L-amino acids.

[0086] The hydrophobicity of amino acid residues can be measured and / or calculated using a variety of techniques. In one embodiment, the hydrophobicity of an amino acid residue can be determined by calculating its consensus value on the consensus scale of D. Eisenberg et al., using the method described in D. Eisenberg et al., “Hydrophobic Moments and Protein Structure,” Faraday Symp. Chem. Soc. 1982, 17, 109-120 (e.g., D. Eisenberg et al.). A hydrophobic amino acid is an amino acid with a hydrophobic side chain.

[0087] Amino acid residues having side chains containing a guanidine group, a guanidine substitution group, or their protonated form As used in this article, guanidine refers to the following structure: .

[0088] As used in this article, the protonated form of guanidine refers to the following structure: .

[0089] Guanidinium substitution groups are functional groups on the side chains of amino acids that carry a positive charge at or above physiological pH, or can reproduce the hydrogen bond donation and acceptance activities of guanidinium groups.

[0090] While not wishing to be bound by theory, it is believed that guanidine substitution groups can facilitate cell penetration and therapeutic delivery while reducing toxicity associated with guanidine groups or their protonated forms. cCPP may contain at least one amino acid with a side chain containing a guanidine or guanidine-ionon substitution group. cCPP may contain two amino acids with a side chain containing a guanidine or guanidine-ionon substitution group. cCPP may contain three amino acids with a side chain containing a guanidine or guanidine-ionon substitution group. cCPP may contain four amino acids with a side chain containing a guanidine or guanidine-ionon substitution group. cCPP may contain five amino acids with a side chain containing a guanidine or guanidine-ionon substitution group. cCPP may contain six amino acids with a side chain containing a guanidine or guanidine-ionon substitution group.

[0091] The guanidine or guanidinium group can be an isosteric state of guanidine or guanidinium. The basicity of the guanidine or guanidinium substitution group can be lower than that of guanidine.

[0092] As used in this article, guanidine substitution group refers to... , , , , , Or their protonated forms.

[0093] In embodiments, cCPP comprising 6 to 10 amino acid residues is provided, wherein: (i) at least two amino acids have side chains comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid residue does not have a side chain or has a guanidine-substituted group or a protonated form thereof; and (iii) at least two amino acid residues independently have side chains comprising an aryl or heteroaryl group. In embodiments, two amino acids independently have side chains comprising an aryl or heteroaryl group. In embodiments, three amino acids independently have side chains comprising an aryl or heteroaryl group. In embodiments, at least two amino acids do not have side chains or have side chains comprising a guanidine-substituted group or a protonated form thereof. As used herein, when no side chain is present, the amino acid has two hydrogen atoms (e.g., -CH2-) on one or more carbon atoms linking the amine and carboxylic acid. In embodiments, the amino acid without a side chain may be glycine.

[0094] In some embodiments, cCPP may contain 2, 3, 4, 5, or 6 amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or their protonated form. In some embodiments, cCPP may contain 2 amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or their protonated form. In some embodiments, cCPP may contain 3 amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or their protonated form. In some embodiments, cCPP may contain 4 amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or their protonated form. In some embodiments, cCPP may contain 5 amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or their protonated form. In some embodiments, cCPP may contain 6 amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or their protonated form. In some embodiments, cCPP may contain 2, 3, 4, or 5 amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or their protonated form. In an embodiment, cCPP may comprise two, three, or four amino acid residues that independently have a side chain containing a guanidine group, a guanidine substitution group, or a protonated form thereof.

[0095] In an embodiment, an amino acid residue independently having a side chain comprising a guanidine group, a guanidine-substituted group, or their protonated form may be an L-amino acid. In an embodiment, an amino acid residue independently having a side chain comprising a guanidine group, a guanidine-substituted group, or their protonated form may be a D-amino acid. In an embodiment, an amino acid residue independently having a side chain comprising a guanidine group, a guanidine-substituted group, or their protonated form may be a mixture of L-amino acids or D-amino acids.

[0096] In the embodiments, each amino acid residue having a side chain containing a guanidine group or its protonated form can independently be arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid, or residues in their protonated forms. In the embodiments, each amino acid residue having a side chain containing a guanidine group or its protonated form can independently be arginine or its protonated form. In the embodiments, an amino acid residue having a side chain containing a guanidine substitution group or its protonated form can independently be a citrulline residue.

[0097] In the embodiments, cCPP may contain residues of asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine. In the embodiments, cCPP may contain residues of asparagine. In the embodiments, cCPP may contain residues of glutamine.

[0098] Chirality While not wishing to be bound by theory, it is believed that the chirality of amino acids in cCPP can affect cytoplasmic uptake efficiency. In some embodiments, cCPP may contain at least one D amino acid. In some embodiments, cCPP may contain 1 to 20 D amino acids. In some embodiments, cCPP may contain 1 to 15 D amino acids. In some embodiments, cCPP may contain 1 to 10 D amino acids. In some embodiments, cCPP may contain 1 to 8 D amino acids. In some embodiments, cCPP may contain 1, 2, 3, 4, 5, 6, 7, or 8 D amino acids. In some embodiments, cCPP may contain all D amino acids. In some embodiments, cCPP may contain at least one L amino acid. In some embodiments, cCPP may contain 1 to 20 L amino acids. In some embodiments, cCPP may contain 1 to 15 L amino acids. In some embodiments, cCPP may contain 1 to 10 L amino acids. In some embodiments, cCPP may contain 1 to 8 L amino acids. In some embodiments, cCPP may contain 1, 2, 3, 4, 5, 6, 7, or 8 L amino acids. In some embodiments, cCPP may contain all L amino acids. In some embodiments, cCPP may contain 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids having alternating D and L chirality. In some embodiments, cCPP may contain three adjacent amino acids having the same chirality. In some embodiments, cCPP may contain two adjacent amino acids having the same chirality. In some embodiments, at least two amino acids may have opposite chirality. In some embodiments, at least two amino acids with opposite chirality may be adjacent to each other. In some embodiments, at least three amino acids may have alternating stereochemistry relative to each other. In some embodiments, at least three amino acids with alternating chirality relative to each other may be adjacent to each other. In some embodiments, at least four amino acids have alternating stereochemistry relative to each other. In some embodiments, at least four amino acids with alternating chirality relative to each other may be adjacent to each other. In some embodiments, at least two amino acids may have the same chirality. In some embodiments, at least two amino acids with the same chirality may be adjacent to each other. In the embodiments, at least two amino acids have the same chirality and at least two amino acids have opposite chirality. In the embodiments, the at least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Therefore, adjacent amino acids in cCPP may have any of the following sequences: DL; LD; DLD, LDL, DLLD; LDDL; LDLLD; DLDDL; DLLDL; or LDDLD. In the embodiments, all amino acid residues forming cCPP may be L-amino acids. In the embodiments, all amino acid residues forming cCPP may be D-amino acids.

[0099] In an embodiment, one or more amino acid residues forming the cCPP may be achiral. In an embodiment, the cCPP may contain a motif of 3, 4, or 5 amino acids, wherein two amino acids having the same chirality may be separated by an achiral amino acid. The cCPP may contain the following sequences: D / LXD / L; D / LXD / LX; D / LXD / LXD / L; DXD; DXDX; DXDXD; LXL; LXLX; or LXLXL; wherein the D / L indicator amino acid may be a D or L amino acid and X is an achiral amino acid. The achiral amino acid may be glycine.

[0100] In embodiments, an amino acid having a side chain containing a guanidine substitution group or its protonated form may be adjacent to an amino acid having a side chain containing an aryl or heteroaryl group. In embodiments, an amino acid having a side chain containing a guanidine substitution group or its protonated form may be adjacent to at least one amino acid having a side chain containing a guanidine or its protonated form. In embodiments, an amino acid having a side chain containing a guanidine or its protonated form may be adjacent to an amino acid having a side chain containing an aryl or heteroaryl group. In embodiments, two amino acids having a side chain containing a guanidine substitution group or its protonated form may be adjacent to each other. In embodiments, two amino acids having a side chain containing a guanidine or its protonated form may be adjacent to each other. In embodiments, cCPP may contain at least two adjacent amino acids having a side chain that may contain an aryl or heteroaryl group and at least two non-adjacent amino acids having a side chain containing a guanidine substitution group or its protonated form. In embodiments, cCPP may contain at least two adjacent amino acids having a side chain containing an aryl or heteroaryl group and at least two amino acids having a side chain containing an aryl or heteroaryl group. Or, non-adjacent amino acids in the side chain of their protonated form. In embodiments, adjacent amino acids may have the same chirality. In embodiments, adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, for example, any sequence described in the preceding paragraphs.

[0101] In the embodiments, at least two amino acids having side chains containing a guanidine substitution group or its protonated form are alternated with at least two amino acids having side chains containing a guanidine group or its protonated form.

[0102] In the implementation scheme, cCPP may include the structure of formula (1): Equation (1): (1), or its protonated form, in: R 1 R 2 R3、 R4, R 5 R 6 and R 7 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; and q can be 1, 2, 3 or 4.

[0103] The cCPP of formula (1) may have any configuration and / or amino acid side chains, including but not limited to those configurations and / or amino acid side chains described in the following PCT publications: WO 2015 / 179691 filed on 21 May 2015 (titled “CELL PENETRATING PEPTIDES AND METHODS OF MAKING AND USING THEREOF”); WO 2021 / 127650 filed on 21 December 2020 (titled “COMPOSITIONS FOR DELIVERY OF ANTISENSE COMPOUNDS”); WO 2022 / 213118 filed on 31 March 2022 (titled “CYCLICCELL PENETRATING PEPTIDES”); WO 2022 / 241408 filed on 9 May 2022 (titled “COMPOSITIONS AND METHODS FOR CYCLICCELL PENETRATING PEPTIDES”). The disclosures of the following patents are hereby incorporated herein by reference in their entirety: MODULATING TISSUE DISTRIBUTION OF INTRACELLULARTHERAPEUTICS; U.S. Patent Publication No. 2023 / 0312653, filed March 30, 2023 (titled “CYCLIC CELL PENETRATING PEPTIDES”); and U.S. Patent No. 11,225,506, filed April 20, 2020 (titled “CELL PENETRATING PEPTIDES AND METHODS OF MAKING AND USINGTHEREOF”).

[0104] In the implementation scheme, cCPP is formula (1) or its protonated form, wherein: R 1 R 2 and R 3 Each is an aryl or heteroaryl side chain of H or an amino acid; R 1 R 2 and R 3At least two of them are aryl or heteroaryl side chains of amino acids; R 4 R 5 R 6 R 7 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; and q can be 1, 2, 3 or 4.

[0105] In the implementation scheme, cCPP is formula (1) or its protonated form, wherein: R 1 R 2 and R 3 Each is an aryl or heteroaryl side chain of H or an amino acid; R 1 R 2 and R 3 At least two of them are aryl or heteroaryl side chains of amino acids; R 4 R 5 R 6 R 7 Independent of H or amino acid side chains; R 4 R 5 R 6 R 7 At least two of them are independently arginine side chains; AA SC It is an amino acid side chain; and q can be 1, 2, 3 or 4.

[0106] In the implementation scheme of formula (1), R 1 R 2 and R 3 Each is independently H or a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are side chains of phenylalanine; R 4、 R5, R 6 and R 7 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; and q can be 1, 2, 3 or 4.

[0107] In the implementation scheme of formula (1), R 1 R2 and R 3 One of them is H; R 1 R 2 and R 3 Both of them are CH2Ph, where Ph is a phenyl group; and R 4 R 5 R 6 and R 7 It can be an H or amino acid side chain independently.

[0108] In the implementation scheme of formula (1), R 1 R 2 and R 3 One of them is H; R 1 R 2 and R 3 Both of them are CH2Ph; and R 4 R 5 R 6 and R 7 An amino acid side chain that is independently H or arginine.

[0109] In the implementation scheme of formula (1), R 1 R 2 and R 3 -CH2Ph; R 4 R 5 R 6 and R 7 It can be an H or amino acid side chain independently.

[0110] In the implementation scheme of formula (1), R 1 R 2 and R 3 Each is an independent side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are side chains of phenylalanine; R 4 R 5 R 6 and R 7 Independently, it is an H or amino acid side chain; and q can be 1, 2, 3 or 4.

[0111] In the implementation scheme of formula (1), R 1R 2 and R 3 Each is an independent H or amino acid side chain; R 1 R 2 and R 3 At least one of them is an aryl or heteroaryl side chain of an amino acid; R 1 R 2 and R 3 At least one of them is a side chain of arginine; R 4 R 5 R 6 and R 7 Independently, it is an H or amino acid side chain; and q can be 1, 2, 3 or 4.

[0112] In the implementation scheme of formula (1), R 1 R 2 and R 3 Each is an independent H or amino acid side chain; R 1 R 2 and R 3 At least two of them are arginine side chains; R 4 R 5 R 6 and R 7 Independently, it is an H or amino acid side chain; and q can be 1, 2, 3 or 4.

[0113] In the implementation of formula (I), R 1 R 2 and R 3 Each is an independent side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least one of them is a side chain of naphthylalanine; R 4 R 5 R 6 and R 7 Independently, it is an H or amino acid side chain; and q can be 1, 2, 3 or 4.

[0114] In the implementation of formula (I), R 1 R 2 and R 3 Each is an independent side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are side chains of naphthylalanine; R 4 R 5 R 6 and R 7 Independently, it is an H or amino acid side chain; and q can be 1, 2, 3 or 4.

[0115] In the implementation of formula (I), R 1 R 2 and R 3 Each is an independent side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are side chains of phenylalanine; R 1 R 2 and R 3 At least one of them is a side chain of naphthylalanine; R 4 R 5 R 6 and R 7 Independently, it is an H or amino acid side chain; and q can be 1, 2, 3 or 4.

[0116] AAsc can be a side chain or terminus of an amino acid residue on cCPP. Non-limiting examples of AAsc include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or modified side chains of glutamine or asparagine (e.g., reduced side chains having an amino group). In embodiments, AA... SC Can be or , where t can be an integer from 0 to 5. AA SC Can be , where t can be 0 or an integer from 1 to 5. In the implementation, t can be 1 to 5. In the implementation, t is 2 or 3. In the implementation, t can be 2. In the implementation, t can be 3. In the implementation, AA SC It can be conjugated with a linker. In an embodiment, AAsc is a side chain of a glutamine residue. In an embodiment, AAsc is a side chain of a glutamic acid residue. In an embodiment, when AA... SC When the side chain is glutamine, the carboxamide nitrogen of the glutamine side chain forms a bond with the -(CH2)y′- group.

[0117] In the implementation scheme, cCPP is defined by equation (1), where R 4 R 5 R 6 R 7 At least one of them is an uncharged, non-aromatic side chain of an amino acid. In the embodiment, R 4 R 5 R 6 and R 7 At least one of them is independently a side chain of H or serine, histidine, or citrulline. In the embodiment, R 4 R 5 R 6 and R 7 One of them is independently an H or a side chain of serine, histidine, or citrulline. In the implementation scheme, R 4 R 5 R 6 and R 7 At least two of them are independently H or serine, histidine, or citrulline side chains. In the embodiments, R 4 R 5 R 6 and R 7 Both of them are H or serine, histidine or citrulline side chains.

[0118] In one embodiment, an EEV comprising a cCPP having 6 to 10 amino acids is provided, wherein at least two amino acids of the cCPP are charged amino acids, at least two amino acids of the cCPP are aromatic hydrophobic amino acids, and at least two amino acids of the cCPP are uncharged non-aromatic amino acids. In one embodiment, at least two charged amino acids of the cCPP are arginine. In another embodiment, two charged amino acids of the cCPP are arginine. In another embodiment, at least two aromatic hydrophobic amino acids of the cCPP are independently phenylalanine, 2-naphthylalanine, or combinations thereof. In another embodiment, two or three aromatic hydrophobic amino acids of the cCPP are independently phenylalanine, 2-naphthylalanine, or combinations thereof. In another embodiment, at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, histidine, serine, glycine, or combinations thereof. In another embodiment, two amino acids of the cCPP are citrulline, histidine, serine, glycine, or combinations thereof. In the implementation scheme, cCPP has 6 to 10 amino acids, wherein two amino acids of cCPP are arginine, two or three amino acids are independently selected from phenylalanine, 2-naphthylalanine, β-homophenylalanine aromatic hydrophobic amino acids, and two amino acids are independently selected from citrulline, serine, histidine and glycine uncharged non-aromatic amino acids.

[0119] In the implementation scheme, cCPP is defined by equation (1), where R 4 R 5 R 6 and R 7 At least two of them are positively charged side chains of amino acid residues. In the embodiment, R 4 R 5 R 6 and R 7 Both of these are positively charged side chains of amino acid residues. In the implementation scheme, R 4 R 5 R 6 and R 7 At least three of them are positively charged side chains of amino acid residues. In the implementation scheme, R 4 R 5 R 6 R 7 The three components are positively charged side chains of amino acid residues. In the implementation scheme, R 4 R 5 R 6 and R 7 It is a positively charged side chain of an amino acid residue.

[0120] cCPP can contain the structure of equation (2): Equation (2): (2), or its protonated form, in: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are aryl or heteroaryl side chains of amino acids; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; m′ is an integer from 0 to 3; m′′ is an integer from 0 to 3; and q can be 1, 2, 3 or 4.

[0121] In the implementation scheme, cCPP is equation (1) or (2), where R 1 R 2 and R 3Each can be independently an H, -alkylene-aryl, -alkylene-heteroaryl, or an amino acid side chain containing a guanidine, a guanidine substitution group, or their protonated form. R 1 R 2 and R 3 Each can be independently H or -C 1-3 alkylene-aryl, -C 1-3 An alkylene-heteroaryl group or an amino acid side chain containing a guanidine group or its protonated form. In embodiments, cCPP is of formula (1) or (2), wherein R 1 R 2 and R 3 Each can be independently H, -alkylene-aryl, or -alkylene-heteroaryl. 1 R 2 and R 3 Each can be independently H or -C 1-3 alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R 1 R 2 and R 3 Each can be independently H or -alkylene-aryl. R 1 R 2 and R 3 Each can be H or -C independently. 1-3 Alkylene-aryl. 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 phenyl, naphthyl, or anthracene. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolinyl, or isoquinolinyl. The amino acid side chain containing the guanidine group may be arginine. R 1 R 2 and R 3 Each can be independently H or -C 1-3 Alkylene-Ph or -C 1-3 Alkylene-naphthyl. R 1 R 2 and R 3 Each can be independently H, -CH2Ph, or -CH2-naphthyl. 1 R 2 and R 3 Each can be H or -CH2Ph ​​independently. R 1 R 2 and R 3 One of them could be arginine.

[0122] In the implementation scheme, cCPP is equation (1) or (2), where R 1 R 2 and R 3Each of these can independently be a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothiophene-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-anthrayl)-alanine, or arginine. R 1 R 2 and R 3 Each can be an independent side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, arginine, or tryptophan.

[0123] In the implementation scheme, cCPP is equation (1) or (2), where R 1 It can be a side chain of tyrosine. R 1 It can be a side chain of phenylalanine. R 1 It can be a side chain of 1-naphthylalanine. R 1 It can be a side chain of 2-naphthylalanine. R 1 It can be a side chain of tryptophan. R 1 It can be a side chain of 3-benzothiophene alanine. R 1 It can be a side chain of 4-phenylphenylalanine. R 1 It can be a side chain of 3,4-difluorophenylalanine. 1 It can be a side chain of 4-trifluoromethylphenylalanine. 1 It can be a side chain of 2,3,4,5,6-pentafluorophenylalanine. 1 It can be a side chain of high-phenylalanine. R 1 It can be a side chain of β-homophenylalanine. R 1 It can be a side chain of 4-tert-butyl-phenylalanine. R 1 It can be a side chain of 4-pyridylalanine. R 1 It can be a side chain of 3-pyridylalanine. R 1 It can be a side chain of 4-methylphenylalanine. R 1 It can be a side chain of 4-fluorophenylalanine. R 1 It can be a side chain of 4-chlorophenylalanine. R 1 It can be a side chain of 3-(9-anthrayl)-alanine. R 1 It can be H. R 1 It can be a side chain of arginine.

[0124] In the implementation scheme, cCPP is equation (1) or (2), where R 2 It can be a side chain of tyrosine. R 2 It can be a side chain of phenylalanine. R 2It can be a side chain of 1-naphthylalanine. R 2 It can be a side chain of 2-naphthylalanine. R 2 It can be a side chain of tryptophan. R 2 It can be a side chain of 3-benzothiophene alanine. R 2 It can be a side chain of 4-phenylphenylalanine. R 2 It can be a side chain of 3,4-difluorophenylalanine. 2 It can be a side chain of 4-trifluoromethylphenylalanine. 2 It can be a side chain of 2,3,4,5,6-pentafluorophenylalanine. 2 It can be a side chain of high-phenylalanine. R 2 It can be a side chain of β-homophenylalanine. R 2 It can be a side chain of 4-tert-butyl-phenylalanine. R 2 It can be a side chain of 4-pyridylalanine. R 2 It can be a side chain of 3-pyridylalanine. R 2 It can be a side chain of 4-methylphenylalanine. R 2 It can be a side chain of 4-fluorophenylalanine. R 2 It can be a side chain of 4-chlorophenylalanine. R 2 It can be a side chain of 3-(9-anthrayl)-alanine. R 2 It can be H. R 2 It can be a side chain of arginine.

[0125] In the implementation scheme, cCPP is equation (1) or (2), where R 3 It can be a side chain of tyrosine. R 3 It can be a side chain of phenylalanine. R 3 It can be a side chain of 1-naphthylalanine. R 3 It can be a side chain of 2-naphthylalanine. R 3 It can be a side chain of tryptophan. R 3 It can be a side chain of 3-benzothiophene alanine. R 3 It can be a side chain of 4-phenylphenylalanine. R 3 It can be a side chain of 3,4-difluorophenylalanine. 3 It can be a side chain of 4-trifluoromethylphenylalanine. 3 It can be a side chain of 2,3,4,5,6-pentafluorophenylalanine. 3 It can be a side chain of high-phenylalanine. R 3 It can be a side chain of β-homophenylalanine. R 3 It can be a side chain of 4-tert-butyl-phenylalanine. R 3 It can be a side chain of 4-pyridylalanine. R 3 It can be a side chain of 3-pyridylalanine. R 3 It can be a side chain of 4-methylphenylalanine. R3 It can be a side chain of 4-fluorophenylalanine. R 3 It can be a side chain of 4-chlorophenylalanine. R 3 It can be a side chain of 3-(9-anthrayl)-alanine. R 3 It can be H. R 3 It can be a side chain of arginine.

[0126] In the implementation scheme, cCPP is equation (1) or (2), where R 4 It can be an H or a side chain of arginine, citrulline, serine, or histidine. R 4 It can be a side chain of H or arginine. R 4 It can be H. R 4 It can be a side chain of arginine. R 4 It can be a side chain of citrulline. R 4 It can be a side chain of serine. R 4 It can be a side chain of histidine. R 4 It can be a side chain of valine. R 4 It can be a side chain of leucine.

[0127] In the implementation scheme, cCPP is defined by equation (1), where R 5 It can be an H or a side chain of arginine, citrulline, serine, or histidine. R 5 It can be a side chain of H or arginine. R 5 It can be H. R 5 It can be a side chain of arginine. R 5 It can be a side chain of citrulline. R 5 It can be a side chain of serine. R 5 It can be a side chain of histidine.

[0128] In the implementation scheme, cCPP is equation (1) or (2), where R 6 It can be an H or a side chain of arginine, citrulline, serine, or histidine. R 6 It can be a side chain of H or arginine. R 6 It can be H. R 6 It can be a side chain of arginine. R 6 It can be a side chain of citrulline. R 6 It can be a side chain of serine. R 6 It can be a side chain of histidine. R 6 It can be a side chain of valine. R 6 It can be a side chain of leucine.

[0129] In the implementation scheme, cCPP is represented by equation (2), where R 7 It can be an H or a side chain of arginine, citrulline, serine, or histidine. R 7 It can be a side chain of H or arginine. R 7 It can be H. R7 It can be a side chain of arginine. R 7 It can be a side chain of citrulline. R 7 It can be a side chain of serine. R 7 It can be a side chain of histidine.

[0130] In the implementation scheme, cCPP is equation (1) or (2), where R 1 R 2 R 3 R 4 R 5 R 6 and R 7 One, two, or all three of them can be H. 1 R 2 R 3 R 4 R 5 R 6 and R 7 At least one of them can be H. R 1 R 2 R 3 R 4 R 5 R 6 and R 7 One of them can be H. R 1 R 2 R 3 R 4 R 5 R 6 and R 7 Both of them can be H. R 1 R 2 R 3 R 5 R 6 and R 7 The three can be H. R 1 R 2 and R 3 One of them can be H. R 4 R 5 R 6 and R 7 At least one of them can be H. R 4 R 5 R 6 and R 7 One of them can be H. R 4 R 5 R 6 and R 7 Both of them can be H. R 4 R 5 R 6 and R7 The three can be H. R 4 R 5 R 6 and R 7 The four elements in the equation can be H.

[0131] In the implementation scheme, cCPP is equation (1) or (2), where R 4 R 5 R 6 and R 7 At least one of them can be H or a side chain of arginine, citrulline, serine, or histidine. 4 R 5 R 6 and R 7 At least one of them is H. R 4 R 5 R 6 and R 7 At least one of them can be a side chain of arginine. R 4 R 5 R 6 and R 7 At least one of them can be a side chain of citrulline. R 4 R 5 R 6 and R 7 At least one of them can be a serine side chain. R 4 R 5 R 6 and R 7 At least one of them can be a side chain of histidine. R 4 R 5 R 6 and R 7 One of them can be H or a side chain of arginine, citrulline, serine, or histidine. 4 R 5 R 6 and R 7 One of them is H. R 4 R 5 R 6 and R 7 One of them could be the side chain of arginine. R 4 R 5 R 6 and R 7 One of them can be the side chain of citrulline. R 4 R 5 R 6 and R 7 One of them could be a side chain of serine. R 4 R 5 R 6 and R7 One of them can be the side chain of histidine.

[0132] In the implementation scheme, cCPP is equation (1) or (2), where R 4 R 5 R 6 and R 7 Both of these can be H or side chains of arginine, citrulline, serine, or histidine. 4 R 5 R 6 and R 7 Both of them can be H. R 4 R 5 R 6 and R 7 Both of these can be side chains of arginine. R 4 R 5 R 6 and R 7 Both of these can be side chains of citrulline. R 4 R 5 R 6 and R 7 Both of these can be side chains of serine. R 4 R 5 R 6 and R 7 Both of them can be side chains of histidine.

[0133] In the implementation scheme, cCPP is equation (1) or (2), where R 4 R 5 R 6 and R 7 The three components can be H or the side chain arginine, citrulline, serine, or histidine. 4 R 5 R 6 and R 7 The three can be H. R 4 R 5 R 6 and R 7 The three components in R can be side chains of arginine. 4 R 5 R 6 and R 7 The three components in R can be side chains of citrulline. 4 R 5 R 6 and R 7 The three components in R can be side chains of serine. 4 R 5 R 6 and R 7The three components can be the side chains of histidine.

[0134] In the implementation scheme, cCPP is formula (1) or (2), where AA SC It can be a side chain of asparagine, glutamine, or high-glutamine residues. AA SC It can be a side chain of a glutamine residue. In the embodiments, AA SC Can be or , where t can be an integer from 0 to 5. AA SC Can be , where t can be 0 or an integer from 1 to 5. In an embodiment, t can be 1 to 5. In an embodiment, t is 2 or 3. In an embodiment, t can be 2. In an embodiment, t can be 3. In an embodiment, AAsc is a side chain of a glutamine residue. In an embodiment, AAsc is a side chain of a glutamic acid residue. In an embodiment, when AA SC When the side chain is glutamine, the carboxamide nitrogen of the glutamine side chain forms a bond with the -(CH2)y′- group.

[0135] In the implementation scheme, cCPP is equation (1) or (2), where q can be 1, 2, or 3. q can be 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.

[0136] In the implementation scheme, cCPP is formula (1) or (2), where m′ can be 1 to 3. m′ can be 1 or 2. m′ can be 0. m′ can be 1. m′ can be 2. m′ can be 3.

[0137] In the implementation scheme, cCPP is formula (1) or (2), where m′′ can be 1 to 3. m′′ can be 1 or 2. m′′ can be 0. m′′ can be 1. m′′ can be 2. m′′ can be 3.

[0138] In the implementation scheme, cCPP includes the structure of formula (2) or its protonated form; wherein: R 1 R 2 and R 3 One of them is H; R 1 R 2 and R 3 Both of them are -CH2Ph; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; q is 1, 2, 3, or 4; m′ is an integer from 0 to 3; and m′′ is an integer from 0 to 3.

[0139] In the implementation scheme, cCPP includes the structure of formula (2) or its protonated form, wherein: R 1 R 2 and R 3 -CH2Ph; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; q is 1, 2, 3, or 4; m′ is an integer from 0 to 3; and m′′ is an integer from 0 to 3.

[0140] In the implementation scheme, cCPP includes the structure of formula (2) or its protonated form, wherein: R 1 R 2 and R 3 Each is an independent side chain of an amino acid containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least one of them is a side chain of naphthylalanine; AA SC It is an amino acid side chain; R 4 and R 6 An amino acid side chain that is independently H or arginine, serine, histidine, or citrulline; q is 1, 2, 3, or 4; m′ is an integer from 0 to 3; and m′′ is an integer from 0 to 3.

[0141] In the implementation scheme, cCPP comprises the structure of formula (2) or its protonated form, and R 4 and R 6 The side chain is independently H or arginine, histidine, serine, or citrulline. In the embodiments, R... 4 and R 6 The side chain is independently H or arginine, histidine, or serine. In the implementation, R 4 For H. In the implementation scheme, R 4 It is the side chain of arginine. In the implementation scheme, R 4 It is a histidine side chain. In the implementation scheme, R 4 It is a serine side chain. In the implementation scheme, R 4 It is a side chain of citrulline. In the implementation scheme, R6 For H. In the implementation scheme, R 6 It is the side chain of arginine. In the implementation scheme, R 6 It is a histidine side chain. In the implementation scheme, R 6 It is a serine side chain. In the implementation scheme, R 6 It is the side chain of citrulline.

[0142] The cCPP of equation (2) may contain the structure of equation (2-a) or equation (II-b): Equation (2-a): (2-a); Equation (2-b): (2-b), or their protonated forms, Among them AA SC R 1 R 2 R 3 R 4 R 6 m′ and m′′ are defined in this paper for equation (2).

[0143] The cCPP of equation (2) may contain the structure of equation (2-a1), (2-b1), (2-a2), (2-b2), (2-a3), or (2-b3): Equations (2-a1), (2-b1), (2-a2), (2-b2), (2-a3), or (2-b3): (2-a1); (2-b1); (2-a2); (2-b2); (2-a3); (2-b3); or their protonated forms, where AA SC m′ and m′′ are defined in this paper for equation (2).

[0144] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: FGFGRGR; GfFGrGr; FfFGRGR; FGFGRRR; or FGFRRRR.

[0145] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: FGFGHGH or FGFSHSH.

[0146] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: FfFSRSR or FGFSRSR.

[0147] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: Ff-Nal-RrRr; Ff-Nal-GrGr; Ff-Nal-GRGR; Ff-Nal-HrHr; or Ff-Nal-SrSr.

[0148] In the implementation, the cCPP of formula (1) or (2) may include one of the following sequences: Ff-Nal-Cit-r-Cit-r; Ff-Nal-Rr-Cit-r; Ff-Nal-Cit-rRr; or Ff-Nal-R-cit-R-cit.

[0149] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: FGFGRGRQ; GfFGrGrQ; FfFGRGRQ; FGFGRRRQ; or FGFRRRRQ.

[0150] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: FGFGHGHQ or FGFSHSHQ.

[0151] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: FfFSRSRQ or FGRSRSRQ.

[0152] In an implementation, the cCPP of formula (1) or (2) may include those cCPPs having one of the following sequences: Ff-Nal-RrRrQ; Ff-Nal-GrGrQ; Ff-Nal-GRGRQ; Ff-Nal-HrHrQ; or Ff-Nal-SrSrQ.

[0153] In the implementation scheme, the cCPP of formula (1) or (2) may contain one of the following sequences: Ff-Nal-Cit-r-Cit-rQ; Ff-Nal-Rr-Cit-rQ; Ff-Nal-Cit-rRrQ; or Ff-Nal-R-cit-R-cit-Q.

[0154] Where Nal = L-naphthylalanine; nal = D-naphthylalanine; Ω = L-leucine.

[0155] In the implementation scheme, cCPP of formula (1) or (2) can be selected from Ff-Nal-GrGrQ; FfFGRGRQ; FGFGRGRQ; GfFGrGrQ; FfFGRGRQ; FGFGRRRQ and FGFRRRRQ.

[0156] In the implementation scheme, cCPP of formula (1) or (2) can be selected from fNalRrRrQ, Ff-Nal-Cit-r-Cit-rQ and Ff-Nal-GrGrQ.

[0157] In the implementation, the cCPP of formula (1) or (2) has the FfFGRGRQ sequence.

[0158] In the implementation scheme, the cCPP of formula (1) or (2) has an FGFGRGRQ sequence or a GfFGrGrQ sequence.

[0159] In the implementation scheme, cCPP of formula (1) or (2) can be FAFARARQ.

[0160] In the implementation scheme, cCPP of formula (1) or (2) can be Ff-Nal-GrGrQ.

[0161] In the implementation scheme, cCPP of formula (1) or (2) can be selected from FfFGRGRQ, FfFRRRRQ, FfFRrRrQ, fffrrrrQ or FfFRrRrQ.

[0162] In the implementation scheme, cCPP of formula (1) or (2) can be FG-Nal-GRGRQ.

[0163] In the implementation scheme, cCPP of formula (1) or (2) can be selected from FGFGRGRQ, fGfGrGrQ, fGfGrGrQ, FGFGRGRQ, FGFGRQ, fGfrrrrQ, FGFRRRRQ or FGFRRRRQ.

[0164] In the implementation scheme, cCPP of formula (I) or (II) can be selected from FFFRRRRQ, FFFGRRRQ, FFFFRGRRQ; FFFRRGRQ, FFFFRRRGQ, GFFRRRRQ or FFGRRRRQ.

[0165] In the implementation scheme, cCPP of formula (I) or (II) may be FFFRRRRQ.

[0166] In the implementation scheme, cCPP of formula (I) or (II) may be FFFGRRRQ.

[0167] In the implementation scheme, cCPP of formula (I) or (II) may be FFFRGRRQ.

[0168] In the implementation scheme, cCPP of formula (I) or (II) may be FFFRRGRQ.

[0169] In the implementation scheme, cCPP of formula (I) or (II) may be FFFRRRGQ.

[0170] In the implementation scheme, cCPP of formula (I) or (II) may be GFFRRRRQ.

[0171] In the implementation scheme, cCPP of formula (I) or (II) may be FFGRRRRQ.

[0172] In the implementation scheme, cCPP of formula (I) or (II) can be selected from Nal-G-Nal-GRGRQ; FGFKRKRQ; FGFRRRRQ; FfGFRRRRQ; or FGFRRGRRQ.

[0173] In the implementation scheme, cCPP of formula (I) or (II) may be Nal-G-Nal-GRGRQ.

[0174] In the implementation scheme, cCPP of formula (I) or (II) may be FGFKRKRQ.

[0175] In the implementation scheme, cCPP of formula (I) or (II) may be FGFRRRRQ.

[0176] In the implementation scheme, cCPP of formula (I) or (II) may be FfGFRRRRQ.

[0177] In the implementation scheme, cCPP of formula (I) or (II) may be FGFRRGRRQ.

[0178] In the implementation scheme, cCPP of formula (I) or (II) can be selected from RGRGRGRQ; FFFGRGRQ; FGFRRRRQ; FGFRGRGQ; FRGRGRGQ; FRFGRGRQ; FRFRFRFQ; FGFLRLRQ; or FGFVRVRQ.

[0179] In the implementation scheme, cCPP of formula (I) or (II) may be RGRGRGRQ.

[0180] In the implementation scheme, cCPP of formula (I) or (II) may be FFFGRGRQ.

[0181] In the implementation scheme, cCPP of formula (I) or (II) may be FGFRRRRQ.

[0182] In the implementation scheme, cCPP of formula (I) or (II) may be FGFRGRGQ.

[0183] In the implementation scheme, cCPP of formula (I) or (II) may be FRGRGRGQ.

[0184] In the implementation scheme, cCPP of formula (I) or (II) may be FRFGRGRQ.

[0185] In the implementation scheme, cCPP of formula (I) or (II) may be FRFRFRFQ.

[0186] In the implementation scheme, cCPP of formula (I) or (II) may be FGFLRLRQ.

[0187] In the implementation scheme, cCPP of formula (I) or (II) may be FGFVRVRQ. In the implementation scheme, cCPP of formula (1) or (2) may be FGWRGRQ.

[0188] In the implementation scheme, cCPP in formula (1) or (2) can be fFRGRQ.

[0189] The cCPP of equation (2) can have the structure of equation (II-c): Equation (2-c): (2-c), or its protonated form, Where R 4 R 6 , q, m′, m′′ and AAsc are as defined in this paper.

[0190] The cCPP of equation (2) can have the structure of equation (2-d): Equation (2-d): (2-d), or its protonated form, Where R 4 R 6 , q, m′, m′′ and AAsc are as defined in this paper.

[0191] The cCPP of equation (2) can have the structure of equation (2-e): Equation (2-e): (2-e), or its protonated form, Where: R 2 R 4 R 6 , m′, m′′, and AAsc are as defined herein. In the implementation scheme, R2 is H.

[0192] In the implementation scheme, cCPP can be Equation (3): Equation (3): (3), or its protonated form in: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are aryl or heteroaryl side chains of amino acids; R 4 R 5 R 6 and R 7 Independent of H or amino acid side chains; R 4 R 5 R 6 and R 7 At least two of them are independently arginine side chains; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q can be 1, 2, 3 or 4.

[0193] In the implementation scheme, cCPP is defined by equation (3), where R 1 R 2 Or R 3 At least one of them is H. In an embodiment, the amino acid residue having a side chain containing an aryl or heteroaryl group is phenylalanine, homophenylalanine, or 2-naphthylalanine. In an embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least two of the components are each independently an amino acid residue having a side chain containing a charged group. In an embodiment, the amino acid residue having a side chain containing a charged group is arginine. In an embodiment, cCPP is of formula (3), where q is 1.

[0194] In the implementation scheme, cCPP is defined by equation (3), where n X =1, where R 5 and R 7 It is the side chain of arginine. In the implementation scheme, cCPP is of formula (3), where n X The value is 1, wherein the aryl or heteroaryl group is phenylalanine, β-homophenylalanine, or 2-naphthylalanine, and wherein R 4 R 5R 6 and R 7 At least two of them are arginine side chains. In the embodiment, cCPP is of formula (3), where n X =1, where R 4 R 5 R 6 and R 7 It is the side chain of arginine. In the implementation scheme, cCPP is of formula (3), where n X =1, where R 5 and R 7 It is the side chain of arginine, and R 4 and R 6 It is H.

[0195] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 R 6 Or R 7 At least one of them is an amino acid side chain of serine or histidine. In the embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least two of them are independently amino acid side chains of serine, citrulline, or histidine. In the embodiments, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least three of the components are independently serine or histidine amino acid side chains. In the embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least four of them are independently serine or histidine amino acid side chains.

[0196] In the implementation of cCPP in equation (3): R 1 R 2 and R 3 At least two of them are independently phenylalanine or 2-naphthylalanine side chains; R 4 R 5 R 6 Or R 7 At least two of them are independently arginine side chains; R 4 R 5 R 6 Or R 7At least two of them are independently H or side chains of arginine, serine, citrulline or histidine; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q is 1.

[0197] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 R 6 Or R 7 Both of these are serine side chains. In the implementation scheme, cCPP is of formula (3), where R 4 R 5 R 6 Or R 7 Both of these are histidine side chains. In the embodiment, cCPP is of formula (3), where R 4 R 5 R 6 Or R 7 Both of these are side chains of citrulline. In the embodiment, cCPP is of formula (3), where R 4 R 5 R 6 Or R 7 Both of them are H independently.

[0198] In the implementation scheme, cCPP is defined by equation (3), where: R 1 R 2 and R 3 At least two of them are side chains of phenylalanine or naphthylalanine; R 4 R 5 R 6 Or R 7 At least two of them are arginine side chains; R 4 R 5 R 6 Or R 7 At least two of them are independently H or uncharged non-aryl amino acids, wherein the uncharged non-aryl amino acids are selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, citrulline and 3-(4-thienyl)-alanine; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q is 1.

[0199] In the implementation scheme, cCPP is defined by equation (3), where: R 1 R 2 and R 3 At least two of them are independently side chains of phenylalanine or naphthylalanine; R 4 R 5 R 6 Or R 7 At least two of them are arginine side chains; R 4 R 5 R 6 Or R 7 At least two of them are independently serine, citrulline or histidine side chains; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q is 1.

[0200] In the implementation scheme, cCPP is defined by equation (3), where R 1 R 2 Or R 3 At least one of them is H. In the implementation scheme, cCPP is Equation (3), where R 1 R 2 Or R 3 At least one of them is a side chain of phenylalanine. In the embodiment, cCPP is of formula (3), wherein R 1 R 2 Or R 3 At least two of them are phenylalanine side chains. In the embodiment, cCPP is of formula (3), wherein R 1 R 2 Or R 3 At least one of them is a side chain of 2-naphthylalanine.

[0201] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 R 6 Or R 7 At least two of them are independently serine, citrulline or histidine side chains.

[0202] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 R 6 Or R 7 At least one of them is independently an uncharged, non-aryl side chain of an amino acid. In the embodiment, R4 R 5 R 6 Or R 7 At least two of the components are independently side chains of uncharged non-aryl amino acids (e.g., histidine, citrulline, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine). In an embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least two of the components are independently uncharged non-aryl amino acid side chains, wherein the uncharged non-aryl amino acids are selected from histidine, citrulline, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine. In an embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least two of them are independently uncharged non-aryl amino acid side chains, said uncharged non-aryl amino acid being selected from histidine, citrulline and serine.

[0203] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 R 6 Or R 7 At least one of them is H independently. In the implementation, cCPP is Equation (3), where R 4 R 5 R 6 Or R 7 Both of them are H independently.

[0204] In the implementation scheme, cCPP can be Equation (3), where: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are phenylalanine; R 4 R 5 R 6 R 7Independent of H or amino acid side chains; R 4 R 5 R 6 R 7 Both of them are independent side chains of arginine; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q can be 1, 2, 3 or 4.

[0205] In the implementation scheme, cCPP is defined by equation (3), where R 1 R 2 Or R 3 At least one of them is H independently. In the implementation, cCPP is Equation (3), where R 1 R 2 and R 3 At least one of them is an amino acid residue having a side chain containing an aryl or heteroaryl group. In an embodiment, the amino acid residue having a side chain containing an aryl or heteroaryl group is phenylalanine, β-homophenylalanine, or 2-naphthylalanine. In an embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 and R 7 At least two of the components are each independently an amino acid residue having a side chain containing a charged group. In an embodiment, the amino acid residue having a side chain containing a charged group is arginine. In an embodiment, cCPP is of formula (3), where q is 1.

[0206] In the implementation scheme, cCPP is defined by equation (3), where n X It is 1, and where R 5 and R 7 It is the side chain of arginine. In the implementation scheme, cCPP is of formula (3), where n X The value is 1, wherein the aryl or heteroaryl group is phenylalanine, β-homophenylalanine, or 2-naphthylalanine, and wherein R 4 R 5 R 6 and R 7 At least two of them are arginine side chains. In the embodiment, cCPP is of formula (3), where n X =1, where R 4 R 5 R 6 and R 7 It is the side chain of arginine. In the implementation scheme, cCPP is of formula (3), where n X =1, where R 5and R 7 It is the side chain of arginine, and R 4 and R 6 It is H. In the implementation scheme, cCPP is Equation (3), where R 4 R 5 (if it exists), R 6 Or R 7 At least one of (if present) is an amino acid side chain of H or serine, citrulline, or histidine. In an embodiment, cCPP is of formula (3), wherein R 4 R 5 (if it exists), R 6 Or R 7 At least two of (if present) are independently H or serine or histidine amino acid side chains. In an embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least three of them are independently H or amino acid side chains of serine, citrulline or histidine. In the embodiment, cCPP is of formula (3), wherein R 4 R 5 R 6 Or R 7 At least four of them are independently H or amino acid side chains of serine, citrulline or histidine.

[0207] In the implementation scheme of cCPP in equation (3): R 1 R 2 and R 3 At least two of them are independently side chains of phenylalanine, β-homophenylalanine, or 2-naphthylalanine; R 4 R 5 R 6 Or R 7 At least two of them are independently arginine side chains; R 4 R 5 R 6 Or R 7 At least two of them are independently H or side chains of arginine, serine, citrulline or histidine; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q is 1. It should be understood that when R... 1 When the side chain is β-hydroxyphenylalanine, n X The value is 1.

[0208] In the implementation scheme of cCPP in equation (3): R 1 R 2 and R 3 At least two of them are independently phenylalanine or 2-naphthylalanine side chains; R4 and R 6 Each is an independent side chain of H or arginine, serine, citrulline, or histidine; R 5 and R 7 It is the side chain of arginine; AA SC It is an amino acid side chain; n X It is 0 or 1; and q is 1.

[0209] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 (if it exists), R 6 Or R 7 Both (if present) are independently serine side chains. In the embodiment, cCPP is of formula (3), where R 4 R 5 (if it exists), R 6 Or R 7 Both (if present) are independently histidine side chains. In the embodiment, cCPP is of formula (3), where R 4 R 5 (if it exists), R 6 Or R 7 Both of them (if they exist) are H independently.

[0210] In the implementation scheme, cCPP is Equation (3), where: R 1 R 2 and R 3 At least two of them are independently side chains of phenylalanine, β-homophenylalanine, or naphthylalanine; R 4 R 5 R 6 Or R 7 At least two of them are independently arginine side chains; R 4 R 5 R 6 Or R 7 At least two of the components are independently H or uncharged non-aryl amino acids, wherein the uncharged non-aryl amino acids are selected from histidine, threonine, serine, citrulline, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q is 1. It should be understood that when R... 1 When the side chain is β-hydroxyphenylalanine, n X The value is 1.

[0211] In the implementation scheme, cCPP is Equation (3), where: R 1 R 2 and R 3 At least two of them are independently side chains of phenylalanine, 2-naphthylalanine, or β-homophenylalanine; R 4 and R 6 The side chain is independently H or an uncharged non-aryl amino acid, wherein the uncharged non-aryl amino acid is selected from histidine, threonine, serine, citrulline, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine; R 5 and R 7 It is the side chain of arginine; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q is 1. It should be understood that when R... 1 When the side chain is β-hydroxyphenylalanine, n X The value is 1.

[0212] In the implementation scheme, cCPP is Equation (3), where: R 1 R 2 and R 3 At least two of them are independently side chains of phenylalanine, β-homophenylalanine, or 2-naphthylalanine; R 4 R 5 R 6 Or R 7 At least two of them are independently arginine side chains; R 4 R 5 R 6 Or R 7 At least two of them are independently serine, citrulline, or histidine side chains; AA SC It is an amino acid side chain; n X It is either 0 or 1; and q is 1. It should be understood that when R... 1 When the side chain is β-hydroxyphenylalanine, n X The value is 1.

[0213] In the implementation scheme, cCPP is Equation (3), where: R 1 R 2 and R 3 At least two of them are independently side chains of phenylalanine, β-homophenylalanine, or naphthylalanine; R 4 and R 6 A side chain that is independently serine, citrulline, or histidine; R 5 and R 7 It is the side chain of arginine; AA SC It is an amino acid side chain; nX It is either 0 or 1; and q is 1. It should be understood that when R... 1 When the side chain is β-hydroxyphenylalanine, n X The value is 1.

[0214] In the implementation scheme, cCPP is defined by equation (3), where R 1 R 2 and R 3 At least one of them is H. In the implementation scheme, cCPP is Equation (3), where R 1 R 2 and R 3 At least one of them is a side chain of phenylalanine. In the embodiment, CPP is of formula (3), wherein R 1 R 2 and R 3 At least two of them are side chains of phenylalanine. In the embodiment, CPP is of formula (3), wherein R 1 R 2 and R 3 Both of these are side chains of phenylalanine. In the embodiment, CPP is of formula (3), where R 1 R 2 and R 3 The three components are side chains of phenylalanine. In the implementation scheme, cCPP is of general formula (3), wherein R 1 R 2 and R 3 One of them is the side chain of naphthylalanine.

[0215] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 R 6 Or R 7 At least two of them are independently serine or histidine side chains. In the embodiments, cCPP is of general formula (3), wherein R 4 and R 6 The side chain is independently H or serine, citrulline, or histidine. In the embodiments, cCPP is of formula (3), wherein R 4 and R 6 It is a serine side chain. In the embodiment, cCPP is of formula (3), wherein R 4 and R 6 The side chain is histidine. In the embodiment, cCPP is of formula (3), where R is the side chain of histidine. 4 and R 6 The side chain is citrulline. In the embodiment, cCPP is of formula (3), where R is the side chain of citrulline. 4 and R 6 For H. In the implementation scheme, cCPP is Equation (3), where R 5 and R7 It is the side chain of arginine.

[0216] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 (if it exists), R 6 R 7 At least one of (if present) is independently an uncharged, non-aryl side chain of an amino acid. In an embodiment, cCPP is of formula (3), wherein R 4 R 5 (if it exists), R 6 R 7 At least two of the following (if present) are independently uncharged non-aryl amino acids (e.g., histidine, threonine, serine, citrulline, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine). In an embodiment, cCPP is of formula (3), wherein R 4 R 5 (if it exists), R 6 R 7 At least two of the following (if present) are independently uncharged non-aryl amino acid side chains, said uncharged non-aryl amino acid being selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine. In an embodiment, CPP is of formula (3), wherein R 4 R 5 (if it exists), R 6 R 7 At least two of the following (if present) are independently uncharged non-aryl amino acid side chains selected from histidine and serine. In an embodiment, cCPP is of formula (3), wherein R 5 and R 7 It is the side chain of arginine.

[0217] In the implementation scheme, cCPP is defined by equation (3), where R 4 R 5 R 6 Or R 7 At least one of them is H independently. In the implementation, cCPP is Equation (3), where R 4 R 5 R 6 Or R 7Both of them are H independently. In the implementation scheme, cCPP is Equation (3), where R 4 Or R 6 At least one of them is H independently. In the implementation, cCPP is Equation (3), where R 4 and R 6 For H. In the implementation scheme, cCPP is Equation (3), where R 5 and R 7 It is the side chain of arginine.

[0218] In one embodiment, an EEV comprising a cCPP having 6 to 10 amino acids is provided, wherein at least two amino acids of the cCPP are charged amino acids, at least two amino acids of the cCPP are aryl or heteroaryl hydrophobic amino acids, and at least two amino acids of the cCPP are uncharged non-aryl amino acids. In one embodiment, the at least two charged amino acids of the cCPP are arginine. In one embodiment, the at least two aryl or heteroaryl hydrophobic amino acids of the cCPP are phenylalanine, naphthylalanine (3-naphth-2-yl-alanine), β-homophenylalanine, or combinations thereof. In one embodiment, the at least two uncharged non-aryl amino acids of the cCPP are glycine. In one embodiment, two of the uncharged amino acids are serine, citrulline, histidine, or combinations thereof.

[0219] In an implementation, the cCPP of formula (3) may include those cCPPs having one of the following sequences: hFf-Nal-GrGr; bhF-F-Nal-SRSR; bhF-F-Nal-GRGR; bhF-F-Nal-HRHR; bhF-f-Nal-GrGr; bhF-f-Nal-SRSR; bhF-f-Nal-SrSr or bhFf-Nal-HrHr (where bhF – b-homophenylalanine; hFf – holophenylalanine).

[0220] In an implementation, the cCPP of formula (3) may include those cCPPs having one of the following sequences: hFf-Nal-GrGrQ; bhF-F-Nal-SRSRQ; bhF-F-Nal-GRGRQ; bhF-F-Nal-HRHRQ; bhF-f-Nal-GrGrQ; bhF-f-Nal-SRSRQ; bhF-f-Nal-SrSrQ or bhFf-Nal-HrHrQ (where bhF – b-homophenylalanine; hFf – holophenylalanine).

[0221] In an implementation, the cCPP of formula (3) may include those cCPPs having one of the following sequences: hFf-Nal-GrGr; bhF-F-Nal-SRSR; bhF-F-Nal-GRGR; bhF-F-Nal-HRHR; bhF-f-Nal-GrGr; bhF-f-Nal-SRSR; bhF-f-Nal-SrSr or bhFf-Nal-HrHr (where bhF – β-homophenylalanine; hFf – holophenylalanine).

[0222] In an implementation, the cCPP of formula (3) may include those cCPPs having one of the following sequences: hFf-Nal-GrGrQ; bhF-F-Nal-SRSRQ; bhF-F-Nal-GRGRQ; bhF-F-Nal-HRHRQ; bhF-f-Nal-GrGrQ; bhF-f-Nal-SRSRQ; bhF-f-Nal-SrSrQ; or bhFf-Nal-HrHrQ.

[0223] connector An EEV may contain one or more connector arms. The connector can attach the cargo to the cCPP. The connector can attach the EP to the cCPP. The connector can attach to the side chain of an amino acid in the cCPP, and the cargo can be attached to the appropriate location on the connector.

[0224] The linker can be any suitable portion that conjugates the cCPP to one or more additional portions (e.g., exocyclic peptides (EPs) and / or cargo). Prior to conjugation (e.g., to the cCPP and / or one or more additional portions), the linker has two or more functional groups, each capable of independently forming covalent bonds (e.g., to the cCPP and / or one or more additional portions). If the cargo is an oligonucleotide, the linker can be covalently bound to the 5' end of the cargo or the 3' end of the oligonucleotide cargo. The linker can be covalently bound to the 5' end of the oligonucleotide cargo. The linker can be covalently bound to the 3' end of the oligonucleotide cargo. If the cargo is a peptide, the linker can be covalently bound to the N-terminus or C-terminus of the peptide cargo. The linker can be covalently bound to the backbone of the oligonucleotide or peptide cargo (e.g., at some point in the middle, rather than at one or more ends). The linker can be any suitable portion that conjugates the cCPP described herein to a therapeutic portion (such as an oligonucleotide, peptide, or small molecule).

[0225] The linker can be covalently attached to the cargo at any suitable location on the cargo. The linker can be covalently attached to the 3' end or 5' end of the oligonucleotide cargo. The linker can be covalently attached to the N-terminus or C-terminus of the peptide cargo. The linker can be covalently attached to the backbone of the oligonucleotide or peptide cargo. The linker can be covalently attached to the cargo via the bonding group M′.

[0226] The linker can bind to the side chain of an amino acid (AA) on cCPP. SC This includes, for example, aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or modified side chains of glutamine or asparagine (e.g., reduced side chains having amino groups). The linker may bind to the lysine side chain on the cCPP.

[0227] In an embodiment, the connector may comprise one or more polyethylene glycol (PEG) components. Each (PEG) component may have, for example, 0 to 12 repeating (PEG) units. A (PEG) unit refers to the group -(CH2CH2O)-, which may be repeated any number of times.

[0228] While not wishing to be bound by theory, it is believed that joints can affect the efficacy and durability of EEV-cargo conjugates, for example, by increasing efficacy. In embodiments, efficacy increases without decreasing durability. In embodiments, one or more hydrophobic components (X) are added to the joint of the EEV. In embodiments, the length of the joint can be varied. The length of the joint can affect efficacy and durability. In some cases, as the joint length decreases, efficacy increases and durability decreases; while as the joint length increases, efficacy decreases and durability increases. In embodiments, increasing the hydrophobicity of the joint can increase efficacy.

[0229] The hydrophobic component X can be added to one or more sites on the joint. For example, X can be present in the joint between CPP and EP; or in the joint between CPP and cargo. In an embodiment, X can be a bonding group between CPP and EP. In an embodiment, X is not a bonding group between CPP and EP. In an embodiment, X can be a bonding group between CPP and cargo. In an embodiment, X is not a bonding group between CPP and cargo. X can form part of the backbone of the joint between CPP and EP. X can form part of the backbone of the joint between CPP and cargo. X can be attached to the joint. X can be attached to the side chain of lysine residues in the joint. X can be attached to the joint between CPP and EP. X can be attached to the joint between CPP and cargo.

[0230] In the implementation, the hydrophobic component (X) in the connector may be aliphatic, olefinic, alkyneic, aromatic (e.g., carbocyclic or heteroaromatic), or a combination of aliphatic and aromatic.

[0231] X can be a D-amino acid residue or an L-amino acid residue with a hydrophobic side chain. X can be a naturally occurring or non-naturally occurring amino acid residue with a hydrophobic side chain. X can be an amino acid residue with an aromatic side chain. X can be an amino acid residue with a heteroaromatic side chain. X can be selected from phenylalanine, 3-(4',4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or combinations thereof. X can be 2-naphthylalanine. X can be Na1. X can be d-N1 (nal). X can be 3-(4',4-biphenyl)-L-alanine. X can be Bip. X can be D-Bip (bip). X can be a C4-C8 alkyl hydrocarbon. X can be a C6 alkyl hydrocarbon.

[0232] In this embodiment, the hydrophobic component X comprises an optionally substituted alkyl group. The alkyl group may comprise a branched alkyl group. In this embodiment, the alkyl group comprises a double bond. In this embodiment, the alkyl group comprises an ethyl or propyl group. In this embodiment, the alkyl group is a butyl, pentyl, or hexyl group. In this embodiment, the alkyl group is -CH2(CH2). n CH2-, CH3(CH2) n CH2-、C(H)C(CH2) n CH2-, CH3(CH2) n CH2NH-、C(H)C(CH2) n CH2NH-, CH3(CH2) n CO-, CH3(CH2) n CH2O-, CH3(CH2) n CH2S-, -CH2(CH2) n CH2SH-、-OC(CH2) n CH2-, -OC(CH2) n CH2NH-, -OC(CH2) n CO-, -OC(CH2) n CH2O-、-OC(CH2) n CH2S-、-HNC(CH2) n CH2-、-HNC(CH2) n CH2NH-、-HNC(CH) n CO-, -HNC(CH2) n CH2O-、-HNC(CH2) n CH2S-、-OCH2(CH2) n CH2-、-OCH2(CH2) n CH2NH-、-OCH2(CH2) n CO-, -OCH2(CH2) nCH2O-、-OCH2(CH2) n CH2S-, NH2(CH2) n CH2-, SH(CH2) n CH2- or N3-C(O)CH2(CH2) n CH2- group, where "n" is 4-34, including the terminal value.

[0233] In the embodiments, the hydrophobic component X may be an optionally substituted aromatic group. The aromatic group may be a carbocyclic heteroaromatic, monocyclic, bicyclic, or polycyclic. Carbocyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, anthracene, etc. Heteroaromatic groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0234] In the embodiments, hydrophobic component X may comprise one or more hydrophobic amino acid residues. The amino acid residues may be D or L. The amino acid residues may be native or non-native hydrophobic amino acids. In the embodiments, the hydrophobic amino acid residues comprise substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, or aralkyl side chains, wherein the alkyl, alkenyl, and alkynyl side chains comprise at most one heteroatom per six carbon atoms. X may be a D or L amino acid residue with a hydrophobic side chain. X may be a native or non-native amino acid residue with a hydrophobic side chain. X may be an amino acid residue with an aromatic side chain. X may be an amino acid residue with a heteroaromatic side chain. Hydrophobic component X may comprise an amino acid residue with a hydrophobic side chain, which may be selected from valine, proline, alanine, leucine, isoleucine, phenylalanine, cysteine, glycine, histidine, methionine, and tryptophan. The amino acid residue with a hydrophobic side chain may be selected from the group consisting of glycine, phenylglycine, alanine, valine, leucine, isoleucine, ortholeucine, phenylalanine, tryptophan, naphthylalanine, proline, and combinations thereof, wherein the aromatic side chains on phenylglycine, phenylalanine, tryptophan, or naphthylalanine may be optionally substituted. The amino acid residue with a hydrophobic side chain may be naphthylalanine, lysine-naphthylalanine, biphenylalanine, or lysine-biphenylalanine. X may be an aliphatic hydrocarbon. X may be C4-C. 12 Aliphatic hydrocarbons. X can be C4-C8 aliphatic hydrocarbons. X can be C6 aliphatic hydrocarbons. X can be selected from phenylalanine, 3-(4',4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or combinations thereof. X can be an amino acid selected from tryptophan, tyrosine, isoleucine, leucine, histidine, phenylalanine, or combinations thereof.

[0235] X can be 2-naphthylalanine. X can be Na1. X can be d-Nal (nal). X can be 3-(4',4-biphenyl)-L-alanine (Bip). X can be 3-(4',4-biphenyl)-D-alanine (d-Bip or bip).

[0236] In the embodiments, the hydrophobic component X may comprise a modified amino acid subunit. For example, an amino-terminal or carboxyl-terminal amino acid subunit may be modified. Such modification involves capping the amino-terminus or carboxyl-terminus with a group to make the amino acid subunit more hydrophobic. For example, the amino terminus may be capped with an acyl group (e.g., acetyl, benzoyl, or stearoyl moiety). For example, the amino terminus of a modified amino acid subunit may be illustrated as follows: .

[0237] In the implementation scheme, X can be derived via lysine or SH(CH2). n CH2- or N3-C(O)CH2(CH2) n CH2- is attached to the main chain of the connector.

[0238] In the implementation scheme, the size of the aromatic or heteroaromatic groups present in the hydrophobic component X can be selected to improve the cytoplasmic delivery efficiency of the cargo. While not wishing to be bound by theory, it is believed that the presence of the hydrophobic component X in the joint of an EEV may improve the cytoplasmic delivery efficiency of the cargo compared to an EEV that is otherwise identical and does not contain the hydrophobic component X.

[0239] Connections may include hydrocarbon connections.

[0240] The linker may contain a cleavage site. The cleavage site may be a disulfide or cysteine ​​cleavage site (e.g., Val-Cit-). p -Aminobenzyloxycarbonyl, also known as Val-Cit-PABC).

[0241] The linker may contain: (i) one or more D or L amino acids, each optionally substituted; (ii) one or more -(U) amino acids. 1 -JU 2 )i′-subunit, where U 1 and U 2 Each of the elements in each instance is independently selected from alkylene groups, and each J is independently C, N, U. 3 -NU 3 C(O)-, S and O, where U 3 The groups are independently selected from H, alkyl, alkenyl, alkynyl, carbocyclic and heterocyclic groups, each of which is optionally substituted, and i′ is an integer from 1 to 50.

[0242] The linker may contain one or more D or L amino acids and / or -(OCH2CH2). i -, where i is an integer from 0 to 60; or a combination thereof. i can be an integer from 0 to 12. i can be 0, 2, 4, 8, or 12. i can be 0. i can be 2. i can be 4. i can be 8. i can be 12. "-(OCH2CH2) i It can also be called polyethylene glycol (PEG).

[0243] The connector may contain one or more PEG components - (OCH2CH2) i - where i is an integer from 0 to 12. PEG components can be represented as (PEG)x′, (PEG)z′, and / or (PEG)z′′. The connector may contain one or more hydrophobic components (X... o (X') and (Xo''). Hydrophobic components can attach to the side chains of amino acids in the linker. Hydrophobic component (X'') o (K(Xo′)) can be attached to the side chain (K(Xo′)) of lysine in the linker. The linker may contain a bonding group (M).

[0244] The linker may contain (i) β-alanine residues and lysine residues; (ii) -(JU 1 ) i′ ; or (iii) combinations thereof. Each U 1 It can be independently alkylene, alkenylene, ynylene, carbocyclic, or heterocyclic, and each J can be independently C or N. 3 -NU 3 C(O)-, S or O, where U 3 The group can be H, alkyl, alkenyl, ynyl, carbocyclic, or heterocyclic, each optionally substituted, and z′′ can be an integer from 1 to 50. Each U 1 It can be an alkylene group, and each J can be O.

[0245] The linker may contain (i) residues of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or combinations thereof; and (ii) -(U 1- J) i′ or -(JU 1 ) i′ Each U 1 It can be independently alkylene, alkenylene, ynylene, carbocyclic, or heterocyclic, and each J can be independently C or N. 3 -NU 3 C(O)-, S or O, where U 3 The group can be H, alkyl, alkenyl, alkynyl, carbocyclic, or heterocyclic, each optionally substituted, and i′ can be an integer from 1 to 50. Each U 1It may be an alkylene group, and each J may be O. The linker may contain glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or a combination thereof.

[0246] The connector can also incorporate cleavage sites, including disulfides [NH2-(CH2O)]. n -SS-(CH2O) n -COOH] or caspase cleavage site (Val-Cit-PABC).

[0247] The linker may contain residues of glycine or β-alanine.

[0248] The adapter can be divalent and connects the cCPP to the cargo. The adapter can be divalent and connects the cCPP to the exopeptide (EP).

[0249] The connector can be trivalent and can connect cCPP to cargo and EP.

[0250] The connector can be either bivalent or trivalent C1-C. 50 Alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclic, heteroaryl, cycloalkyl, or cycloalkenyl. The linker may be divalent or trivalent C1-C 50 Alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)- or combinations thereof.

[0251] The cargo can be coupled to the glutamate of the cyclic peptide, which converts the glutamate to glutamine. A linker (L) can couple the cargo to the glutamine / glutamate of the cyclic peptide. In an embodiment, the linker (L) is covalently bound to the backbone of the cargo.

[0252] The connector can be a trivalent connector, which has the structure shown in formula (A′): Equation (A′) (A′); in: **For attachment sites to linear exocyclic peptides (EPs) as defined herein; *This is the attachment site for cell-penetrating peptides (CPPs) as defined herein; L 1 and L 2 Independently designed as a connector arm; ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; and M contains a reaction handle.

[0253] In the implementation plan, L 1 and L 2 Each component independently does not exist, or contains either a hydrocarbon component, a polyethylene glycol (PEG) component, a hydrophobic component (X), an amino acid (AA) component containing one or more amino acid residues, or a combination thereof. In the embodiments, L 1 It contains a polyethylene glycol (PEG) x′ component, where x′ is an integer from 1 to 12. In the embodiment, L 2 It contains a polyethylene glycol (PEG) z′ component, where z′ is an integer from 1 to 12. In an embodiment, L2 contains a hydrophobic component (Xo′).

[0254] In the implementation plan, L 1 L 2 Or cCPP may contain one or more hydrophobic components (X).

[0255] In the implementation plan, L 1 Contains PEG components, and L 2 It contains PEG components, hydrophobic components (X), or combinations thereof.

[0256] In the implementation plan, L 1 It does not exist. In the implementation scheme, when x′ is 0, L 1 It does not exist. In the implementation plan, when L 1 In its absence, the lysine residue linked to the cyclic peptide is acylated (Ac). In the implementation scheme, L... 1 It does not exist, and L 2 Contains PEG component (PEG)z′, hydrophobic component (X) o ′), or a combination thereof.

[0257] In the implementation plan, L 2 Contains (PEG)z′, hydrophobic component (X o ′), or a combination thereof. In the implementation, L 2 It contains a PEG component (PEG) z′. In the embodiment, L 2 Contains hydrophobic components (X) o In the implementation plan, L 2 Contains (PEG)z′ and hydrophobic component (X) o In the implementation scheme, the hydrophobic component (X)o ′) is attached from the side chain of a lysine amino acid residue.

[0258] M contains a functional group (sometimes referred to herein as a reaction handle) that reacts with a corresponding functional group on the cargo to form an EEV-cargo conjugate or a connector-cargo conjugate. Upon conjugation, the EEV-cargo or connector-cargo conjugate contains a bonding group M′, which is the reaction product of the two functional groups.

[0259] In the embodiments, M may be -OH (e.g., as part of the C-terminus of an amino acid). In the embodiments, M comprises -OH, or , where y′′ is an integer from 1 to 4. y′′ can be 1. y′′ can be 2. y′′ can be 3. y′′ can be 4.

[0260] In the implementation scheme, the connector type (A′) may have a form selected from (B′), (C′), or (D′): Equation (B′): (B′); Equation (C′): (C′); or Equation (D′): (D′); x′ is an integer from 1 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; j′′ is 0, 1 or 2, where j′′ is 0 when z′ is 0; X o ′ is a hydrophobic component; and K # It is a D-lysine or L-lysine residue.

[0261] Apart from glycine (G), which has an H as a side chain, each amino acid has an asymmetric or chiral α-carbon atom and exists in two enantiomers, referred to as “D-” and “L-”. The “L-” isomer is most commonly found in naturally occurring proteins. While not wishing to be bound by theory, it is believed that the chirality of amino acids in cell-penetrating peptides (CPPs) or endosome escape mediators (EEVs) can affect cytoplasmic delivery efficiency and toxicity. This paper discloses EEVs in which the stereochemistry of amino acids within the peptide sequence of the EEV has been modified to affect cytoplasmic delivery efficiency and / or reduce toxicity.

[0262] In the embodiments, the linker comprises a structure selected from formula (B′), (C′), or (D′), wherein EP comprises all D-amino acids; and cCPP comprises a D-amino acid, a chiral amino acid, and AAsc, wherein AAsc is an amino acid side chain, and AA SC Connect the cCPP to the connector.

[0263] In one embodiment, cCPP contains at least two D-amino acid residues with hydrophobic side chains. In another embodiment, cCPP contains two D-amino acid residues with hydrophobic side chains. In another embodiment, cCPP contains three D-amino acid residues with hydrophobic side chains. In another embodiment, cCPP contains at least two D-arginine amino acid residues. In another embodiment, cCPP contains two D-arginine amino acid residues. In another embodiment, cCPP contains three D-arginine amino acid residues. In another embodiment, cCPP contains four D-arginine amino acid residues. In another embodiment, cCPP contains five D-arginine amino acid residues. In another embodiment, cCPP contains six D-arginine amino acid residues. In another embodiment, cCPP contains at least two D-phenylalanine residues. In another embodiment, cCPP contains two D-phenylalanine residues. In another embodiment, cCPP contains three D-phenylalanine residues. In another embodiment, cCPP contains at least two glycine residues. In another embodiment, cCPP contains two glycine residues. In one embodiment, cCPP contains 3 glycine residues. In another embodiment, cCPP contains 4 glycine residues. In another embodiment, cCPP contains 5 glycine residues. In another embodiment, cCPP contains 6 glycine residues. In another embodiment, cCPP contains at least 2 D-amino acid residues with hydrophobic side chains and at least 2 D-arginine residues. In another embodiment, cCPP contains at least 2 D-phenylalanine residues and at least 2 D-arginine residues. In yet another embodiment, AA... SC It is a side chain of L-glutamine.

[0264] The connector can be of type (B′): Equation (B′): (B′); Where **, *, x′, j′, ^, y′, z′, j′′ and M are as defined in this paper.

[0265] The connector can be of type (C′): Equation (C′): (C′); Among them, **, *, x′, j′, ^, y′, z′, j′′, X o ′ and M are as defined in this paper.

[0266] The connector can be of type (D′): Equation (D′): (D′); Among them, **, *, x′, j′, ^, y′, z′, j′′, X o ′、K # M is as defined in this paper.

[0267] In the implementation scheme, M includes -OH, , , where y′′ is an integer from 1 to 4. y′′ can be 1. y′′ can be 2. y′′ can be 3. y′′ can be 4.

[0268] x′ can be an integer from 0 to 12. x′ can be an integer from 1 to 12. x′ can be an integer from 2 to 12. x′ can be 0, 2, 4, 8, or 12. x′ can be 0 or 2. x′ can be 0, 2, or 12. x′ can be 0 or 12. x′ can be 2 or 12. x′ can be 0. x′ can be 1. x′ can be 2. x′ can be 3. x′ can be 4. x′ can be 5. x′ can be 6. x′ can be 7. x′ can be 8. x′ can be 9. x′ can be 10. x′ can be 11. x′ can be 12.

[0269] j′ can be 0, 1, or 2. j′ can be 1. j′ can be 2. It should be understood that when x′ is 0, j′ is 0.

[0270] ^Can indicate L-stereochemistry. ^Can indicate D-stereochemistry.

[0271] y′ can be an integer from 1 to 5, such as 1, 2, 3, 4, or 5, including all ranges and subranges in between. y′ can be an integer from 2 to 5. y′ can be an integer from 3 to 5. y′ can be 3 or 4. y′ can be 4 or 5. y′ can be 1. y′ can be 2. y′ can be 3. y′ can be 4. y′ can be 5.

[0272] z′ can be an integer from 0 to 12. z′ can be an integer from 1 to 12. z′ can be an integer from 2 to 12. z′ can be 0, 2, 4, 8, or 12. z′ can be 2 or 12. z′ can be 0, 2, or 12. z′ can be 0 or 12. z′ can be 2 or 12. z′ can be 0. z′ can be 1. z′ can be 2. z′ can be 3. z′ can be 4. z′ can be 5. z′ can be 6. z′ can be 7. z′ can be 8. z′ can be 9. z′ can be 10. z′ can be 11. z′ can be 12.

[0273] j′′ can be 0, 1, or 2. j′′ can be 0. j′′ can be 1. j′′ can be 2. It should be understood that when z′ is 0, j′′ is 0.

[0274] K # Indicates an L-lysine residue. K # Indicates D-lysine residues.

[0275] In the implementation scheme, ^ indicates L-stereochemistry. In the implementation scheme, ^ indicates D-stereochemistry.

[0276] In the implementation scheme, M′ includes -NH-, -C(O)-, -O-, ; ;or , or , where y′′ is an integer from 1 to 4, and t′ is an integer from 0 to 10. In the implementation, M′ contains -C(O)-. Where y′′ is an integer from 1 to 4, and in the implementation, M′ contains - , where t′ is an integer from 0 to 10. In the implementation, M′ contains .

[0277] AAsc can be a side chain or terminus of an amino acid residue on cCPP. Non-limiting examples of AAsc include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or modified side chains of glutamine or asparagine (e.g., reduced side chains having an amino group). In an embodiment, AAsc is a side chain of a glutamine residue. In an embodiment, AAsc is a side chain of a glutamic acid residue. In an embodiment, when AAsc is a side chain of glutamine, the carboxamide nitrogen of the glutamine side chain is combined with -(CH2). y′ - Groups form bonds.

[0278] y′′ can be an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. y′′ can be 0, 1, 2, 3, or 4. y′′ can be 0. y′′ can be 1. y′′ can be 2. y′′ can be 3. y′′ can be 4.

[0279] X o It can be an aliphatic hydrocarbon. X o ′ can be C4-C 12 Aliphatic hydrocarbons. X o It can be a C4-C8 aliphatic hydrocarbon. o It can be a C6 aliphatic hydrocarbon. X o ′ can be selected from phenylalanine, 3-(4′,4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or any combination thereof. X o ′ can be an amino acid residue selected from tryptophan, tyrosine, isoleucine, leucine, histidine, phenylalanine, or any combination thereof.

[0280] X o ′ can be a D-amino acid residue or an L-amino acid residue with a hydrophobic side chain. X o ′ can be a naturally occurring or non-natural amino acid residue with a hydrophobic side chain. X o ′ can be an amino acid residue with an aromatic side chain. X o ′ can be an amino acid residue with a heteroaromatic side chain. X o ′ can be selected from phenylalanine; 2-naphthylalanine (Nal or nal); 3-(4′,4-biphenyl)-L-alanine (Bip or bip); tryptophan; tyrosine; valine; isoleucine; leucine; or histidine or any combination thereof. X o ′ could be 2-naphthylalanine (Nal or nal). X o ′ could be L-2-naphthylalanine (L-Nal or Naal). X o ′ could be D-2-naphthylalanine (d-nal or nal). X o ′ could be 3-(4′,4-biphenyl)-L-alanine. X o ′ can be 3-(4′,4-biphenyl)-L-alanine (L-Bip or Bip). X o ′ can be 3-(4′,4-biphenyl)-D-alanine (d-bip or bip). X o ′ can be a C4-C8 alkyl or dialkyl hydrocarbon. X o ′ can be a C6 alkyl or dialkyl hydrocarbon.

[0281] Escape medium (EEV) An EEV containing a cyclic cell-penetrating peptide (cCPP) and one or more adapters is provided. In one embodiment, the EEV contains an exocyclic peptide (EP). In another embodiment, EP is absent.

[0282] EEV can include the structure of formula (A): Formula (A): (A); in: EP is a linear exocyclic peptide; cCPP is a cell-penetrating peptide; L 1 and L 2 Independently designed as a connector arm; ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; and M contains a reaction handle.

[0283] In this embodiment, cCPP is linked to the -(CH2)y′- group via the AAsc group of cCPP. y The ′-group makes the connection a structure Where n is an integer from 1 to 5. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, when n is 1, AAsc is a side chain of a glutamine residue. In an embodiment, when n is 1, AAsc is a side chain of a glutamic acid residue. In an embodiment, when AAsc is a side chain of glutamine, the carboxamide nitrogen of the glutamine side chain forms a bond with the -(CH2)y′- group.

[0284] In the implementation scheme, the EEV of formula (A) includes a structure selected from formulas (B), (C), or (D): Formula (B): (B); Formula (C): (C); or Formula (D): (D); in: EP is a linear exocyclic peptide; cCPP is a cyclic cell-penetrating peptide; x′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; z′ is an integer from 0 to 12; j′′ is 0, 1 or 2, where j′′ is 0 when z′ is 0; X o It contains hydrophobic components; K # It is a D-lysine or L-lysine residue; and M contains a reaction handle.

[0285] EEV can include the structure of formula (A): Formula (A): (A); in EP is a linear exocyclic peptide; cCPP is a cell-penetrating peptide; L 1 and L 2 Independently designed as a connector arm; ^Indicator D-stereochemistry; y′ is an integer from 1 to 5; M contains a reaction handle; and EP contains all D-amino acids; and cCPP contains D-amino acids, achiral amino acids, and AAsc, where AAsc is an amino acid side chain and AAsc connects cCPP to a linker.

[0286] In the implementation, the EEV of formula (A) comprises a structure selected from formula (B), formula (C) or formula (D), wherein the EP comprises all D-amino acids; and the cCPP comprises a D-amino acid, an achiral amino acid and AAsc, wherein AAsc is an amino acid side chain and AAsc conjugates the cCPP to a linker.

[0287] X may be a D-amino acid residue or an L-amino acid residue with a hydrophobic side chain. X may be a naturally occurring or non-naturally occurring amino acid residue with a hydrophobic side chain. X may be an amino acid residue with an aromatic side chain. X may be an amino acid residue with a heteroaromatic side chain. X may be 2-naphthylalanine. X may be Na1. X may be d-N1 (nal). X may be 3-(4-biphenyl)-D-alanine. X may be Bip. X may be bip. X may be a C4-C8 alkyl hydrocarbon. X may be a C6 alkyl hydrocarbon. X may be an amino acid selected from tryptophan, tyrosine, isoleucine, leucine, histidine, phenylalanine, or combinations thereof.

[0288] In the implementation plan, L 1 and L 2 When present, they are each independent hydrocarbon connectors (e.g., NR). h H-(CH2) n -COOH), PEG components (e.g., NR) h H-(CH2O) n -COOH, where Rh is H, methyl or ethyl), a linker containing a hydrophobic component, one or more amino acid residues or combinations thereof.

[0289] In one embodiment, AAsc is a side chain of glutamine. In another embodiment, AAsc is a side chain of L-glutamine. In yet another embodiment, AAsc is a side chain of D-glutamine.

[0290] In the implementation scheme, M contains -OH or or , where y′′ is an integer from 1 to 4.

[0291] In the implementation scheme, EEV includes formula (B): Formula (B): (B); EP, cCPP, x′, j′, ^, y′, z′, j′′, and M are as defined in this paper.

[0292] In the implementation scheme, EEV includes formula (C): Formula (C): (C); Among them, EP, cCPP, x′, j′, ^, y′, z′, j′′, X o ′ and M are as defined in this paper.

[0293] In the implementation scheme, EEV includes formula (D): Formula (D): (D); Among them, EP, cCPP, x′, j′, ^, y′, z′, j′′, X o ′、K # M is as defined in this paper.

[0294] In the implementation scheme, the EEV may include formula (A-2): Equation (A-2): (A-2) Among them, EP, L 1 L 2 ,M,y′,^,n,q,R1,R2,R 3、 R4, R 5、 R6 and R7 are as defined in this paper.

[0295] In one embodiment, the EEV of formula (A-2) may include the connector of formula (B′). In another embodiment, the EEV of formula (A-2) may include the connector of formula (C′). In yet another embodiment, the EEV of formula (A-2) may include the connector of formula (D′).

[0296] In the implementation scheme, the EEV may include formula (A-2a): Equation (A-2a): (A-2a) Among them, EP, L 1 L 2 ,M,y′,^,n,m′,m′′,R 1 R 2 R 3R4 and R 6 As defined in this article.

[0297] In one embodiment, the EEV of formula (A-2a) may include the connector of formula (B′). In another embodiment, the EEV of formula (A-2a) may include the connector of formula (C′). In yet another embodiment, the EEV of formula (A-2a) may include the connector of formula (D′).

[0298] In an implementation, the EEV may have the structure shown in equation (B), where x′ is 0 and z′ is 12. In an implementation, the EEV may have the sequence shown in Table 2a: Table 2a: EEV sequences

[0299] In an implementation, the EEV may have the structure shown in equation (B), where x′ is 2 and z′ is 12. In an implementation, the EEV may have the sequence shown in Table 2b: Table 2b: EEV sequences

[0300] In an implementation, the EEV may have the structure shown in equation (B), where x′ is 2 and z′ is 2. In an implementation, the EEV may have the sequence shown in Table 2c: Table 2c: EEV sequences

[0301] In an implementation, the EEV may have the structure shown in equation (B), where x′ is 2 and z′ is 12. In an implementation, the EEV may have the sequence shown in Table 3a: Table 3a: EEV sequences

[0302] In an implementation, the EEV may have the structure shown in equation (D), where x′ is 2 and z′ is 12. In an implementation, the EEV may have the sequence shown in Table 3b: Table 3b: EEV sequences

[0303] In an implementation, the EEV may have the structure shown in equation (C), where x′ is 2 and z′ is 12. In an implementation, the EEV may have the sequence shown in Table 3c: Table 3c: EEV sequences

[0304] In the implementation scheme, EEV can be selected from Table 4, where cCPP has the formula: FGFGRGRQ.

[0305] Table 4: Internal escape media of cCPP with formula FGFGRGRQ

[0306] In the implementation scheme, EEV can be selected from Table 5, where cCPP has the formula: GfFGrGrQ.

[0307] Table 5: Intravolumetric escape media of cCPP with the formula GfFGrGrQ

[0308] In the implementation scheme, the EEV construct can be selected from Table 6, where cCPP has the formula: FfFGRGRQ.

[0309] Table 6: Internal escape medium of cCPP with formula FfFGRGRQ

[0310] In the implementation scheme, EEV can be selected from Table 7, where cCPP has the formula: Ff-Nal-GrGrQ.

[0311] Table 7: Intravolumetric escape media of cCPP with formula Ff-Nal-GrGrQ

[0312] In the implementation scheme, the EEV can be selected from Table 8A, wherein the connector includes a hydrophobic group.

[0313] Table 8A: Endogenous escape mediators of cCPP containing hydrophobic groups

[0314] In the implementation scheme, the EEV can be selected from Table 8B, wherein cCPP includes citrulline (where Cit = L-citrulline and cit = D-citrulline).

[0315] Table 8B: Endosome escape mediators of cCPP containing citrulline

[0316] In the EEV provided herein, the cCPP can be connected to the connector via AAsc. In the implementation, the cCPP is connected to the connector via AAsc via -(CH2). y′ - The group is attached to the connector. In the embodiment, cCPP is transferred via AAsc through -(CH2). y′ - A group is attached to the connector, making the connection a structure. Where n is an integer from 1 to 5. In an embodiment, cCPP can be linked to the lysine component of the linker via AAsc. In an embodiment, n is 1 when AAsc is glutamic acid. In the EEVs shown in Tables 2a-2c and 3a-3c, cCPP is linked to the lysine component of the linker via AAsc, where AAsc is glutamic acid (E), and n is 1 before conjugation. After conjugation with the linker, AAsc is shown as glutamine (Q).

[0317] In the provided EEV sequence, the lysine may contain a protecting group (not shown). In one embodiment, the protecting group is a trifluoroacetyl (Tfa) group. In another embodiment, the protecting group is an acetyl (Ac) group. It should be understood that other protecting groups may also be used, and the protecting groups may be removed after the EEV is conjugated with the cargo. In one embodiment, the EEV is deprotected after conjugation with the PMO.

[0318] Exocyclic peptides An exopeptide (EP) may contain 2 to 10 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. An EP may contain 2 to 8 amino acid residues or 2 to 6 amino acid residues. In one embodiment, the EP contains 2 amino acid residues. In another embodiment, the EP contains 3 amino acid residues. In yet another embodiment, the EP contains 4 amino acid residues. In a third embodiment, the EP contains 5 amino acid residues. In a fourth embodiment, the EP contains 6 amino acid residues. In a fifth embodiment, the EP contains 7 amino acid residues. In a sixth embodiment, the EP contains 8 amino acid residues.

[0319] The amino acids in EP can have D- or L-stereochemistry. The amino acid residues in EP can be all D-amino acids. The amino acid residues in EP can be all L-amino acids. The amino acid residues in EP can be a combination of D- and L-amino acids.

[0320] Exocyclic peptides can be acylated at the N-terminus (Ac-EP). It should be understood that EP and Ac-EP are used interchangeably throughout the application. For example, EP may contain Ac-PKKKRKV.

[0321] Each amino acid in an exocyclic peptide can be either natural or non-natural. The term "non-natural amino acid" refers to an organic compound that is a homolog of a natural amino acid because it has a similar structure to the natural amino acid, thus mimicking its structure and reactivity. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of the 20 commonly found naturally occurring amino acids, nor are they the rare natural amino acids selenocysteine ​​or pyrrolidone. Non-natural amino acids can also be D-isomers of natural amino acids. Amino acids and / or amino acid residues can be referred to using their full name, their conventional three-letter abbreviation, or their conventional single-letter abbreviation. When using single-letter abbreviations, uppercase letters indicate L amino acids or residues, and lowercase letters indicate D amino acids or residues. For example, arginine can be referred to as Arg, R, or r.

[0322] The EP may contain at least one amino acid residue that is positively charged at a physiologically relevant pH. In embodiments, the EP contains at least one amino acid residue comprising a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. In embodiments, the EP contains at least one amino acid residue comprising a guanidine group or a protonated form thereof. In embodiments, the EP contains at least one amino acid residue comprising a terminal amine or a protonated form thereof. In embodiments, the EP contains at least one amino acid residue comprising an imidazole or a protonated form thereof. The protonated form may refer to its salt throughout this disclosure.

[0323] An EP may contain 1 to 5 amino acid residues containing a side chain with a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. An EP may contain 1 to 4 amino acid residues containing a side chain with a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. An EP may contain 1 to 3 amino acid residues containing a side chain with a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. An EP may contain 1 or 2 amino acid residues containing a side chain with a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. An EP may contain 2 or 3 amino acid residues containing a side chain with a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. An EP may contain 2 to 4 amino acid residues containing a side chain with a guanidine group, a terminal amine, an imidazole, or a protonated form thereof.

[0324] In one embodiment, the amino acid containing the guanidine group is arginine. In one embodiment, the amino acid containing the terminal amine is lysine. In one embodiment, the amino acid containing the imidazole is histidine.

[0325] An EP may contain 1, 2, 3, 4, or 5 amino acid residues, including a side chain containing a guanidine group or its protonated form. An EP may contain 1 amino acid residue, including a side chain containing a guanidine group or its protonated form. An EP may contain 3 amino acid residues, including a side chain containing a guanidine group or its protonated form. An EP may contain 4 amino acid residues, including a side chain containing a guanidine group or its protonated form. An EP may contain 5 amino acid residues, including a side chain containing a guanidine group or its protonated form. The amino acid residue containing the guanidine group side chain may be an arginine residue. An EP may contain 1, 2, 3, 4, or 5 arginine residues. An EP may contain 1 arginine residue. An EP may contain 2 arginine residues. An EP may contain 3 arginine residues. An EP may contain 4 arginine residues. An EP may contain 5 arginine residues.

[0326] An EP may contain one, two, three, four, or five amino acid residues containing a terminal amine or its protonated form. An EP may contain one amino acid residue containing a terminal amine or its protonated form. An EP may contain two amino acid residues containing a terminal amine or its protonated form. An EP may contain three amino acid residues containing a terminal amine or its protonated form. An EP may contain four amino acid residues containing a terminal amine or its protonated form. An EP may contain five amino acid residues containing a terminal amine or its protonated form. The amino acid containing the terminal amine may be lysine. An EP may contain one, two, three, four, or five lysine residues. An EP may contain one lysine residue. An EP may contain two lysine residues. An EP may contain three lysine residues. An EP may contain four lysine residues. An EP may contain five lysine residues. The amino group on the side chain of each lysine residue may be replaced by a protecting group, such as a trifluoroacetyl (-COCF3), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl (Dde), or (4,4-dimethyl-2,6-dioxocyclohexyl-1-ylidene-3)-methylbutyl (ivDde) ​​group. The amino group on the side chain of each lysine residue may be replaced by a trifluoroacetyl (-COCF3). Protecting groups may be included to achieve amide conjugation. The protecting groups may be removed after EP and cCPP conjugation.

[0327] An EP may contain one, two, three, four, or five amino acid residues containing imidazole or its protonated form. An EP may contain one amino acid residue containing imidazole or its protonated form. An EP may contain two amino acid residues containing imidazole or its protonated form. An EP may contain three amino acid residues containing imidazole or its protonated form. An EP may contain four amino acid residues containing imidazole or its protonated form. An EP may contain five amino acid residues containing imidazole or its protonated form. The imidazole-containing amino acid may be histidine. An EP may contain one, two, three, four, or five histidine residues. An EP may contain one histidine residue. An EP may contain two histidine residues. An EP may contain three histidine residues. An EP may contain four histidine residues. An EP may contain five histidine residues.

[0328] An EP may contain one, two, three, or four amino acid residues with uncharged side chains. The uncharged side chains may include hydrophobic side chains. An EP may contain one, two, three, or four amino acid residues with hydrophobic side chains. An EP may contain one, two, three, or four amino acid residues with uncharged hydrophobic side chains. An EP may contain one amino acid residue with an uncharged hydrophobic side chain. An EP may contain two amino acid residues with uncharged hydrophobic side chains. An EP may contain three amino acid residues with uncharged hydrophobic side chains. An EP may contain four amino acid residues with uncharged hydrophobic side chains. The amino acid residues with uncharged hydrophobic side chains may be selected from valine, proline, β-alanine, and glycine. The amino acid residues with hydrophobic side chains may be valine or proline. The amino acid residues with hydrophobic side chains may be valine. The amino acid residues with hydrophobic side chains may be proline. The amino acid residue with a hydrophobic side chain can be β-alanine. The amino acid residue with a hydrophobic side chain can be glycine.

[0329] An EP may contain at least one positively charged amino acid residue and at least one uncharged hydrophobic residue. An EP may contain two positively charged amino acid residues and one uncharged hydrophobic residue. An EP may contain three positively charged amino acid residues and one uncharged hydrophobic residue. An EP may contain four positively charged amino acid residues and one uncharged hydrophobic residue. An EP may contain two positively charged amino acid residues and two uncharged hydrophobic residues. An EP may contain three positively charged amino acid residues and two uncharged hydrophobic residues. An EP may contain four positively charged amino acid residues and two uncharged hydrophobic residues.

[0330] An EP may contain at least one lysine residue and at least one arginine residue. An EP may contain two, three, four, or five lysine residues and / or arginine residues. An EP may contain two lysine residues and / or arginine residues. An EP may contain three lysine residues and / or arginine residues. An EP may contain four lysine residues and / or arginine residues. An EP may contain five lysine residues and / or arginine residues.

[0331] EP may contain one or more amino acid residues selected from lysine (K), arginine (R), histidine (H), glycine (G), β-alanine (B), phenylalanine (F), proline (P), valine (V), or combinations thereof. EP may contain one proline (P). EP may contain one valine (V). EP may contain one glycine (G). EP may contain one or more lysine (K). EP may contain one lysine (K). EP may contain two lysine (K). EP may contain three lysine (K). EP may contain four lysine (K). EP may contain one or more arginine (R). EP may contain one arginine (R). EP may contain two arginine (R). EP may contain three arginine (R). EP may contain four arginine (R). EP may contain five arginine (R). EP may contain six arginine (R). An EP may contain one or more histidine (H). An EP may contain one histidine (H). An EP may contain two histidine (H). An EP may contain three histidine (H). An EP may contain four histidine (H). An EP may contain five histidine (H). An EP may contain six histidine (H). An EP may contain one or more β-alanine (B). An EP may contain two β-alanine (B). An EP may contain two β-alanine (B). The amino acid residues in an EP can have D stereochemistry or L stereochemistry.

[0332] EP may contain KK, KR, RR, RK, RF, HH, HK, HR, RH, KKK, KFK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, RHR, RRV, FRR, RFR, RBR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH, PKKK, KHKK, KKHK, KKK H, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, KRKF, RKKR, RRRR, RBRB, BRBR, HRHR, RHRH, HBHB, BHBH, KGKK, KKGK, HBHBH, RBHBH, BBRRB, HBRBH, BHRHR, BRHRH, HBKBH, RRRRR, KKKKK, KKK RK, RKKKK, KRKKK, KKRKK, KKKKR, KKFRK, KBKBK, KRKIL, RBFBR, RBRBR, RBHBR, RFRRF, RIRRI, RKKKKG, KRKKKG, KKRKKG, KKKKRG, KKKRKG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKKKP, PKKKRK, KRKRKR, RKRKRK, RBRBRB, RBRRBR, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, where B is β-alanine. EP can contain PKKKRKV. EP can contain RR, RRR, RHR, RBR, RBRBR, RBRRBR, RBGRBR, RBRGBR, RBRRBG, RBHBR, or HBRBH, where B is β-alanine. The amino acids in EP can have D stereochemistry or L stereochemistry.

[0333] The EP may include kk, kr, rr, rk, rf, hh, hk, hr, rh, kkk, kfk, kgk, kbk, kbr, krk, krr, rkk, rrr, rhr, rrv, frr, rfr, rbr, kkh, khk, hkk, hrr, hrh, hhr, hbh, hhh, hhhh, pkkk, khkk, kkhk, kkkh, khkh, hkhk, kkkk, kkrk, krkk, krrk, krkf, rkkr, rrrr, rbrb, brbr, hrhr, rhrh, hbhb, bhbh, kgkk, kkgk, hbhbh, rbhbh, brbrb, hbrbh, bhrhr, brhrh, hbkbh, rrrrr, kkkkk, kkkrk, rkkkk, krkkk, kkrkk, kkkkr, kkfrk, kbkbk, krkil, rbfbr, rbrbr, rbhbr, rfrrf, rirri, rkkkkg, krkkkg, kkrkkg, kkkkrg, rkkkkb, krkkkb, kkrkkb, kkkkrb, kkkrkv, rrrrrr, hhhhhh, rhrhrh, hrhrhr, krkkkp, pkkkrk, krkrkr, rkrkrk, rbrbrb, rbrrbr, kbkbkb, pkkkrkv, pgkkrkv, pkgkrkv, pkkgrkv, pkkkgkv, pkkkrgv or pkkkrkg.

[0334] EP can be composed of: KK, KR, RR, RK, RF, HH, HK, HR, RH, KKK, KFK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, RHR, RRV, FRR, RFR, RBR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH, PKKK, KHKK, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, KRKF, RKKR, RRRR, RBRB, BRBR, HRHR, RHRH, HBHB, BHBH, KGKK, KKGK, HBHBH, RBHBH, BBRRB, HBRBH, BHRHR, BRHRH, HBKBH, RRRRR, KKKKK, K KKRK, RKKKK, KRKKK, KKRKK, KKKKR, KKFRK, KBKBK, KRKIL, RBFBR, RBRBR, RBHBR, RFRRF, RIRRI, RKKKKG, KRKKKG, KKRKKG, KKKKRG, KKKRKG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKKKP, PKKKRK, KRKRKR, RKRKRK, RBRBRB, RBRRBR, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, where B is β-alanine. EP can be composed of PKKKRKV. EP can also be composed of the following: RR, RRR, RHR, RBR, RBRBR, RBRRBR, RBGRBR, RBRGBR, RBRRBG, RBHBR, or HBRBH, where B is β-alanine. The amino acids in EP can have D-stereochemistry or L-stereochemistry.

[0335] EP can be composed of: kk, kr, rr, rk, rf, hh, hk, hr, rh, kkk, kfk, kgk, kbk, kbr, krk, krr, rkk, rr r, rhr, rrv, frr, rfr, rbr, kkh, khk, hkk, hrr, hrh, hhr, hbh, hhh, hhhh, pkkk, khkk, k khk, kkkh, khkh, hkhk, kkkk, kkrk, krkk, krrk, krkf, rkkr, rrrr, rbrb, brbr, hrhr, rhrh, hbhb, bhbh, kgkk, kkgk, hbhbh, rbhbh, brbrb, hbrbh, bhrhr, brhrh, hbkbh, rrrr r, kkkkk, kkkrk, rkkkk, krkkk, kkrkk, kkkkr, kkfrk, kbkbk, krkil, rbfbr, rbrbr, rbhbr, rfrrf, rirri, rkkkkg, krkkkg, kkrkkg, kkkkrg, rkkkkb, krkkkb, kkrkkb, kkkkr b, kkkrkv, rrrrrr, hhhhhh, rhrhrh, hrhrhr, rkkkp, pkkkrk, krkrkr, rkrkrk, rbrbrb, rbrrbr, kbkbkb, pkkkrkv, pgkkrkv, pkgkrkv, pkkgrkv, pkkkgkv, pkkkrgv or pkkkrkg.

[0336] EP can contain KK, KR, RR, RK, RF, KKK, KGK, KFK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, KRKF, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKFRK, KRKIL, RFRRF, RIRRI, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP can also contain PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B represents β-alanine.

[0337] EP can contain kk, kr, rr, rk, rf, kkk, kgk, kfk, kbk, kbr, krk, krr, rkk, rrr, kkkk, kkrk, krkk, krrk, krkf, rkkr, rrrr, kgkk, kkgk, kkkkk, kkkrk, kbkbk, kkfrk, krkil, rfrrf, rirri, kkkrkv, pkkkrkv, pgkkrkv, pkgkrkv, pkkgrkv, pkkkgkv, pkkkrgv, or pkkkrkg. EP can contain pkkkrkv, rr, rrr, rhr, rbr, rbrbr, rbhbr, or hbrbh.

[0338] EP can be composed of the following: KK, KR, RR, RK, RF, KKK, KGK, KFK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, KRKF, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKFRK, KRKIL, RFRRF, RIRRI, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP can also be composed of the following: PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B represents β-alanine.

[0339] EP can be composed of the following: kk, kr, rr, rk, rf, kkk, kgk, kbk, kfk, kbr, krk, krr, rkk, rrr, kkkk, kkrk, krkk, krrk, krkf, rkkr, rrrr, kgkk, kkgk, kkkkk, kkkrk, kbkbk, kkfrk, krkil, rfrrf, rirri, kkkrkv, pkkkrkv, pgkkrkv, pkgkrkv, pkkgrkv, pkkkgkv, pkkkrgv or pkkkrkg.

[0340] An EP may contain one of the following sequences: HBH, HBHBH, or HBRBH. An EP may contain HHRBFBR. An EP may contain one of the following sequences: KBK, KBKBK, or KBR. An EP may contain one of the following sequences: KGK or KGKK. An EP may contain one of the following sequences: KKGK, KKKK, KKKKR, KKKRK, KKKRKG, or KKRK. An EP may contain one of the following sequences: KR, KRK, or KRKKK. An EP may contain PGKKRKV. An EP may contain one of the following sequences: PKGKRKV, PKK, PKKGRKV, PKKK, PKKKGKV, PKKKRGV, PKKKRKG, PKKRGV, PKKRKG, or PKKRKV. An EP may contain one of the following sequences: RBHBH, RBHBR, RBR, RBRBR, or RBRRBR. EP may contain one of the following sequences: RFR or RHR. EP may contain RKKK. EP may contain one of the following sequences: RR, RRR, or RRRRFFF. EP may contain one of the following sequences: RK or RF. EP may contain one of the following sequences: KFK or KRKF. EP may contain one of the following sequences: KKFRK or KRKIL. EP may contain one of the following sequences: RFRRF or RIRRI. EP may contain PKKKRKV. B is β-alanine.

[0341] An EP may contain 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). An EP may contain an NLS containing the amino acid sequence PKKKRKV. An EP may consist of an NLS containing the amino acid sequence PKKKRKV.

[0342] An EP contains 2 to 10, 6 to 9, or 4 to 8 consecutive amino acid residues. An EP may contain 2 to 10, 6 to 9, or 4 to 8 amino acids, wherein not all amino acids are consecutive. For example, the amino acid residues of an EP may be separated from one or more components of an EEV. For example, the amino acid residues of an EP may be separated from a linker, cCPP, or a combination thereof, thereby forming a “split” EP.

[0343] EEV-Cargo Conjugate In the implementation scheme, the EEV can be combined with cargo to form an EEV-cargo conjugate, which includes the structure of formula (A-1): Equation (A-1): (A-1) in: EP is a linear exocyclic peptide as defined in this paper; cCPP is the cyclic cell-penetrating peptide defined in this paper; L 1 and L 2 Independently designed as a connector arm; ^Indicates D-stereochemistry or L-stereochemistry; y′ is an integer from 1 to 5; M′ is the bonding group defined in this paper; and The goods are peptides, oligonucleotides, small molecules, or any combination thereof.

[0344] In the implementation scheme, the EEV-cargo conjugate of formula (A-1) may have a formula selected from (B-1), (C-1), and (D-1): Equation (B-1): (B-1); Equation (C-1): (C-1); and Equation (D-1): (D-1); Among them, EP, cCPP, x′, j′, ^, y′, z′, j′′, X o ′、K # M′ and goods are as defined herein.

[0345] In the embodiments, the EEV-cargo conjugate has the structure of formula (B-1), formula (C-1) or formula (D-1), wherein EP comprises all D-amino acids; cCPP comprises D-amino acids, achiral amino acids and AAsc, wherein AAsc is an amino acid side chain and AAsc conjugates cCPP to a linker.

[0346] In the implementation scheme, the EEV-cargo conjugate has the structure of formula (A-3): Equation (A-3): (A-3) in: EP is a linear exocyclic peptide; L 1 and L 2 For the connector arm, M′ is a bonding group; and y′ is an integer between 1 and 5. ^Indicates D-stereochemistry or L-stereochemistry; R 1 R 2 and R3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 4 R 5 R 6 and R 7 A side chain that is independently H or an amino acid; q is an integer between 1 and 4; n is an integer from 1 to 4; and The cargo consists of peptides, oligonucleotides, small molecules, or combinations thereof.

[0347] In one embodiment, the EEV-cargo conjugate of formula (A-3) has a joint structure of formula (B′). In another embodiment, the EEV-cargo conjugate of formula (A-3) has a joint structure of formula (C′). In yet another embodiment, the EEV-cargo conjugate of formula (A-3) has a joint structure of formula (D′).

[0348] In the implementation scheme, the EEV-cargo conjugate has the structure of formula (A-3a): Equation (A-3a): (A-3a) Among them, EP, M′, L 1 L 2 ,^,y′,n,R 1 R 2 R 3 R 4 R 5 R 6 and R 7 As defined herein, and the goods are peptides, oligonucleotides, small molecules, or combinations thereof.

[0349] In one embodiment, the EEV-cargo conjugate of formula (A-3a) has a joint structure of formula (B′). In another embodiment, the EEV-cargo conjugate of formula (A-3a) has a joint structure of formula (C′). In yet another embodiment, the EEV-cargo conjugate of formula (A-3a) has a joint structure of formula (D′).

[0350] In the implementation scheme, the EEV-cargo conjugate has the structure of formula (A-3b): Equation (A-3b): (A-3b) Among them, EP, L 1 L 2 ,M′,^,y′,n,m′,m′′,q,R 1 R 2 R3 R 4 and R 6 As defined herein, and the goods are peptides, oligonucleotides, small molecules, or combinations thereof.

[0351] In one embodiment, the EEV-cargo conjugate of formula (A-3b) has a joint structure of formula (B′). In another embodiment, the EEV-cargo conjugate of formula (A-3b) has a joint structure of formula (C′). In yet another embodiment, the EEV-cargo conjugate of formula (A-3b) has a joint structure of formula (D′).

[0352] In the implementation scheme, the EEV-cargo conjugate has the structure of formula (A-3c): Formula (A-3c): (A-3c); Among them, EP, L 1 L 2 ,M′,^,y′,n,m′,m′′,R 1 R 2 R 3 R 4 and R 6 As defined herein, and the goods are peptides, oligonucleotides, small molecules, or combinations thereof.

[0353] In one embodiment, the EEV-cargo conjugate of formula (A-3c) has a joint structure of formula (B′). In another embodiment, the EEV-cargo conjugate of formula (A-3c) has a joint structure of formula (C′). In yet another embodiment, the EEV-cargo conjugate of formula (A-3c) has a joint structure of formula (D′).

[0354] In this embodiment, the goods are oligonucleotides. In this embodiment, the oligonucleotides are therapeutic oligonucleotides. In this embodiment, the oligonucleotides are antisense oligonucleotides. In this embodiment, the oligonucleotides are diaminophosphate morpholino oligonucleotides (PMOs).

[0355] M′ may contain: -NH-, -C(O)-, -O-, ; ; ; or , where y′′ is an integer from 1 to 4, and t′ is from 0 to 10.

[0356] M′ may contain: -NH-, -C(O)-, -O-, ; ; ; where y′′ is an integer from 1 to 4.

[0357] In the implementation scheme, M′ includes or , where t′ is between 0 and 10.

[0358] M′ may contain -NH-. M′ may contain -C(O)-. M′ may contain -O-.

[0359] M′ may contain a structure selected from the following: and , where R is an alkyl, alkenyl, alkynyl, carbocyclic or heterocyclic group.

[0360] M′ contains a structure selected from the following: , or M′ may contain M′ may contain M′ may contain .

[0361] M′ may contain -C(O)-. M′ may contain -NH-. M′ may contain -O-.

[0362] M′ may contain , or M′ may contain Where t′ is between 0 and 10. M′ may contain M′ can be -O-. M′ can be -NH-. M′ can be -C(O)-.

[0363] This article provides an EEV-cargo conjugate comprising an EEV conjugated to a cargo via a bonding group (M′), wherein M′ may include -NH-, -C(O)-, -O-, ... , , , or .

[0364] The linker can be covalently attached to the cargo at any suitable location on the cargo. The linker can be covalently attached to the 3' or 5' end of the oligonucleotide cargo. The linker can be covalently attached to the main chain of the cargo.

[0365] M′ forms a bond with the free secondary amine of the morpholine ring of the 3′ terminal nucleotide of the PMO cargo. In an embodiment, M′ forms a bond with the free secondary amine of the morpholine ring of the 3′ terminal nucleotide of the PMO. In an embodiment, M′ forms a bond with the free hydroxyl group of the 5′ terminal nucleotide of the PMO.

[0366] In an embodiment, the EEV-cargo conjugate has the structure shown in formula (B-1), where x′ is 0 and z′ is 12. In an embodiment, the EEV-cargo conjugate may have the sequence shown in Table 9a.

[0367] Table 9a: EEV-cargo conjugate sequence

[0368] In an embodiment, the EEV-cargo conjugate has the structure shown in formula (B-1), where x′ is 2 and z′ is 12. In an embodiment, the EEV-cargo conjugate may have the sequence shown in Table 9b.

[0369] Table 9b: EEV-Cargo Conjugate Sequence

[0370] In an embodiment, the EEV-cargo conjugate has the structure shown in formula (B-1), where x′ is 2 and z′ is 2. In an embodiment, the EEV-cargo conjugate may have the sequence shown in Table 9c.

[0371] Table 9c: EEV-cargo conjugate sequence

[0372] In an embodiment, the EEV-cargo conjugate has the structure shown in formula (B-1), where x′ is 2 and z′ is 12. In an embodiment, the EEV-cargo conjugate may have the sequence shown in Table 9d.

[0373] Table 9d: EEV-Cargo Conjugate Structure

[0374] In an embodiment, the EEV-cargo conjugate has the structure shown in formula (D-1), where x′ is 2 and z′ is 12. In an embodiment, the EEV-cargo conjugate may have the sequence shown in Table 10a.

[0375] Table 10a: EEV-Cargo Conjugate Structure

[0376] In an embodiment, the EEV-cargo conjugate has the structure shown in formula (C-1), where x′ is 2 and z′ is 12. In an embodiment, the EEV-cargo conjugate may have the sequence shown in Table 10b.

[0377] Table 10b: EEV-Cargo Conjugate Structure

[0378] The linker can be covalently attached to the cargo at any suitable location on the cargo. The linker can be covalently attached to the 3′ or 5′ end of the oligonucleotide cargo. The linker can be covalently attached to the main chain of the cargo.

[0379] In an embodiment where the cargo is PMO, M′ forms a bond with the free secondary amine of the morpholine ring of the terminal nucleotide of the PMO cargo. In another embodiment, M′ forms a bond with the free secondary amine of the morpholine ring of the 3′ terminal nucleotide of the PMO. In yet another embodiment, M′ forms a bond with the free hydroxyl group of the 5′ terminal nucleotide of the PMO.

[0380] The structure of the EEV-cargo conjugate is as follows: Figure 27-29 As shown.

[0381] goods In one embodiment, an EEV is provided comprising a cyclic peptide, such as a cyclic cell-penetrating peptide (cCPP) conjugated to a cargo. In another embodiment, the cargo is an oligonucleotide. In yet another embodiment, the oligonucleotide is a therapeutic oligonucleotide. In yet another embodiment, the oligonucleotide is an antisense oligonucleotide. In yet another embodiment, the oligonucleotide targets a target polynucleotide. In yet another embodiment, the antisense oligonucleotide is a diaminophosphate morpholino oligonucleotide (PMO). In yet another embodiment, the oligonucleotide comprises siRNA, RNAi, microRNA, antagomir, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, supermir, miRNA mimics, miRNA inhibitors, or combinations thereof. For example, see Chery, J., “RNAtherapeutics: RNAi and antisense mechanisms and clinical applications,” Postdoc J, July 2016, 4(7):35-50, and Zhu, et al., “RNA-based therapeutics: an overview and prospectus,” Cell Death & Disease, July 23, 2022, 12(644) (doi.org / 10.1038 / s41419-022-05075-2).

[0382] The term "antisense oligonucleotide," or simply "antisense," refers to an oligonucleotide that is complementary to a target polynucleotide sequence. Antisense oligonucleotides are single-stranded DNA or RNA that are complementary to a selected sequence (such as the mRNA of a target gene).

[0383] Antisense oligonucleotides can regulate one or more aspects of protein transcription, translation, and expression. In embodiments, antisense oligonucleotides can target a target sequence in a target precursor mRNA and regulate one or more aspects of precursor mRNA splicing. As used herein, splicing regulation refers to altering the processing of a precursor mRNA transcript such that the spliced ​​mRNA molecule contains a different combination of exons (due to exon skipping or exon insertion), one or more exons are deleted, or sequences not normally present in the spliced ​​mRNA (e.g., intron sequences) are deleted or added. In embodiments, hybridization of an antisense oligonucleotide with a target sequence in a precursor mRNA molecule restores the mutated precursor mRNA sequence to its natural splicing. In embodiments, hybridization of an antisense oligonucleotide results in alternative splicing of the target precursor mRNA. In embodiments, hybridization of an antisense oligonucleotide results in exon inclusion or exon skipping of one or more exons. In embodiments, the skipped exon sequence contains frameshift mutations, nonsense mutations, or missense mutations. In embodiments, the skipped exon sequence contains deletions, substitutions, or insertions of nucleic acids. In one implementation, the skipped exon itself does not contain sequence mutations, but adjacent exons contain mutations that result in frameshift or nonsense mutations. In another implementation, hybridization of the antisense oligonucleotide with the target sequence in the target precursor mRNA prevents the exon sequence from being included in the mature mRNA molecule. In yet another implementation, hybridization of the antisense oligonucleotide with the target sequence in the target precursor mRNA leads to preferential expression of the wild-type target protein isoform. Finally, hybridization of the antisense oligonucleotide with the target sequence in the target precursor mRNA leads to expression of a respliced ​​target protein containing the active fragment of the wild-type target protein.

[0384] The mechanism of antisense oligonucleotides is the hybridization of the antisense oligonucleotide with its target nucleic acid. In one embodiment, the antisense oligonucleotide hybridizing with its target sequence inhibits the expression of the target protein. In another embodiment, the hybridization of the antisense oligonucleotide with its target sequence inhibits the expression of one or more wild-type target protein isoforms. In yet another embodiment, the hybridization of the antisense oligonucleotide with its target sequence upregulates the expression of the target protein. And finally, the hybridization of the antisense oligonucleotide with its target sequence increases the expression of one or more wild-type target protein isoforms.

[0385] Methods for generating antisense oligonucleotides are known in the art and can be readily applied to generate antisense oligonucleotides targeting any polynucleotide sequence. The selection of antisense oligonucleotide sequences specific to a given target sequence is based on analysis of the selected target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or inhibit specific binding to target mRNA in the host cell. Target regions of mRNA may include regions at or near the AUG translation start codon, as well as those sequences substantially complementary to the 5' region of the mRNA. For example, these secondary structure analyses and target site selection considerations can be performed using OLIGO primer analysis software v.4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0386] In implementation methods, oligonucleotides (e.g., antisense oligonucleotides) can alter one or more aspects of the splicing, translation, or expression of a target gene, for example, by altering the splicing of eukaryotic target precursor mRNA. Oligonucleotides comprise nucleic acid sequences complementary to sequences found in the target precursor mRNA sequence, such as sequences containing at least a portion of exons, at least a portion of introns, or both. The use of these oligonucleotides provides a direct genetic approach capable of modulating the splicing of specific disease-causing genes. The principle behind antisense technology is that antisense oligonucleotides hybridizing with target nucleic acids regulate gene expression activity through one of a variety of antisense mechanisms, such as splicing or translation. The sequence specificity of oligonucleotides makes this technology highly attractive as a therapeutic approach, selectively modulating the splicing of precursor mRNAs involved in the pathogenesis of any of a variety of diseases. Antisense technology is an effective means of altering the expression of one or more specific gene products and can therefore prove useful in many therapeutic, diagnostic, and research applications.

[0387] Oligonucleotides may contain one or more asymmetric centers, and thus produce enantiomers, diastereomers, and other stereoisomers, which can be defined by absolute stereochemistry as (R) or (S), α or β or (D) or (L). All these possible isomers, as well as their racemic and optically pure forms, can be included in oligonucleotides.

[0388] Oligonucleotide hybridization sites The antisense mechanism relies on the hybridization of an antisense oligonucleotide with a target nucleic acid. In one embodiment, an antisense oligonucleotide complementary to the target nucleic acid is provided. In another embodiment, the target nucleic acid sequence is present in the precursor mRNA molecule. In yet another embodiment, the target nucleic acid sequence is present in an exon of the precursor mRNA molecule. Finally, in yet another embodiment, the target nucleic acid sequence is present in an intron of the precursor mRNA molecule.

[0389] Precursor mRNA molecules are produced in the cell nucleus and processed before or during their transport to the cytoplasm for translation. Precursor mRNA processing involves adding a 5′ methylation cap and a poly(A) tail of approximately 200-250 bases to the 3′ end of the transcript. The next step in mRNA processing is precursor mRNA splicing, which occurs during the maturation of 90-95% of mammalian mRNA. Introns (or insertion sequences) are regions in the primary transcript (or the DNA encoding it) that are not included in the coding sequence of the mature mRNA. Exons are regions in the primary transcript that are retained in the mature mRNA; these regions remain in the mature mRNA when it reaches the cytoplasm. Exons are spliced ​​together to form the mature mRNA sequence. The splice junction is also called the splice site; the 5′ side of the junction is often called the “5′ splice site” or “splicing donor site,” and the 3′ side is called the “3′ splice site” or “splicing acceptor site.” During splicing, the 3′ end of the upstream exon joins the 5′ end of the downstream exon. Therefore, unspliced ​​RNA (or precursor mRNA) has an exon / intron junction at the 5' end of an intron and an intron / exon junction at the 3' end of an intron. After the intron is removed, the exons in mature mRNA are continuous at what are sometimes called exon / exon junctions or boundaries. Causal splicing sites are those that are less commonly used but can be used when the usual splicing sites are blocked or unavailable. Alternative splicing, which splices together different combinations of exons, usually results in a single gene producing multiple mRNA transcripts.

[0390] In some embodiments, the oligonucleotide can hybridize with sequences at splice sites. In some embodiments, the oligonucleotide can hybridize with sequences containing partial splice sites. In some embodiments, the oligonucleotide can hybridize with sequences containing partial or complete splice sites. In some embodiments, the oligonucleotide can hybridize with sequences containing partial or complete splice donor sites. In some embodiments, the oligonucleotide can hybridize with sequences containing partial or complete splice acceptor sites. In some embodiments, the oligonucleotide can hybridize with sequences containing partial or complete cryptic splice sites. In some embodiments, the oligonucleotide can hybridize with sequences containing exon / intron junctions.

[0391] Precursor mRNA splicing involves two consecutive biochemical reactions. Both reactions involve spliceosome transesterification between RNA nucleotides. In the first reaction, the 2′-OH (defined during spliceosome assembly) of a specific branching point nucleotide in the intron nucleophilically attacks the first nucleotide of the intron at the 5′ splice site, forming a lasso intermediate. In the second reaction, the 3′-OH of the released 5′ exon nucleophilically attacks the last nucleotide of the intron at the 3′ splice site, thereby linking the exon and releasing the intron lasso. Precursor mRNA splicing is regulated by intron silencer sequences (ISS) and terminal stem-loop (TSL) sequences. As used herein, the terms “intron silencer sequence (ISS)” and “terminal stem-loop (TSL)” refer to sequence elements in the intron and exon, respectively, that control alternative splicing by binding to trans-acting protein factors in the precursor mRNA, resulting in differential use of splice sites. Typically, intron silencer sequences are between 8 and 16 nucleotides long and are less conserved than splice sites at exon-intron junctions. Terminal stem-loop sequences are typically between 12 and 24 nucleotides long and form secondary loop structures due to complementarity, thus binding within a 12-24 nucleotide sequence.

[0392] In one embodiment, the oligonucleotide hybridizes with a sequence containing some or all of the intron silencer sequences. In another embodiment, the oligonucleotide hybridizes with a sequence containing some or all of the terminal stem-loop sequences.

[0393] Up to 50% of human genetic diseases caused by point mutations are due to aberrant splicing. These point mutations can disrupt existing splicing sites or create new ones, resulting in mRNA transcripts with different exon combinations or exon deletions. Point mutations can also lead to the activation of cryptic splicing sites or the disruption of regulatory cis elements (i.e., splice enhancers or silencers).

[0394] In one embodiment, the oligonucleotide hybridizes with a sequence containing some or all of an aberration splicing site caused by a mutation in the target gene. In another embodiment, the oligonucleotide hybridizes with a sequence containing some or all of a regulatory element. Additionally, antisense oligonucleotides targeting cis-regulatory elements are also provided. In one embodiment, the regulatory element is located in an exon. In another embodiment, the regulatory element is located in an intron.

[0395] In the embodiments, oligonucleotides can specifically hybridize with sequences in the translation start codon region, 5′ cap region, intron / exon junction, coding sequence, translation stop codon region, or 5′- or 3′-untranslated region. In the embodiments, oligonucleotides can hybridize partially or completely with precursor mRNA splice sites, exon-exon junctions, or intron-exon junctions. In the embodiments, oligonucleotides can hybridize with aberrant fusion junctions due to rearrangements or deletions. In the embodiments, oligonucleotides can hybridize with specific exons in alternatively spliced ​​mRNA.

[0396] In some embodiments, the oligonucleotide can hybridize with a sequence of 5 to 50 nucleotides in length, which may also be referred to as the length of the oligonucleotide. In some embodiments, the length of the oligonucleotide is 5 to 50, 10 to 40, 15 to 30, 20 to 30, or 20 to 25 nucleotides. In some embodiments, the length of the oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the oligonucleotide is at least 15, 16, 17, 18, 19, or 20, and at most 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the oligonucleotide is 10 nucleotides. In some embodiments, the length of the oligonucleotide is 15 nucleotides. In one embodiment, the oligonucleotide is 16 nucleotides long. In another embodiment, the oligonucleotide is 17 nucleotides long. In another embodiment, the oligonucleotide is 18 nucleotides long. In another embodiment, the oligonucleotide is 19 nucleotides long. In another embodiment, the oligonucleotide is 20 nucleotides long. In another embodiment, the oligonucleotide is 21 nucleotides long. In another embodiment, the oligonucleotide is 22 nucleotides long. In another embodiment, the oligonucleotide is 23 nucleotides long. In another embodiment, the oligonucleotide is 24 nucleotides long. In another embodiment, the oligonucleotide is 25 nucleotides long. In another embodiment, the oligonucleotide is 26 nucleotides long. In another embodiment, the oligonucleotide is 27 nucleotides long. In another embodiment, the oligonucleotide is 28 nucleotides long. In another embodiment, the oligonucleotide is 29 nucleotides long. In another embodiment, the oligonucleotide is 30 nucleotides long.

[0397] In embodiments, the oligonucleotide may be less than 100% complementary to the target nucleic acid sequence. As used herein, the term "complementarity percentage" refers to the number of nucleobases of the oligonucleotide that is nucleobase complementary to the corresponding nucleobases of the oligonucleotide or nucleic acid divided by the total length (nucleobase number) of the oligonucleotide. Those skilled in the art will recognize that it is possible to include mismatches without eliminating the activity of the antisense oligonucleotide. In embodiments, the oligonucleotide may contain up to 20% nucleotides that disrupt the base pairing between the oligonucleotide and the target nucleic acid. In embodiments, the oligonucleotide contains no more than 15%, no more than 10%, no more than 5% mismatches, or no mismatches. In embodiments, the oligonucleotide contains no more than 1, 2, 3, 4, or 5 mismatches. In embodiments, the oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target nucleic acid. The complementarity percentage of the oligonucleotide is calculated by dividing the number of complementary nucleobases by the total number of nucleobases in the oligonucleotide. The complementarity percentage of an oligonucleotide region is calculated by dividing the number of complementary nucleobases in the region by the total number of nucleobase regions.

[0398] In implementations, nucleotide affinity modifications allow for a greater number of mismatches compared to unmodified oligonucleotides. Similarly, some oligonucleotide sequences may be more tolerant of mismatches than others. Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, for example, by determining the melting temperature (Tm). Tm or ΔTm can be calculated using techniques familiar to those skilled in the art. For example, the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allows those skilled in the art to evaluate the ability of nucleotide modifications to increase the melting temperature of the RNA:DNA duplex.

[0399] antisense mechanism Oligonucleotides can be antisense oligonucleotides that regulate one or more aspects of protein transcription, translation, and expression. In embodiments, oligonucleotides that hybridize with a target sequence in a target precursor mRNA can regulate one or more aspects of precursor mRNA splicing. As used herein, splicing regulation refers to altering the processing of a precursor mRNA transcript such that the spliced ​​mRNA molecule contains a different combination of exons (due to exon skipping or exon insertion), one or more exons are deleted, or sequences not normally present in the spliced ​​mRNA (e.g., intron sequences) are deleted or added. In embodiments, hybridization of an oligonucleotide with a target sequence in a precursor mRNA molecule restores the mutated precursor mRNA sequence to its natural splicing. In embodiments, oligonucleotide hybridization results in alternative splicing of the target precursor mRNA. In embodiments, oligonucleotide hybridization results in exon inclusion or exon skipping of one or more exons. In embodiments, the skipped exon sequence contains frameshift mutations, nonsense mutations, or missense mutations. In embodiments, the skipped exon sequence contains a deletion, substitution, or insertion of nucleic acid. In one implementation, the skipped exon itself does not contain a sequence mutation, but the adjacent exon contains a mutation that results in a frameshift or nonsense mutation. In another implementation, the deletion of an exon that does not contain a sequence mutation restores the reading frame of the mature mRNA. In yet another implementation, hybridization of the oligonucleotide with the target sequence in the target precursor mRNA leads to preferential expression of the wild-type target protein isoform. Finally, hybridization of the oligonucleotide with the target sequence in the target precursor mRNA leads to expression of a respliced ​​target protein containing the active fragment of the wild-type target protein.

[0400] The mechanism of antisense oligonucleotides involves hybridization of the antisense oligonucleotide with a target nucleic acid. In one embodiment, the oligonucleotide hybridizing with its target sequence inhibits the expression of the target protein. In another embodiment, the oligonucleotide hybridizing with its target sequence inhibits the expression of one or more wild-type target protein isoforms. In yet another embodiment, the oligonucleotide hybridizing with its target sequence upregulates the expression of the target protein. And in yet another embodiment, the oligonucleotide hybridizing with its target sequence increases the expression of one or more wild-type target protein isoforms.

[0401] The efficacy of an oligonucleotide 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 the hybridization of the antisense oligonucleotide with its target nucleic acid. Such detection and / or measurement can be direct or indirect. In embodiments, antisense activity is assessed by detecting and / or measuring the amount of the target protein. In embodiments, antisense activity is assessed by detecting and / or measuring the amount of the respliced ​​target protein. In embodiments, antisense activity is assessed by detecting and / or measuring the amount of the target nucleic acid and / or the cleaved target nucleic acid and / or the alternatively spliced ​​target nucleic acid.

[0402] Antisense oligonucleotide design The design of oligonucleotides will depend on the sequence being targeted. Targeting oligonucleotides to specific target nucleic acid molecules can be a multi-step process. This process typically begins with the identification of the target nucleic acid whose expression needs to be regulated. 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 precursor mRNA and mRNA), and cDNA derived from such RNA. For example, a target nucleic acid could be a cellular gene (or mRNA transcribed from that gene) whose expression is associated with a specific condition or disease state, or a nucleic acid molecule derived from an infectious agent.

[0403] Those skilled in the art will be able to design, synthesize, and screen antisense oligonucleotides with different nucleobase sequences to identify sequences that produce antisense activity. For example, antisense oligonucleotides can be designed to alter the splicing of target precursor mRNA or inhibit the expression of target proteins. Methods for designing, synthesizing, and screening antisense oligonucleotides with antisense activity against preselected target nucleic acids can be found, for example, in "Antisense Drug Technology, Principles, Strategies, and Applications" edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida, which is incorporated herein by reference in its entirety for any purpose.

[0404] In the implementation scheme, the antisense oligonucleotide comprises a modified nucleoside, a modified inter-nucleoside linker, and / or a conjugate group.

[0405] In the implementation scheme, the antisense oligonucleotide is "tricyclic DNA (tc-DNA)," which refers to a class of restricted DNA analogs in which each nucleotide is modified by introducing a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry at the twist angle γ. tc-DNA containing the same basic adenine and thymine forms highly stable AT base pairs with complementary RNA.

[0406] nucleosides In the embodiments, antisense oligonucleotides comprising a linker nucleoside are provided. In the embodiments, some or all of the nucleosides are modified nucleosides. In the embodiments, one or more nucleosides comprise modified nucleobases. In the embodiments, one or more nucleosides comprise modified sugars. Chemically modified nucleosides are commonly used to be incorporated into antisense oligonucleotides to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for target RNA.

[0407] Generally, a nucleobase is any group containing one or more atoms or groups of atoms capable of bonding with hydrogen atoms of another nucleic acid. Besides the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), which are considered "unmodified" or "natural" nucleobases, many modified nucleobases or nucleobase mimics known to those skilled in the art also apply to the oligonucleotides described herein. The terms modified nucleobase and nucleobase mimic can overlap, but generally, a modified nucleobase refers to a nucleobase that is structurally similar to the parental nucleobase, such as, for example, 7-denitropurine, 5-methylcytosine, or G-clamp, while a nucleobase mimic will include more complex structures, such as, for example, tricyclic phenoxazine nucleobase mimics. Methods for preparing the aforementioned modified nucleobases are well known to those skilled in the art.

[0408] In the embodiments, the oligonucleotides provided herein comprise one or more nucleosides having modified sugar moieties. In the embodiments, the furanyl glycan ring of the natural nucleoside can be modified in various ways, including but not limited to, adding substituent groups, bridging two non-twinned ring atoms to form bicyclic nucleic acids (BNAs), and using atoms or groups such as -S-, -N(R)-, or -C(U)-. 1 (U) 2 ) replaces the epoxide at the 4'-position, wherein U, U 1 and U 2 Each sugar moiety is independently selected from H or any suitable substituent. Modified sugar moieties are well known and can be used to alter (generally increase) the affinity of antisense oligonucleotides for their targets and / or increase nuclease resistance. Representative lists of modified sugars include, but are not limited to, non-bicyclic substituted sugars, especially non-bicyclic 2'-substituted sugars having 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituent groups; and 4'-thio-modified sugars. Sugars may also be substituted with sugar mimic groups, etc. In embodiments, the sugar is substituted with a six-membered morpholine ring. Methods for preparing modified sugars are well known to those skilled in the art.

[0409] In the embodiments, the nucleosides comprise bicyclic modified sugars (BNAs), including LNA (4'-(CH2)-O-2' bridge), 2'-thio-LNA (4'-(CH2)-S-2' bridge), 2'-amino-LNA (4'-(CH2)-NR-2' bridge), ENA (4'-(CH2)2-O-2' bridge), 4'-(CH2)3-2' bridged BNA, 4'-(CH2CH(CH3))-2' bridged BNA" cEt (4'-(CH(CH3)-O-2' bridge) and cMOE BNA (4'-(CH(CH2OCH3)-O-2' bridge).

[0410] This article also provides "locked nucleic acids" (LNAs), in which the 2'-hydroxy group of the ribosyl sugar ring is attached to the 4' carbon atom of the sugar ring, forming a 2'-C,4'-C-oxymethylene bond to form a bicyclic sugar moiety. The bond can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom. The bicyclic moiety is referred to by the term LNA; if the position is vinyl, it is referred to by the term ENA™ (Singh et al., Chem. Commun., 1998, 4, 455-456; ENA™: Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNAs and other bicyclic sugar analogs exhibit very high double-strand thermal stability (Tm = +3 °C to +10 °C), stability towards 3'-exonuclease degradation, and good solubility with complementary DNA and RNA. Effective and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0411] Another LNA isoform studied is α-L-LNA, which has been shown to have improved stability against 3'-exonucleases. α-L-LNA has been incorporated into antisense nick polymers and chimeras that exhibit effective antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0412] The synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as 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.

[0413] Analogs of LNA, phosphate thio-LNA and 2'-thio-LNA, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). The synthesis of a novel conformationally restricted high-affinity oligonucleotide analog, 2'-amino-LNA, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino-LNA and 2'-methylamino-LNA have been prepared, and their thermal stability with duplexes of complementary RNA and DNA strands has been previously reported.

[0414] Nucleoside interlinking This article describes internucleotide linking groups that connect nucleosides or other modified monomeric units together to form antisense oligonucleotides. Two main categories of internucleotide linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleotide links include, but are not limited to, phosphodiesters, phosphotriesters, methyl phosphates, amino phosphates (including diamino phosphates), and thiophosphates. Representative phosphorus-free internucleotide linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), aminomethylthiocarbonyl esters (-OC(O)(NH)-S-), siloxanes (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Antisense oligonucleotides with non-phosphodiester linking groups are called oligonucleotides. Compared to natural phosphodiester links, modified internucleotide links can be used to alter (and often increase) the nuclease resistance of antisense oligonucleotides. Nucleoside linkages with chiral atoms can be prepared as racemic, chiral, or mixtures. Representative chiral nucleoside linkages include, but are not limited to, alkyl phosphonates and thiophosphates. Methods for preparing phosphorus-containing and phosphorus-free linkages are well known to those skilled in the art.

[0415] In the implementation, the phosphate group can be attached to the 2', 3', or 5' hydroxyl portion of the sugar. In the formation of oligonucleotides, phosphate ester groups covalently link adjacent nucleosides to form linear polymeric oligonucleotides. Within the oligonucleotide, the phosphate ester group is typically referred to as the inter-nucleoside backbone that forms the oligonucleotide. Normal bonds or backbones of RNA and DNA are 3' to 5' phosphodiester bonds.

[0416] Conjugate group In this embodiment, the oligonucleotide is modified by covalent attachment of one or more conjugation groups. Typically, the conjugation groups modify one or more properties of the oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. The conjugation groups are conventionally used in the chemical field and are attached directly to the oligonucleotide or, optionally, via a linker or linker group. Conjugation groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinones, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In this embodiment, the conjugation group is polyethylene glycol (PEG), and the PEG is conjugated to the oligonucleotide or cyclic peptide.

[0417] Conjugated groups include lipid moieties, such as cholesterol moieties (Letsinge et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); bile acids (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); and aliphatic chains, such as dodecyl glycol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111). Kabanov et al., FEBSLett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids such as di-hexadecyl-racemic-glycerol or triethylammonium-1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. AcidsRes., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxocholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0418] The oligonucleotides provided herein may include linker groups or bifunctional linker moieties, such as those known in the art. Linker groups can be used to attach chemical functional groups, conjugation groups, reporter groups, and other groups to a selective site in an oligonucleotide (e.g., an antisense oligonucleotide). In embodiments, the bifunctional linker moieties comprise a hydrocarbon moiety having two functional groups. Any linker described herein may be used. In embodiments, the linker comprises a chain structure or oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups used in bifunctional linker moieties include, but are not limited to, electrophilic agents for reaction with nucleophilic groups and nucleophilic agents for reaction with electrophilic groups. In embodiments, the bifunctional linker moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturated bonds (e.g., double or triple bonds), etc. Some non-limiting examples of bifunctional linker moieties include 8-amino-3,6-dioxanoic acid (ADO), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C groups. 10 Alkyl, substituted or unsubstituted C2-C 10 alkenyl or substituted or unsubstituted C2-C 10 Alkynyl, wherein a non-limiting list of substituents includes hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0419] In the implementation, the oligonucleotide may be linked to a 10-arginine-serine dipeptide repeat sequence. Oligonucleotides linked to 10-arginine-serine dipeptide repeat sequences have been used in vitro to artificially recruit splicing enhancer factors to induce the inclusion of mutated BRCA1 and SMN2 exons, which would otherwise be skipped. See Cartegni and Kraner 2003, incorporated herein by reference.

[0420] In this embodiment, the goods are oligonucleotides. In this embodiment, the oligonucleotides are therapeutic oligonucleotides. In this embodiment, the oligonucleotides are antisense oligonucleotides. In this embodiment, the goods are capable of binding to human proteins encoding dystrophin. DMD Oligonucleotides that hybridize with the nucleic acid sequence of a gene. In the implementation scheme, the oligonucleotides can be hybridized with... DMD Hybridization with at least a portion of exon 50 of the gene. In the implementation scheme, with DMD Oligonucleotides that hybridize to at least a portion of exon 50 can also hybridize to at least a portion of the 5' flanking introns of the exon or to at least a portion of the 3' flanking introns of exon 50. In an embodiment, with DMDThe length of the oligonucleotide that hybridizes at least a portion of exon 50 of the gene is 5 to 50, 10 to 40, 15 to 35, or 20 to 30 nucleotides, for example, at least 15, 16, 17, 18, 19, or 20 nucleotides, and at most 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. In the embodiment, with DMD The length of the oligonucleotide that hybridizes at least a portion of exon 50 of the gene is 15 to 35 nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.

[0421] In the implementation scheme, the oligonucleotide can be combined with a compound containing... DMD The nucleic acid sequence of at least a portion of the 5' flanking intron of exon 50 of the gene hybridizes. In the implementation scheme, the oligonucleotide may be coupled with a sequence containing... DMD The nucleic acid sequence of at least a portion of the 3' flanking intron of exon 50 of the gene hybridizes. In the implementation scheme, the oligonucleotide can be hybridized with... DMD Hybridization with nucleic acid sequences within exon 50 of a gene. In the implementation plan, antisense oligonucleotides can hybridize with sequences spanning [exons / regions]. DMD Hybridization of nucleic acid sequences at intron-exon or exon-intron junctions in gene exon 50.

[0422] In the sequences shown in this article, the last nucleotide of the recipient intron sequence preceding exon 50 is denoted as nucleotide "-1", the first nucleotide of exon 50 is denoted as nucleotide "0", and the first nucleotide of the donor intron sequence following exon 50 is denoted as "-1".

[0423] In the implementation plan, DMD The nucleic acid sequence of exon 50 is shown in SEQ ID NO:1 below (from 5' to 3'): In the implementation scheme, the oligonucleotide comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides complementary to the consecutive nucleotides of SEQ ID NO:1 (e.g., the oligonucleotide is a 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, or 30-mer), wherein the first nucleotide of the oligonucleotide is complementary to the consecutive nucleotides of SEQ ID NO:1. DMD Exon 50 of the gene in SEQ ID NO: Position 1: -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +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, +50, +51, +52, +53 Nucleotide hybridization at positions +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, +99, +100, +101, +102, +103, +104, +105, +106, +107, +108, +109. In the implementation scheme, the oligonucleotide comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides complementary to the consecutive nucleotides of SEQ ID NO:1 (e.g., the oligonucleotide is a 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, or 30-mer), wherein the first nucleotide of the oligonucleotide is complementary to the consecutive nucleotides of SEQ ID NO:1. DMDThe positions of exon 50 in SEQ ID NO: 1 are -5, -4, -3, -2, -1, 0, +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, +50, +51, +52, +53, +54, +55. Nucleotide hybridization at positions +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, +99, +100, +101, +102, +103, +104, +105, +106, +107, +108, +109. In the implementation scheme, the oligonucleotide comprises 20, 21, 22, 23, 24, or 25 consecutive nucleotides (e.g., the oligonucleotide is a 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer complementary to the consecutive nucleotides of SEQ ID NO:1, wherein the first nucleotide of the oligonucleotide is... DMD Nucleotide hybridization at positions -5, -4, -3, -2, and -1 in exon 50 of the gene. In an embodiment, the oligonucleotide comprises 20, 21, 22, 23, 24, or 25 consecutive nucleotides (e.g., the oligonucleotide is a 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer complementary to the consecutive nucleotides of SEQ ID NO:1, wherein the first nucleotide of the oligonucleotide is... DMD Nucleotide hybridization occurs at positions 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, or +25 in exon 50 of the gene. In an embodiment, the oligonucleotide comprises 20, 21, 22, 23, 24, or 25 consecutive nucleotides (e.g., the oligonucleotide is a 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer complementary to the consecutive nucleotides of SEQ ID NO:1, wherein the first nucleotide of the oligonucleotide is... DMD Nucleotide hybridization at position +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, or +90 of exon 50 of the gene. As used herein, “first nucleotide” refers to the first 5' nucleotide of the oligonucleotide cargo.

[0424] DMD The nucleic acid sequence of gene exon 50 (uppercase letters in bold and underlined) is shown in SEQ ID NO:2 below. This exon has a 50 nt upstream intron on the 5' flanking side (without underline) and a 50 nt downstream intron on the 3' flanking side (without underline).

[0425] Exon 50 ( Lowercase italics =Intron; uppercase / bold and underlined =Exons). Note: The first nucleotide of an exon sequence is numbered "0".

[0426] 5' The upstream (5') intron sequence (residues -50 to -1) and the downstream (3') intron sequence (residues -1 to -50) are shown in lowercase italics; the exon sequence (residues +0 to +108) is shown in uppercase bold and underlined. In the implementation scheme, human Duchenne muscular dystrophy (Duchenne muscular dystrophy) DMD The nucleic acid sequence of exon 50 of the dystrophin gene contains 109 nucleotides, shown in bold and underlined capital letters.

[0427] In the implementation scheme, the oligonucleotide contains and DMD The oligonucleotide comprises one or more nucleotides complementary to the 5' intron sequence flanking exon 50. In one embodiment, the oligonucleotide comprises one or more nucleotides complementary to the 5' intron sequence flanking exon 50 and one or more nucleotides complementary to the exon sequence of exon 50. In another embodiment, the oligonucleotide comprises one or more nucleotides complementary to the 3' intron sequence following exon 50. In yet another embodiment, the oligonucleotide comprises one or more nucleotides complementary to the 3' intron sequence flanking exon 50 and one or more nucleotides complementary to the exon sequence of exon 50.

[0428] In the implementation plan, oligonucleotides and cross DMD Hybridization of the nucleic acid sequence at the intron-exon or exon-intron junction of gene exon 50. In an embodiment, the oligonucleotide is complementary to the target nucleic acid sequence, which includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more consecutive nucleotides (i.e., starting from position -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2 or -1).

[0429] In the implementation scheme, the oligonucleotide is complementary to the target nucleic acid sequence, which includes at least the first nucleotide at the 5' end (i.e., position 0) of exon 50. In the implementation scheme, the oligonucleotide is complementary to the target nucleic acid sequence, which includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35 or more consecutive nucleotides of exon 50, including the first nucleotide at the 5' end (i.e., starting from position 0).

[0430] In the implementation plan, with DMD Oligonucleotides hybridized to exon 50 of a gene are shown in the nucleic acid sequences in Tables 11A-11D, 12A-12D or 13, their reverse complementary sequences, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity with them.

[0431] In the implementation plan, oligonucleotides and cross DMDHybridization with a portion of the nucleic acid sequence of gene exon 50. In the embodiment, the exon sequence of SEQ ID NO:2 is shown in bold and underlined (residues 0 to +108). In the embodiment, the oligonucleotide has a nucleic acid sequence as shown in Tables 11A-11D, 12A-12D or 13, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it.

[0432] In the implementation, the oligonucleotide comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides complementary to the consecutive nucleotides of SEQ ID NO:1 (e.g., the oligonucleotide is a 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, or 30-mer), wherein the first nucleotide of the oligonucleotide is associated with exon 50 as shown in Tables 11A-11D, 12A-12D, or 13 in SEQ ID NO:1. Hybridization with nucleotides at positions -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +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, their reverse complementary sequences, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity. In the implementation scheme, the first nucleotide of the oligonucleotide is associated with the following as shown in Tables 11A-11D, 12A-12D, or 13. DMDThe exon 50 of the gene in SEQ ID NO: 1 is +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +96, +98, +99. Hybridization occurs at positions +100, +101, +102, +103, +104, +105, +106, +107, +108, and +109, with their reverse complementary sequences or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity. Exon 50 has the following sequence: In the implementation plan, oligonucleotides and DMD Hybridization with the sequence of gene exon 50, which is selected from any of the nucleic acid sequences shown in Tables 11A-11D, 12A-12D, or 13. In the implementation scheme, with DMD The oligonucleotides for exon 50 hybridization are selected from the nucleic acid sequences shown in Tables 11A-11D, 12A-12D, or 13, their reverse complementary sequences, or any sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with them. In the embodiment, with DMD The oligonucleotide hybridizing with exon 50 comprises one or more modified nucleic acids, one or more modified nucleotide links, or combinations thereof. In an embodiment, the oligonucleotide hybridizing with exon 50 comprises one or more morpholine rings, one or more diaminophosphate links, or combinations thereof. In an embodiment, with... DMD The oligonucleotide obtained from exon 50 hybridization of the gene is an antisense diaminophosphate morpholino oligonucleotide. PMO The sequence is selected from any nucleic acid sequence in Tables 11A-11D, 12A-12D or 13, its reverse complementary sequence or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity with it.

[0433] Table 11A: 21-mer oligonucleotides hybridized with exon 50.

[0434] Table 11B: 22-mer oligonucleotides hybridized with exon 50.

[0435] Table 11C: 23-mer oligonucleotides that hybridize with exon 50.

[0436] Table 11D: 24-mer oligonucleotides hybridized with exon 50.

[0437] Table 12A: 21-mer oligonucleotides hybridized with exon 50.

[0438] Table 12B. 22-mer oligonucleotides hybridized with exon 50.

[0439] Table 12C. Trimeric oligonucleotides hybridized with exon 50.

[0440] Table 12D. 24-mer oligonucleotides hybridized with exon 50.

[0441] Table 13. 20-mer oligonucleotides or 30-mer oligonucleotides that hybridize with exon 50.

[0442] In the implementation plan, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-AGTGGTCAGTCCAGGAGCTAGGTC-3' (24-mer-76) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMDThe oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-AGTGGTCAGTCCAGGAGCTAGGTC-3' (24-mer-76). In the embodiment, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-GTGGTCAGTCCAGGAGCTAGG-3' (21-mer-78) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-GTGGTCAGTCCAGGAGCTAGG-3' (21-mer-78). In the embodiment, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-GGTCAGTCCAGGAGCTAGGTCA-3' (22-mer-75) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-GGTCAGTCCAGGAGCTAGGTCA-3' (22-mer-75). In the embodiment, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-TAGTGGTCAGTCCAGGAGCTAGGT-3' (24-mer-77) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMDThe oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-TAGTGGTCAGTCCAGGAGCTAGGT-3' (24-mer-77). In the embodiment, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-GCTCCAATAGTGGTCAGTCCAG-3' (22-mer-86) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-GCTCCAATAGTGGTCAGTCCAG-3' (22-mer-86). In the embodiment, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-ACCGCCTTCCACTCAGAGCTCAGA-3' (24-mer-14) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-ACCGCCTTCCACTCAGAGCTCAGA-3' (24-mer-14). In the implementation scheme, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-TTACCGCCTTCCACTCAGAGCTCA-3' (24-mer-16) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMDThe oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-TTACCGCCTTCCACTCAGAGCTCA-3' (24-mer-16). In the implementation scheme, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-GGAGCTAGGTCAGGCTGCTTTG-3' (22-mer-65) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-GGAGCTAGGTCAGGCTGCTTTG-3' (22-mer-65). In the implementation scheme, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-GTGGTCAGTCCAGGAGCTAGGTC-3' (23-mer-76) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) with the sequence 5'-GTGGTCAGTCCAGGAGCTAGGTC-3' (23-mer-76). In the implementation scheme, with DMD The oligonucleotide hybridized to exon 50 of the gene is an antisense diaminophosphate morpholino oligonucleotide (PMO) having a 5'-AGGAGCTAGGTCAGGCTGCTTT-3' (22-mer-66) sequence, its reverse complementary sequence, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with it. In the implementation scheme, with... DMDThe oligonucleotides hybridized to exon 50 of the gene were antisense diaminophosphate morpholino oligonucleotides (PMOs) with the sequence 5'-AGGAGCTAGGTCAGGCTGCTTT-3' (22-mer-66).

[0443] In the implementation plan, with DMD The oligonucleotide of the exon 50 hybridization of the gene contains a sequence containing 5'-XX ACC GCCXXC CAC XCA GAG CXC AGA-3' (where X = U or T), its reverse complementary sequence, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it. In the embodiment, with DMD The oligonucleotide of the exon 50 hybridization of the gene contains a sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleic acids, wherein the base sequence comprises: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 15 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXCAGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 16 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXCCAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 17 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 18 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMDThe oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 19 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of the exon 50 hybridization of the gene contains a sequence comprising 20 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXCAGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 21 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXCCAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 22 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 23 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of exon 50 hybridization of the gene contains a sequence comprising 24 consecutive nucleic acids with the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T. In an embodiment, with DMD The oligonucleotide of the exon 50 hybridization of the gene contains a sequence comprising 25 consecutive nucleic acids of the following base sequence: 5'-XX ACC GCC XXC CAC XCA GAG CXCAGA-3', where X = U or T. In an embodiment, with DMDThe oligonucleotide of exon 50 hybridization of a gene comprises a sequence of 26 consecutive nucleic acids containing the following base sequence: 5'-XX ACC GCC XXCCAC XCA GAG CXC AGA-3', where X = U or T. In embodiments, the base sequence may vary at up to one, two, three, four, or five positions. In embodiments, the oligonucleotide comprises one or more modified oligonucleotides. In embodiments, the oligonucleotide comprises at least one diaminophosphate morpholine oligonucleotide (PMO). In embodiments, each nucleotide in the oligonucleotide is a diaminophosphate morpholine oligonucleotide (PMO).

[0444] In the implementation scheme, any oligonucleotide described herein, including the oligonucleotides in Tables 11A-11D, 12A-12D, their reverse complementary sequences, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with them, comprises at least one modified nucleotide or nucleic acid selected from: phosphate thioester (PS) nucleotides, diaminophosphate morpholino oligonucleotides (PMO), locked nucleic acids (LNA), peptide nucleic acids (PNA), nucleotides comprising a 2'-O-methyl (2'-OMe) modified backbone, 2'-O-methoxy-ethyl (2'-MOE) nucleotides, 2',4' restricted ethyl (cEt) nucleotides, or 2'-deoxy-2'-fluoro-β-D-arabinose (2'F-ANA). In the implementation scheme, hybridization of the oligonucleotide with the target sequence promotes or induces splicing of exon 50. In the implementation scheme, the oligonucleotide comprises at least one diaminophosphate morpholine oligonucleotide (PMO). In the implementation scheme, 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, or more nucleotides are modified. In the implementation scheme, each nucleotide in the oligonucleotide is a diaminophosphate morpholine oligonucleotide (PMO).

[0445] Cytoplasmic delivery efficiency Modification of endosome escape mediators (EEVs) or their components (e.g., cyclic cell-penetrating peptides (cCPPs), exocyclic peptides (EPs), or linkers) can improve cytoplasmic delivery efficiency. The improved cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of modified EEVs with that of control sequences, where the control does not contain specific modifications (e.g., modifications to cCPPs, EPs, or linkers) but is otherwise identical.

[0446] In this embodiment, the EEV-cargo conjugate exhibits improved cytoplasmic uptake efficiency compared to the cargo alone. Cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of the EEV-cargo conjugate with that of the cargo alone. In this embodiment, the cargo is an oligonucleotide. In this embodiment, the oligonucleotide is a therapeutic oligonucleotide. In this embodiment, the oligonucleotide is an antisense oligonucleotide. In this embodiment, the oligonucleotide is a PMO.

[0447] As used herein, cytoplasmic delivery efficiency refers to the ability of an EEV, EEV-cargo conjugate, or cargo to cross the cell membrane and enter the cytoplasm. The cytoplasmic delivery efficiency of an EEV, EEV-cargo conjugate, or cargo is not necessarily dependent on the receptor or cell type. Cytoplasmic delivery efficiency can refer to absolute cytoplasmic delivery efficiency or relative cytoplasmic delivery efficiency.

[0448] Absolute cytoplasmic delivery efficiency refers to the ratio of the cytoplasmic concentration of EEV, EEV-cargo conjugate, or cargo to the concentration of EEV, EEV-cargo conjugate, or cargo in the growth medium. Relative cytoplasmic delivery efficiency refers to the concentration of EEV, EEV-cargo conjugate, or cargo in the cytoplasm compared to the concentration of control EEV, EEV-cargo conjugate, or cargo in the cytoplasm. Quantification can be achieved by fluorescently labeling EEV, EEV-cargo conjugate, or cargo (e.g., with FITC dyes) and measuring the fluorescence intensity using techniques well known in the art.

[0449] Relative cytoplasmic delivery efficiency is determined by comparing (i) the amount of EEV, EEV-cargo conjugate, or cargo internalized in a cell type (e.g., HeLa cells) with (ii) the amount of control EEV, EEV-cargo conjugate, or cargo internalized in the same cell type. To measure relative cytoplasmic delivery efficiency, cell types can be incubated in the presence of EEV, EEV-cargo conjugate, or cargo for a specific time (e.g., 30 minutes, 1 hour, 2 hours, etc.), after which the amount of cCPP internalized in the cells is quantified using methods known in the art (e.g., fluorescence microscopy). Alternatively, control EEV, EEV-cargo conjugate, or cargo at the same concentration can be incubated for the same period in the presence of the stated cell type, and the amount of control EEV, EEV-cargo conjugate, or cargo internalized in the cells can be quantified.

[0450] The IC50 of modified EEVs, EEV-cargo conjugates, or cargoes against intracellular targets can be measured. 50 And the modified EEV, EEV-cargo conjugate or cargo IC 50 The relative cytoplasmic delivery efficiency was determined by comparing the sequence with a control sequence.

[0451] Resplicing target proteins As used herein, “target protein” refers to the amino acid sequence produced by the transcription and translation of a target gene. As used herein, “re-spliced ​​target protein” refers to a protein encoded by an oligonucleotide bound to target precursor mRNA transcribed from a target gene. “Wild-type target protein” refers to a naturally occurring, correctly translated protein isoform produced by correctly splicing target precursor mRNA encoded by a wild-type target gene. Compared to a wild-type target protein, a re-spliced ​​target protein may contain one or more amino acid substitutions, deletions, and / or insertions. In embodiments, the re-spliced ​​target protein retains some of the activity of the wild-type target protein. In embodiments, the re-spliced ​​target protein is homologous to the wild-type target protein. In embodiments, the re-spliced ​​target protein has an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and up to 100% identical to the wild-type target protein. In one embodiment, the respliced ​​target protein is substantially identical to the wild-type target protein. In another embodiment, the amino acid sequence of the respliced ​​target protein is at least 50% identical to that of the wild-type target protein. In yet another embodiment, the amino acid sequence of the respliced ​​target protein is at least 75% identical to that of the wild-type target protein. In yet another embodiment, the amino acid sequence of the respliced ​​target protein is at least 90% identical to that of the wild-type target protein. In yet another embodiment, the respliced ​​target protein is a shortened version of the wild-type target protein.

[0452] In the implementation scheme, the respliced ​​target protein can rescue one or more phenotypes lost due to diseases associated with the transcription and translation of the target gene, or improve one or more symptoms of diseases associated with the transcription and translation of the target gene. In the implementation scheme, the respliced ​​target protein can rescue one or more phenotypes, or improve one or more symptoms of diseases associated with target protein expression. In the implementation scheme, the respliced ​​target protein is an active fragment of the wild-type target protein. In the implementation scheme, the respliced ​​target protein functions in a manner substantially similar to the wild-type target protein. In the implementation scheme, the respliced ​​target protein enables the cell to function substantially similarly to similar cells expressing the wild-type target protein. In the implementation scheme, the respliced ​​target protein does not cure diseases associated with the target gene or target protein, but improves one or more symptoms of the disease. In the implementation scheme, the resplicing of the target protein can improve the function of the target protein by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and up to 100%.

[0453] In the implementation, the respliced ​​target protein may have an amino acid sequence that is one or more amino acids smaller than that of the wild-type target protein, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 or more amino acids.

[0454] In the embodiments, the respliced ​​target protein may have one or more improved properties relative to the target protein. In the embodiments, the respliced ​​target protein may have one or more improved properties relative to the wild-type target protein. In the embodiments, enzyme activity or stability may be enhanced by promoting different splicing of the target precursor mRNA. In the embodiments, the respliced ​​target protein may have a sequence that is identical or substantially similar to a wild-type target protein isoform, which has improved properties compared to another wild-type target protein isoform.

[0455] In the embodiments, one or more properties of the target protein are either absent (eliminated) or reduced in the resplicing target protein. In the embodiments, one or more properties of the wild-type target protein are either absent (eliminated) or reduced in the resplicing target protein. Non-limiting examples of properties that can be reduced or eliminated include immunogenicity, angiogenesis, thrombotic activity, aggregation, and ligand-binding activity.

[0456] In embodiments, the respliced ​​target protein contains one or more amino acid substitutions compared to the wild-type target protein. These substitutions can be conserved or non-conserved. Examples of conserved amino acid substitutions include replacing one amino acid with another within one of the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small-molecule amino acids (glycine, alanine, serine, threonine, and methionine). In embodiments, structurally similar amino acids are substituted to reverse the charge of the residues (e.g., glutamic acid is replaced with glutamine or vice versa, asparagine is replaced with aspartic acid or vice versa). In embodiments, tyrosine is replaced with phenylalanine, or vice versa. Other non-limiting examples of amino acid substitutions are described, for example, by H. Neurath and RL Hill, 1979. In, The Proteins Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0457] In the implementation scheme, compared to the wild-type target protein, the respliced ​​target protein may contain substitutions, deletions, and / or insertions at one or more (e.g., several) sites. In the implementation scheme, the number of amino acid substitutions, deletions, and / or insertions in the amino acid sequence of the respliced ​​target protein does not exceed 200, 150, 100, 50, 40, 30, 20, or 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0458] Treatment In one embodiment, the EEV-cargo conjugate is administered to a patient diagnosed with Duchenne muscular dystrophy (DMD). In another embodiment, the cargo is an oligonucleotide. In yet another embodiment, the oligonucleotide is a therapeutic oligonucleotide. In yet another embodiment, the oligonucleotide is an antisense oligonucleotide. In yet another embodiment, the EEV-cargo conjugate is administered to the patient at a dose from 0.1 mg / kg to 1000 mg / kg (inclusive of all values ​​and ranges therein and all values ​​and ranges in between).

[0459] A method for treating Duchenne muscular dystrophy (DMD) in subjects of need is provided, comprising administering the EEV-cargo conjugate disclosed herein. In the embodiment, the target gene is... DMD. In the implementation plan, the target sequence includes DMD At least a portion of gene exon 50 DMD At least a portion of the 3' introns flanking the 50th exon of a gene. DMD At least a portion of the 5' introns flanking the 50 exon of a gene, or a combination thereof.

[0460] In the implementation plan, treatment refers to the partial or complete relief, improvement, reduction, suppression, delay of onset, or reduction of the severity and / or incidence of one or more symptoms in the subject.

[0461] In one embodiment, a method for altering target gene expression in a subject in need is provided, comprising administering the EEV-cargo conjugate disclosed herein. In another embodiment, the treatment results in decreased expression of the target protein. In yet another embodiment, the treatment results in resplicing of the target protein's expression. In yet another embodiment, the treatment results in preferential expression of the wild-type target protein isoform.

[0462] In the implementation scheme, compared with the average level of target proteins in the subject before treatment or the average level of target proteins in one or more control individuals with similar diseases who have not received treatment, the treatment results in a reduction of target protein expression in the subject by more than 5%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. In the implementation scheme, compared with the average level of target proteins in the subject before treatment or the average level of target proteins in one or more control individuals with similar diseases who have not received treatment, the treatment results in an increase of respliced ​​target protein expression in the subject by more than 5%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. In the implementation plan, the treatment resulted in an increase in the expression of wild-type target protein isoforms in the subject by more than 5%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%, compared to the average level of target protein in the subject before treatment or the average level of target protein in one or more control individuals with similar diseases but who did not receive treatment.

[0463] As used herein, terms such as “improvement,” “increase,” “decrease,” and “reduction” all refer to values ​​relative to a control. In the implementation plan, a suitable control is a baseline measurement, such as a measurement taken on the same individual prior to the initiation of the treatment described herein, or a measurement taken on a control individual (or multiple control individuals) in the absence of the treatment described herein. A “control individual” is an individual with the same disease whose age and / or sex are substantially the same as the individual receiving treatment (to ensure comparability of disease stages between the treated individual and the control individual).

[0464] The individual receiving treatment (also referred to as the “patient” or “subject”) is an individual who has or is at risk of developing the disease (fetus, infant, child, adolescent, or adult). This individual may have a disease mediated by abnormal gene expression or abnormal gene splicing. In one implementation, the wild-type target protein expression level or activity level in an individual with the disease may be 1% to 99% of the normal protein expression level or activity level in an individual without the disease. In another implementation, this range includes, but is not limited to, 80-99%, 65-80%, 50-65%, 30-50%, 25-30%, 20-25%, 15-20%, 10-15%, 5-10%, or 1-5% of the normal dystrophic protein expression level or activity level. In yet another implementation, the individual's target protein expression level or activity level is 1% to 500% higher than the normal wild-type target protein expression level or activity level. In the implementation scheme, the range includes, but is not limited to, the target protein expression level or activity level being 1-10%, 10-50%, 50-100%, 100-200%, 200-300%, 300-400%, 400-500%, or 500-1000% higher.

[0465] In the implementation plan, the individual is someone who has recently been diagnosed with the disease. Generally speaking, early treatment (starting treatment as soon as possible after diagnosis) is crucial to minimizing the impact of the disease and maximizing the effectiveness of treatment.

[0466] In this implementation, the efficacy of the EEV-cargo conjugate is evaluated in DMD animal models. Animal models are a valuable resource for studying disease pathogenesis and provide a means of testing dystrophin-related activities. In this implementation, mdx mice and Golden Retriever muscular dystrophy (GRMD) dogs (both dystrophin-negative, see, for example, Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003) were used to evaluate the EEV-cargo conjugate. In this implementation, C57BL / 10ScSn-Dmdmdx / J (Bl10 / mdx) or D2.B10-Dmdmdx / J (D2 / mdx) mouse models were used to evaluate the EEV-cargo conjugate. In this implementation, human carriers were used... DMD Genes and lack of mice DmdTransgenic mice (hDMD / Dmd-null mice) were used to evaluate EEV-cargo conjugates. These mice were generated by crossing male hDMD mice (available from Jackson Laboratory, Bar Harbor, ME) with female md-null mice. The following references describe these models, the full contents of which are incorporated herein by reference: J Neuromuscul Dis. 2018; 5(4): 407–417.; Proc Natl Acad Sci US A. 1984;81(4):1189–92.; Am J Pathol. 2010;176(5):2414–24.; J Clin Invest. 2009;119(12):3703–12; International Publication No. WO2019014772. These and other animal models can be used to measure the functional activity of various dystrophin proteins.

[0467] In the embodiments, an in vitro model is used to evaluate the efficacy of the compositions disclosed herein. In the embodiments, the in vitro model is an immortalized muscle cell model. This model is described in its entirety in the following article, which is incorporated herein by reference: Nguyen et al. J Pers Med. 2017 Dec; 7(4):13.

[0468] Preparation method EEV-cargo conjugates can be prepared in a variety of ways known to those skilled in the art of organic synthesis or in variations thereof. EEV-cargo conjugates can be prepared from readily available starting materials. Reaction conditions can vary depending on the specific reactants or solvents used, but these conditions can be determined by those skilled in the art.

[0469] Variations in EEV-cargo conjugates include the addition, reduction, or shifting of various components of the EEV-cargo conjugate. Similarly, the chirality of the molecule can be altered when one or more chiral centers are present. Furthermore, synthesis may involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups, can be determined by those skilled in the art. The chemical properties of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Edition, Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0470] Starting materials and reagents for the preparation of EEV-cargo conjugates and their compositions are 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, IN). The drug delivery system may be prepared using 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, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the drug delivery system described, may be available from commercial sources.

[0471] The reaction to produce the EEV-cargo conjugate can be carried out in a solvent, which can be selected by those skilled in the art of organic synthesis. Under the conditions of the reaction (i.e., temperature and pressure), the solvent should be substantially unreactive with the starting material (reactant), intermediate, or product. The reaction can be carried out in a single solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be achieved by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) Monitoring by infrared spectroscopy, spectrophotometry (e.g., UV-visible light), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin-layer chromatography.

[0472] EEVs and their components (including EP and cCPP), as well as EEV-cargo conjugates, can be prepared via solid-phase peptide synthesis, in which the α-N-terminus of amino acids is protected by an acid or base protecting group. Such protecting groups should be stable to the peptide bonding conditions and readily removable without disrupting the grown peptide chain or racemizing any chiral centers contained therein. Suitable protecting groups include 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, tert-pentyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-tert-butyloxycarbonyl, etc. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group can be used. For side-chain amino groups such as lysine and arginine, other side-chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, tert-butyl (t-Bu), cyclohexyl, cyclopentyl, and acetyl (Ac); for serine, tert-butyl, benzyl, and tetrahydropyranyl; for histidine, triphenylmethyl, benzyl, Cbz, p-toluenesulfonyl, and 2,4-dinitrophenyl; for tryptophan, formyl; for aspartic acid and glutamic acid, benzyl and tert-butyl, and for cysteine, triphenylmethyl (triphenylmethyl). In solid-phase peptide synthesis methods, α-C-terminal amino acids are attached to a suitable solid support or resin. Suitable solid supports for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and insoluble in the medium used. Solid supports for the synthesis of α-C-terminal carboxypeptides are 4-hydroxymethylphenoxymethyl copolymer (styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin, available from Applied Biosystems (Foster City, Calif.). The α-C-terminal amino acid is coupled to the resin via N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or O-benzotriazol-1-yl-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), at a temperature between 10°C and 50°C in a solvent such as dichloromethane or DMF for 1 to 24 hours.When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetaminoethyl resin, the Fmoc group is cleaved with a secondary amine (e.g., piperidine) before coupling with the α-C-terminal amino acid as described above. One method for coupling with deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetaminoethyl resin involves O-benzotriazole-1-yl-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. Continuous coupling of protected amino acids can be performed in an automated peptide synthesizer. In one example, the α-N-terminus of the amino acids in the grown peptide chain is protected with Fmoc. Removal of the Fmoc protecting group from the α-N-terminal side of the grown peptide is accomplished by treatment with a secondary amine (e.g., piperidine). Each protected amino acid is then introduced in a 3-molar excess and coupled in DMF. The coupling agent can be O-benzotriazole-1-yl-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). At the end of solid-phase synthesis, the peptide is removed from the resin and deprotected, either continuously or in a single operation. Removal and deprotection of the peptide can be accomplished in a single operation by treating the resin-bound peptide with a cleavage reagent comprising anisole, water, ethylenedithiol, and trifluoroacetic acid. In the case where the α-C-terminus of the peptide is an alkylamide, the resin is cleaved by ammonolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification (e.g., with methanol), followed by ammonolysis, or by direct transamidation. The protected peptide can then be purified or used directly in the next step. Removal of side-chain protecting groups can be accomplished using the cleavage mixture described above. Completely deprotected peptides can be purified by employing any or all of the following types of chromatographic steps: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underived polystyrene-divinylbenzene (e.g., Amberlite XAD); silica 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 reversed-phase HPLC on octyl-silica or octadecylsilyl-silica bonded phase columns.

[0473] The aforementioned polymers, such as PEG groups, can be attached to oligonucleotides under any suitable conditions for reacting proteins with activated polymer molecules. Any method known in the art can be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation, or other chemically selective conjugation / linking methods through reactive groups (e.g., aldehydes, amino groups, esters, thiols, α-haloacetyl groups, maleimide groups, or hydrazines) on the PEG moiety and reactive groups (e.g., aldehydes, amino groups, esters, thiols, α-haloacetyl groups, maleimide groups, or hydrazines) on the oligonucleotide. Activating groups that can be used to attach water-soluble polymers to one or more proteins include, but are not limited to, sulfones, maleimides, mercapto groups, thiols, trifluoromethanesulfonates, tresylates, azidirines, ethylene oxides, 5-pyridyl groups, and α-haloacyl groups (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). If attachment to oligonucleotides is achieved via reductive alkylation, the selected polymer should have a single reactive aldehyde to control the degree of polymerization. See, for example, Kinstler et al., Adv. Drug Delivery Rev. 54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476 (2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16:157-182 (1995).

[0474] To directly covalently link oligonucleotides to cyclic peptides, suitable amino acid residues of the CPP can react with an organic derivatizing agent capable of reacting with selected side chains, N-termini, or C-termini of the amino acid. Reactive groups on the peptide or conjugate moiety include, for example, aldehydes, amino groups, esters, thiols, α-haloacetyl groups, maleimide groups, or hydrazide groups. Derivatizing agents include, for example, maleimide-benzoylsulfosuccinimide ester (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, or other agents known in the art.

[0475] Methods for synthesizing oligonucleotides are known in the art. This disclosure is not limited to methods for synthesizing oligonucleotides. In embodiments, reactive phosphorus groups that can be used to form nucleoside interlinking include, for example, phosphodiester and thiophosphate nucleoside interlinking. The methods for preparing and / or purifying precursors or oligonucleotides are not limited to the compositions or methods provided herein. Methods for the synthesis and purification of DNA, RNA, and oligonucleotides are well known to those skilled in the art.

[0476] Oligomerization of modified and unmodified nucleosides can be performed according to standard procedures in the literature on DNA (Protocols for Oligonucleotides and Analogs, Agrawal ed. (1993), Humana Press) and / or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically Synthesized RNA in RNA: Protein Interactions, Smith ed. (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

[0477] The oligonucleotides provided herein can be conveniently and routinely prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, CA). Any other methods known in the art for such synthesis may also be used, either alternatively or as an alternative. The preparation of oligonucleotides (such as phosphate thioides and alkylated derivatives) using similar techniques is well known. This invention is not limited to methods of oligonucleotide synthesis.

[0478] Methods for oligonucleotide purification and analysis are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray ionization mass spectrometry. Such synthetic and analytical methods can be performed in multi-well plates. The methods of the present invention are not limited to oligomer purification methods.

[0479] Application method The application of the disclosed EEV-cargo conjugates and compositions containing them can be carried out by any suitable method or technique currently known or anticipated by those skilled in the art. For example, the EEV-cargo conjugates can be formulated into physiologically or pharmaceutically acceptable forms and administered via any suitable route known in the art, including, for example, oral and parenteral administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal, and intrathecal administration, such as by injection. The application of the disclosed EEV-cargo conjugates or compositions can be a single administration or administered at consecutive or varying intervals, as readily determined by those skilled in the art.

[0480] The EEV-cargo conjugates and compositions comprising them disclosed herein can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide a uniform dose over an extended period of time. The EEV-cargo conjugates can also be administered in their salt derivative form or crystalline form.

[0481] EEV-cargo conjugates can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in numerous sources well known to and readily available to those skilled in the art. For example, EW Martin's... Remington's Pharmaceutical Science (1995) describes formulations that can be used in conjunction with the disclosed methods. Typically, EEV-cargo conjugates are formulated such that an effective amount of the EEV-cargo conjugate is combined with a suitable carrier to facilitate the effective administration of the EEV-cargo conjugate. The compositions used may also be in various forms. These forms include, for example, solid, semi-solid, and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusionable solutions, and sprays. The form depends on the intended administration modality and therapeutic application. The composition also contains a conventionally pharmaceutically acceptable carrier and diluent known to those skilled in the art. Examples of carriers or diluents include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide administration of such a dose for the desired therapeutic treatment, the composition may contain one or more EEV-cargo conjugates in total amounts ranging from 0.1% to 100% by weight, based on the total weight of the composition containing the carrier or diluent.

[0482] Suitable formulations for administration include, for example, sterile aqueous solutions for injection, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickeners. Formulations may be available in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored under lyophilized (freeze-dried) conditions requiring only a sterile liquid carrier (e.g., water for injection) prior to use. Temporary injectable solutions and suspensions may be prepared from sterile powders, granules, tablets, etc. It should be understood that, in addition to the components specifically mentioned above, the composition may also contain other conventional agents in the art for the types of formulations discussed.

[0483] EEV-cargo conjugates and compositions comprising them can be delivered to cells through direct contact with cells or via a carrier method. Carrier methods are known in the art and include, for example, encapsulating the composition in a liposome portion. Another delivery method includes attaching the EEV-cargo conjugate to a protein or nucleic acid targeted for delivery to target cells. U.S. Patent No. 6,960,648 and U.S. Application Publications Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow said composition to translocate across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transporting biological portions across cell membranes for intracellular delivery. EEV-cargo conjugates may also be incorporated into polymers, examples of which include poly(DL-lactide-co-glycolic acid) 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.

[0484] EEV-cargo conjugates and compositions thereof, including their pharmaceutically acceptable salts or prodrugs, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth.

[0485] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for ad hoc preparation of sterile injectable or infusionable solutions or dispersions optionally encapsulated in liposomes. The final dosage form should be sterile, fluid, and stable under manufacturing and storage conditions. Liquid carriers or mediators may be solvents or liquid dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by forming liposomes, or, in the case of dispersions, by maintaining the desired particle size, or by using surfactants. Optionally, microbial action may be prevented by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Isotonic agents, such as sugars, buffers, or sodium chloride, are included in many cases. Extended absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0486] A sterile injectable solution is prepared by incorporating the desired amount of the EEV-cargo conjugate with various other components listed above into a suitable solvent, followed by filtration and sterilization as needed. In the case of sterile powders used to prepare the sterile injectable solution, preparation methods include vacuum drying and freeze-drying techniques, which produce powders containing the active ingredient plus any additional desired components present in the previously sterile filtered solution.

[0487] The effective dose of EEV-cargo conjugates and their pharmaceutical compositions can be determined by comparing their in vitro and in vivo activities in animal models. Methods for extrapolating effective doses from mice and other animals to humans are known in the art.

[0488] The dosage range of the composition administered is wide enough to produce the desired effect on symptoms or condition. The dosage should not be high enough to cause adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Generally, the dosage will vary depending on the patient's age, condition, sex, and severity of disease, and can be determined by someone skilled in the art. In the event of any contraindications, the dosage may be adjusted by an individual physician. The dosage may be varied and may be administered once or multiple times daily for one or more days.

[0489] Pharmaceutical compositions comprising an EEV-cargo conjugate and a pharmaceutically acceptable carrier are also disclosed. Pharmaceutical compositions suitable for oral, topical, or parenteral administration are provided, comprising a specific amount of the EEV-cargo conjugate. The dose administered to a patient (particularly a human) should be sufficient to achieve a therapeutic response in the patient within a reasonable timeframe without lethal toxicity and without causing side effects or morbidity not exceeding acceptable levels. Those skilled in the art will recognize that the dose will depend on a variety of factors, including the subject's condition (health), the subject's weight, the type of concurrent treatment (if any), the frequency of treatment, the treatment ratio, and the severity and stage of the pathological condition.

[0490] Kits containing EEV-cargo conjugates in one or more containers are also disclosed. The disclosed kits may optionally include pharmaceutically acceptable carriers and / or diluents. In embodiments, the kit includes one or more other components, excipients, or adjuvants as described herein. In embodiments, the kit includes one or more anticancer agents, such as those described herein. In embodiments, the kit includes instructions or packaging materials describing how to administer the EEV-cargo conjugate or a combination thereof. The container of the kit may be any suitable material, such as glass, plastic, metal, etc., and may be any suitable size, shape, or configuration. In embodiments, the EEV-cargo conjugate is provided in the kit as a solid (such as tablets, pills, or powder). In embodiments, the EEV-cargo conjugate is provided in the kit as a liquid or solution. In embodiments, the kit includes ampoules or syringes containing the EEV-cargo conjugate in liquid or solution form.

[0491] Many embodiments of the invention have been described. However, it should be understood that various modifications may be made without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the following claims.

[0492] Some definitions As used in the specification and appended claims, the singular forms “a / an” and “the” include a plurality of indicators unless the context clearly indicates 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, etc.

[0493] As used herein, the term “circular cell-penetrating peptide” or “cCPP” refers to a peptide that facilitates the delivery of cargo to the cytoplasm of a cell.

[0494] As used herein, the term “endosome escape mediator” (EEV) refers to a cCPP conjugated to a linker and / or an exocyclic peptide (EP) via chemical bonding (i.e., covalent or non-covalent interactions).

[0495] As used herein, the term “EEV-cargo conjugate” refers to an endosome escape mediator (EEV) as defined herein, conjugated to an oligonucleotide via chemical bonding (i.e., covalent or non-covalent interactions). Oligonucleotides can be delivered into cells via EEVs.

[0496] As used herein, the term "exocyclic peptide" (EP) refers to two or more amino acid residues linked by peptide bonds that can be conjugated to a cyclic cell-penetrating peptide (cCPP). When conjugated to a cCPP, an EP can alter the tissue distribution and / or retention of a compound. Typically, an EP contains at least one positively charged amino acid residue, such as at least one lysine residue and / or at least one arginine residue. Non-limiting examples of EPs are described herein. An EP can be a peptide identified in the art as a "nuclear localization sequence" (NLS). Non-restrictive examples of nuclear localization sequences include: the nuclear localization sequence of the SV40 virus large T antigen (its smallest functional unit is the seven-amino acid sequence PKKKRKV), the binomial nucleoplasmic protein NLS (sequence NLSKRPAAIKKAGQAKKKK), the c-myc nuclear localization sequence (amino acid sequence PAAKRVKLD or RQRRNELKRSF), the sequence of the IBB domain of importin-α RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, the sequences of the myoma T protein VSRKRPRP and PPKKARED, the sequence of human p53 PQPKKKPL, and the mouse c-abl The sequence of NLS includes SALIKKKKKMAP of influenza virus IV, DRLRR and PKQKKRK of influenza virus NS1, RKLKKKIKKL of hepatitis virus delta antigen, REKKKFLKRR of mouse Mxl protein, KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase, and RKCLQAGMNLEARKTKK of steroid hormone receptor (human) glucocorticoid. Other examples of NLS are described in International Publication No. 2001 / 038547, which is incorporated herein by reference in its entirety.

[0497] As used herein, "linker" or "L" refers to the portion that covalently binds one or more parts (e.g., exocyclic peptides (EP) and oligonucleotides) to a cyclic cell-penetrating peptide (cCPP). Linkers may contain natural or non-natural amino acids or peptides. Linkers may be synthetic compounds containing two or more suitable functional groups. Linkers may contain a polyethylene glycol (PEG) component. Linkers may contain one or more amino acid (AA) components. Linkers may contain one or more hydrophobic groups (X).

[0498] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising multiple linked nucleotides or nucleosides. One or more nucleotides of an oligonucleotide may be modified. Oligonucleotides may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides may be composed of natural and / or modified nucleobases, sugars, and covalently linked nucleosides, and may further include non-nucleic acid conjugates. In embodiments, the oligonucleotide is a therapeutic oligonucleotide.

[0499] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by a carboxyl group of one amino acid to an α-amino group of another amino acid. Two or more amino acid residues may be linked by a carboxyl group of one amino acid to an α-amino group. Two or more amino acids in a polypeptide may be linked by peptide bonds. A polypeptide may include peptide backbone modifications in which two or more amino acids are covalently attached by bonds other than peptide bonds. A polypeptide may include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules capable of being incorporated into the polypeptide. The term polypeptide includes both naturally occurring and artificially occurring amino acids. The term polypeptide includes, for example, peptides containing 2 to 100 amino acid residues and proteins containing more than 100 or more than 1000 amino acid residues, including but not limited to therapeutic proteins such as antibodies, enzymes, receptors, soluble proteins, etc.

[0500] As used in this article, “polyethylene glycol” and “PEG” are used interchangeably. PEG refers to the group with the chemical formula -CH2CH2O-.

[0501] The term "PEG2" refers to 2-[2-[2-aminoethoxy]ethoxy]acetic acid.

[0502] The term "therapeutic peptide" refers to a peptide that has therapeutic, preventative, or other biological activities. Therapeutic peptides can be produced in any suitable manner. For example, therapeutic peptides can be isolated or purified from naturally occurring environments, chemically synthesized, recombinantly generated, or combinations thereof.

[0503] The term "small molecule" refers to an organic compound that has pharmacological activity and a molecular weight of less than 2,000 Daltons, or less than 1,000 Daltons or less than 500 Daltons. Small molecule therapeutic agents are usually manufactured through chemical synthesis.

[0504] As used herein, the term "adjacent" refers to two amino acids linked by a covalent bond. For example, in representative cyclic cell-penetrating peptides (cCPPs) such as... Examples of adjacent amino acid pairs are given in the cases of AA1 / AA2, AA2 / AA3, AA3 / AA4, and AA5 / AA1.

[0505] As used herein, a residue of a chemical substance refers to a derivative of the chemical substance present in a particular product. In order to form a product, at least one atom of the substance is replaced by a bond with another part, such that the product contains a derivative or residue of the chemical substance. For example, the cyclic cell-penetrating peptide (cCPP) described herein has an amino acid (e.g., arginine) incorporated therein by forming one or more peptide bonds. The amino acid incorporated into the cCPP may be referred to as a residue, or simply as an amino acid. Therefore, arginine or an arginine residue refers to... .

[0506] The term "its protonated form" refers to the protonated form of an amino acid. For example, the guanidinium group on the side chain of arginine can be protonated to form a guanidinium group. The structure of protonated arginine is... As used herein, the term "chirality" refers to a molecule having more than one stereoisomer that differs in the three-dimensional spatial arrangement of atoms, where one stereoisomer is a non-overlapping mirror image of another. Except for glycine, amino acids have a chiral carbon atom adjacent to a carboxyl group. The term "enantiomer" refers to a chiral stereoisomer. Chiral molecules can be amino acid residues with "D" and "L" enantiomers. Molecules lacking a chiral center, such as glycine, can be referred to as "chiral."

[0507] As used herein, the term "hydrophobic" refers to a portion that is insoluble in water or has very low solubility in water. Typically, neutral and / or nonpolar portions, or portions that are predominantly neutral and / or nonpolar, are hydrophobic.

[0508] As used herein, “aromatic” refers to an unsaturated cyclic molecule having 4n + 2 π electrons, where n is any integer. The term “non-aromatic” refers to any unsaturated cyclic molecule that does not fall within the definition of aromatics.

[0509] "alkyl," "alkyl chain," or "alkyl group" refers to a fully saturated straight-chain or branched hydrocarbon chain group having one to forty carbon atoms attached to the remainder of the molecule by single bonds. This includes alkyl groups containing any number of carbon atoms from 1 to 40. Alkyl groups containing up to 40 carbon atoms are C1-C2. 40 Alkyl groups, which contain up to 10 carbon atoms, are C1-C6. 10 Alkyl groups, specifically C1-C6 alkyl groups containing up to 6 carbon atoms and C1-C5 alkyl groups containing up to 5 carbon atoms, are further defined as alkyl groups. C1-C5 alkyl groups include C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl, and C1 alkyl (i.e., methyl). C1-C6 alkyl groups include all the portions described above for C1-C5 alkyl groups, but also include C6 alkyl groups. C1-C 10Alkyl groups include all the portions described above for C1-C5 and C1-C6 alkyl groups, but also include C7, C8, C9, and C6 alkyl groups. 10 Alkyl groups. Similarly, C1-C 12 Alkyl groups include all of the foregoing portions, but also include C4 groups. 11 and C 12 Alkyl group. C1-C 12 Non-limiting examples of alkyl groups include methyl, ethyl, just propyl, different propyl, Zhong propyl, just Butyl, different Butyl, Zhong Butyl, Uncle Butyl, just pentyl, Uncle pentyl, just Jiji, just Gengji, just Sinki, just Renji just guiji, just Undecyl and just Dodecyl group. Unless otherwise specified in the specification, the alkyl group may optionally be substituted.

[0510] "alkylene", "alkylene chain", or "alkylene group" refers to a fully saturated straight-chain or branched divalent hydrocarbon chain group having one to forty carbon atoms. (C2-C) 40 Non-limiting examples of alkylene groups include ethylene, propylene, and... just Butylene, vinylene, propene just Buteneyl, propynyl, just Butyrynyl groups, etc. Unless otherwise specified in the specification, the alkylene chain may optionally be substituted.

[0511] "Alkenyl," "alkenyl chain," or "alkenyl group" refers to a straight-chain or branched hydrocarbon chain group having two to forty carbon atoms and one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. This includes alkenyl groups containing any number of carbon atoms from 2 to 40. Alkenyl groups containing up to 40 carbon atoms are C2-C. 40 Alkenyl groups, which contain up to 10 carbon atoms, are C2-C. 10 Alkenyl groups are categorized into C2-C6 alkenyl groups, containing up to six carbon atoms, and C2-C5 alkenyl groups, containing up to five carbon atoms. C2-C5 alkenyl groups include C5, C4, C3, and C2 alkenyl groups. C2-C6 alkenyl groups include all the portions described above regarding C2-C5 alkenyl groups, but also include C6 alkenyl groups. C2-C 10Alkenyl groups include all the portions described above for C2-C5 and C2-C6 alkenyl groups, but also include C7, C8, C9, and C6 alkenyl groups. 10 Alkenyl. Similarly, C2-C 12 Alkenyl groups include all the aforementioned portions, but also include C. 11 and C 12 Alkenyl. C2-C 12 Non-limiting examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 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 otherwise specifically stated in the specification, the alkyl group may optionally be substituted.

[0512] "Idenoyl", "idenoyl chain", or "idenoyl group" refers to a straight-chain or branched divalent hydrocarbon chain group having two to forty carbon atoms and one or more carbon-carbon double bonds. C2-C 40 Non-limiting examples of alkenyl groups include ethylene, propylene, butene, etc. Unless otherwise specifically stated in the specification, the alkenyl chain may be optional.

[0513] "Alkoxy" or "alkoxy group" means the group -OR, where R is an alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclic group as defined herein. Unless otherwise specified in the specification, the alkoxy group may optionally be substituted.

[0514] "Acyl" or "acyl group" means the group -C(O)R, where R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, or heterocyclic as defined herein. Unless otherwise specified in the specification, the acyl group may optionally be substituted.

[0515] "alkylcarbamoyl" or "alkylcarbamoyl group" refers to the group -OC(O)-NR. a R b , where R a and R b The same or different and independently of alkyl, alkenyl, ynyl, aryl, heteroaryl, or R as defined herein a R b They can be combined to form cycloalkyl or heterocyclic groups as defined herein. Unless otherwise specified in the specification, the alkylcarbamoyl group may optionally be substituted.

[0516] "alkylcarboxamide group" or "alkylcarboxamide group" refers to the group –C(O)-NR a R b , where R a and R b The same or different and independently being an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl or heterocyclic group as defined herein, or R a R b They can be combined to form cycloalkyl groups as defined herein. Unless otherwise specified in the specification, the alkylcarboxamide groups may optionally be substituted.

[0517] "Aryl" refers to a hydrocarbon ring system group containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of this invention, the aryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused ring or bridged ring systems. Aryl groups include, but are not limited to, those derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, and asymmetric indole. as -indacene), symmetrical access province ( s Aryl groups of indacene, indene, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and benzo[a]phenanthrene. Unless otherwise specified in the specification, the term "aryl" refers to an optionally substituted aryl group.

[0518] "Heteroaryl" refers to a 5- to 20-membered ring system group 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 this invention, the heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atom in the heteroaryl group may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples include, but are not limited to, nitrogen-containing heterocyclic heptenyl, acridineyl, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxacyclopentenyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[…]. b [1,4]dioxane-heptenyl, 1,4-benzodioxane, benzonaphthylfuranyl, benzoxazolyl, benzodioxane-pentenyl, benzodioxane-hexenyl, benzopyranyl, benzopyranoneyl, benzofuranyl, benzofuranoneyl, benzothiopheneyl (benzophenylthio), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazoleyl, cenylyl, dibenzofuranyl 1-Franyl, dibenzothiophene, furanyl, furanone, isothiazolyl, imidazolyl, indazole, indole, indazole, isoindole, indolinyl, isoindolinyl, isoquinolinyl, indoleazinyl, isoxazolyl, naphridinyl, oxadiazolyl, 2-oxoazonicyclic heptenyl, oxazolyl, ethylene oxide, 1-oxopyridinyl, 1-pyrimidinyl, 1-pyrazinyl, 1-pyridazinyl, 1-phenyl-1 H -Pyrroloyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purineyl, pyrroloyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quininecycloyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and phenylthioyl (i.e., thiopheneyl). Unless otherwise specified in the specification, heteroaryl groups may optionally be substituted.

[0519] As used herein, the term "substituted" refers to any of the aforementioned groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic, 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, alkoxy, and ester groups; sulfur atoms in groups such as thiols, thioalkyl groups, sulfones, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also refers to any of the aforementioned groups in which one or more atoms are replaced by a higher-order bond (e.g., a double or triple bond) with a heteroatom (such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imine, oxime, hydrazone, and nitrile groups). For example, "substituted" includes groups in which one or more atoms are replaced by -NR. g R h -NR g C(=O)R h -NR g C(=O)NR g R h -NR g C(=O)OR h -NR g SO2R h -OC(=O)NR g R h -OR g -SR g -SOR g -SO2R g -OSO2R g -SO2OR g =NSO2R g and -SO2NR g R h Any of the above groups that are substituted. "Substituted" also refers to one or more hydrogen atoms being replaced by -C(=O)R. g -C(=O)OR g -C(=O)NR g R h -CH2SO2R g -CH2SO2NR g R h Replace any of the aforementioned groups. In the preceding text, R...g and R h The same or different, and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclic, N - Heterocyclic group, heterocyclic alkyl group, heteroaryl group N - Heteroaryl and / or heteroarylalkyl. "Substituted" also refers to one or more atoms being replaced by amino, cyano, hydroxyl, imino, nitro, oxo, thio, halogen, alkyl, alkenyl, alkynyl, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclic, etc. N - Heterocyclic group, heterocyclic alkyl group, heteroaryl group N - Any of the aforementioned substituents that are replaced by a heteroaryl and / or a heteroarylalkyl group. "Substituted" may also mean an amino acid in which one or more atoms on the side chain are replaced by an alkyl, alkenyl, alkynyl, acyl, alkylcarboxamide, alkoxycarbonyl, carbocyclic, heterocyclic, aryl, or heteroaryl group. Additionally, each of the foregoing substituents may optionally be substituted by one or more of the foregoing substituents.

[0520] As used in this article, "phenyl" refers to a cyclic atomic group with the molecular formula C6H5, which can be covalently attached to a molecule as a functional group. In this article, phenyl can be abbreviated as "Ph".

[0521] As used herein, “subject” refers to an individual. Therefore, “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. “Subject” can also include mammals, such as primates or humans. Therefore, a subject can be a human or veterinary patient. The term “patient” refers to a subject under the care of a clinician (e.g., a physician).

[0522] The term "treatment" refers to the medical management of a patient aimed at curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, which is treatment specifically aimed at improving a disease, pathological condition, or symptom, and also includes etiological treatment, which is treatment aimed at eliminating the cause of the related disease, pathological condition, or symptom. Additionally, the term includes palliative treatment, which is treatment aimed at relieving symptoms rather than curing a disease, pathological condition, or symptom; preventative treatment, which is treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological condition, or symptom; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or symptom.

[0523] The term "therapeuticly effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or condition. Such improvement requires only reduction or alteration, not elimination.

[0524] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for contact with tissues in humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0525] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, contributes to or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0526] The term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders intended for reconstitution into sterile injectable solutions or dispersions shortly before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or mediators include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Suitable flowability can be maintained, for example, by using coating materials such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Protection against microorganisms can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents, such as sugars and sodium chloride, may also be desired. Injectable formulations can be sterilized, for example, by filtration through a bacterial trap or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media shortly before use. Suitable inert carriers may include sugars such as lactose.

[0527] The term "sequence identity" refers to the percentage of amino acids that are identical and located at the same relative positions between two polypeptide sequences. Thus, one polypeptide sequence has a certain percentage of sequence identity compared to another. For sequence alignment, typically one sequence is used as a reference sequence, and the test sequence is compared to that reference sequence. Those skilled in the art will understand that if two sequences contain the same residues at corresponding positions, the two sequences are generally considered "substantially identical." In implementations, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) can be used. , J. Mol. Biol. 48: 443-453) to determine sequence identity between two amino acid sequences, this algorithm is used in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet The implementation in the Needle program (16: 276-277) uses the version available up to the application date. The parameters used are: a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (the EMBOSS version of BLOSUM62) replacement matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the identity percentage, calculated as follows: (Number of identical residues × 100) / (Alignment length - Total number of gaps in the alignment). In the implementation plan, the Smith-Waterman algorithm, as of the application date, can be used to determine sequence identity.

[0528] The term "sequence homology" refers to the percentage of amino acids that are homologous and occupy the same relative positions between two polypeptide sequences. Thus, one polypeptide sequence has a certain percentage of sequence homology compared to another polypeptide sequence. As understood by those skilled in the art, two sequences are generally considered "fundamentally homologous" if they contain homologous residues at corresponding positions. Homologous residues may be completely identical residues. Alternatively, homologous residues may be non-completely identical residues with appropriately similar structural and / or functional characteristics. For example, it is well known to those skilled in the art that certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or have "polar" or "nonpolar" side chains, and that the substitution of one amino acid for another of the same type is generally considered a "homologous" substitution.

[0529] As is well known, amino acid sequences can be compared using various algorithms, including those available in commercial computer programs as of the application date (e.g., BLASTP, BLAST with gaps, and PSI-BLAST). Such programs are described in the following literature: Altschul, et al., Basic local alignment search tool. J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al. Methods in Enzymology ; Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al. Bioinformatics A Practical Guide to the Analysis of Genes and Proteins Wiley, 1998; and Misener, et al. (eds.) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above procedures usually provide an indication of the degree of homology.

[0530] The term "antisense oligonucleotide" refers to a polymeric nucleic acid structure (also referred to as an oligonucleotide or polynucleotide) that is at least partially complementary to a target nucleic acid molecule with which it hybridizes. Oligonucleotides can be short (in embodiments, less than 50 base pairs) polynucleotides or polynucleotide homologs containing a sequence complementary to a target sequence in a target precursor mRNA chain. Oligonucleotides can be formed from natural nucleic acids, synthetic nucleic acids, nucleic acid homologs, or any combination thereof. In embodiments, oligonucleotides comprise oligonucleotides. In embodiments, oligonucleotides comprise antisense oligonucleotides. In embodiments, oligonucleotides comprise conjugating groups. Non-limiting examples of oligonucleotides include, but are not limited to, primers, probes, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternative splicing, siRNA, oligonucleotides, oligonucleotides, oligonucleotide analogs, oligonucleotide mimics, and chimeric combinations thereof. Therefore, these compounds can be introduced in single-stranded, double-stranded, circular, branched, or hairpin form and can contain structural elements such as internal or terminal protrusions or loops. Oligomeric double-stranded compounds can be two strands that hybridize to form a double-stranded compound, or single strands with sufficient self-complementarity to allow hybridization and form a complete or partial double-stranded compound. In embodiments, oligonucleotides regulate (increase, decrease, or alter) the expression of target nucleic acids. Various modifications can be made to the polymeric nucleic acid structure, such as diaminophosphate morpholino oligonucleotides (PMOs). Therefore, as used herein, oligonucleotide encompasses any modifications described herein, such as PMOs.

[0531] As used herein, the terms “precursor mRNA” and “primary transcript” refer to newly synthesized eukaryotic mRNA molecules directly after DNA transcription. Precursor mRNA must be capped with a 5' cap, modified with a 3' poly-A tail, and spliced ​​to produce a mature mRNA sequence.

[0532] As used herein, the term "targeting" refers to the association of an oligonucleotide (e.g., a therapeutic oligonucleotide, such as an antisense oligonucleotide) with a target nucleic acid molecule or a region of the target nucleic acid molecule. In embodiments, the oligonucleotide is capable of hybridizing with the target nucleic acid under physiological conditions. In embodiments, the oligonucleotide targets a specific portion or site within the target nucleic acid, for example, a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic, such as a specific exon or intron, or selected nucleobases or motifs within an exon or intron. In embodiments, the oligonucleotide targets a region containing an intron-exon junction of a gene associated with a disease or condition. In embodiments, the oligonucleotide targets exon 50 of the dystrophin gene. In embodiments, the oligonucleotide targets a region containing an intron-exon junction of exon 50 of the dystrophin gene. In embodiments, the oligonucleotide targets a region containing an upstream (or 5') intronic nucleotide sequence of exon 50 of the dystrophin gene. In the implementation scheme, the oligonucleotide targets the region containing the intronic nucleotide sequence downstream (or 3') of exon 50 of the dystrophin gene.

[0533] As used herein, the terms "target nucleic acid" and "target sequence" refer to a nucleic acid molecule having a nucleic acid sequence to which oligonucleotides bind or hybridize. Target nucleic acids include, but are not limited to, RNA (including, but not limited to, precursor mRNA and mRNA or portions thereof), cDNA derived from such RNA, and non-translated RNA, such as miRNA. For example, in embodiments, a target nucleic acid may be a nucleic acid molecule whose expression is associated with a specific symptom or disease state (or mRNA transcribed from such genes) or derived ...

Claims

1. An EEV-cargo conjugate comprising: (a) A cyclic cell-penetrating peptide (cCPP), wherein the cCPP has the structure of formula (2): Equation (2): (2), or its protonated form, in: R 1 R 2 and R 3 Each can be an H or an amino acid residue having a side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are independently aryl or heteroaryl side chains of amino acids; R 4 and R 6 Independent of H or amino acid side chains; AA SC It is an amino acid side chain; q is 1, 2, 3, or 4; and m′ and m′′ are each an independent integer from 0 to 3; (b) Linear exocyclic peptides (EPs) containing 2 to 10 amino acid residues; (c) Connector of type (A′): Equation (A′): (A′) in: ** is the attachment site with the linear exocyclic peptide (EP); * is the attachment site with the cyclic cell-penetrating peptide (cCPP); L 1 and L 2 Independently designed as a connector arm; ^Indicates L-stereochemistry or D-stereochemistry; y′ is an integer from 1 to 5; and M is a reaction handle containing a functional group that reacts with the corresponding functional group on the cargo to form a bonding group (M′); and (d) A cargo containing oligonucleotides, said oligonucleotides binding to the human dystrophin gene ( DMD Exon 50 in the pre-mRNA transcript of ) to induce exon skipping, wherein the oligonucleotide is bound to or contains a nucleic acid sequence shown in Tables 11A-11D, 12A-12D or 13, containing its reverse complementary sequence or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity with it.

2. The EEV-cargo assembly as claimed in claim 1, wherein the connector comprises formula (B′): Equation (B′): (B′)， in: x′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; and j′′ can be 0, 1, or 2, where j′′ is 0 when z′ is 0.

3. The EEV-cargo assembly as claimed in claim 1, wherein the connector comprises formula (C′): Equation (C′): (C′)， x′ is an integer from 1 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; j′′ is 0, 1, or 2, where j′′ is 0 when z′ is 0; and X o ′ is a hydrophobic component.

4. The EEV-cargo assembly as claimed in claim 1, wherein the connector comprises (D′): Equation (D′): (D′)； x′ is an integer from 1 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; j′′ is 0, 1 or 2, where j′′ is 0 when z′ is 0; X o ′ is a hydrophobic component; and K # It is a D-lysine or L-lysine residue.

5. The EEV-cargo conjugate as described in claim 1, comprising formula (A-1): Equation (A-1): (A-1)， in EP is a linear exocyclic peptide; cCPP is a cyclic cell-penetrating peptide; and M′ is a bonding group.

6. The EEV-cargo conjugate as described in claim 5, comprising formula (B-1): Equation (B-1): (B-1)， in x′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; and j′′ can be 0, 1, or 2, where j′′ is 0 when z′ is 0.

7. The EEV-cargo conjugate as described in claim 5, comprising formula (C-1): Equation (C-1): (C-1)， in x′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; j′′ is 0, 1, or 2, where j′′ is 0 when z′ is 0; and X o ′ represents a hydrophobic group.

8. The EEV-cargo conjugate as described in claim 5, comprising formula (D-1): Equation (D-1): (D-1)， in x′ is an integer from 0 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; j′′ is 0, 1 or 2, where j′′ is 0 when z′ is 0; X o ′ is a hydrophobic group; and K # It is a D-lysine or L-lysine residue.

9. The compound of claim 1, comprising formula (A-3): Equation (A-3): (A-3) in: R 1 R 2 and R 3 An aryl or heteroaryl side chain that is independently an H or amino acid residue; R 1 R 2 and R 3 At least two of them are aryl or heteroaryl side chains of amino acid residues; and R 4 R 5 R 6 R 7 It can be an H or amino acid residue side chain independently.

10. The EEV-cargo conjugate as claimed in claim 9, wherein... R 1 R 2 and R 3 Each is an independent side chain containing an aryl or heteroaryl group; R 1 R 2 and R 3 At least two of them are side chains of phenylalanine; R 1 R 2 and R 3 At least one of them is a side chain of naphthylalanine; R 4 R 5 R 6 and R 7 Independently, it is an H or amino acid side chain; and q can be 1, 2, 3 or 4.

11. The EEV-cargo conjugate as claimed in claim 9, wherein... R 1 R 2 and R 3 -CH2Ph; R 4 R 5 R 6 and R 7 It can be an H or amino acid side chain independently.

12. The EEV-cargo conjugate as claimed in claim 9, wherein... R 1 R 2 and R 3 One of them is H; R 1 R 2 and R 3 Both of them are CH2Ph; and R 4 R 5 R 6 and R 7 It can be an H or amino acid side chain independently.

13. The EEV-cargo conjugate as claimed in claim 9, wherein... R 1 R 2 and R 3 One of them is H; R 1 R 2 and R 3 Both of them are CH2Ph; and R 4 R 5 R 6 and R 7 An amino acid side chain that is independently H or arginine.

14. The EEV-cargo conjugate of claim 9, wherein the cCPP has a sequence selected from: fNalRrRrQ, Ff-Nal-Cit-r-Cit-rQ and Ff-Nal-GrGrQ.

15. The EEV-cargo conjugate of claim 9, wherein the cCPP has the FfFGRGRQ sequence.

16. The EEV-cargo conjugate of claim 9, wherein the cCPP has an FGFGRGRQ or GfFGrGrQ sequence.

17. The EEV-cargo conjugate of claim 1, wherein AA SC It contains glutamine (Q).

18. The EEV-cargo assembly of claim 9, wherein the connector is selected from: Equation (B′): (B′)； Equation (C′): (C′); and Equation (D′): (D′)； in: x′ is an integer from 1 to 12; j′ is 0, 1 or 2, where j′ is 0 when x′ is 0; z′ is an integer from 0 to 12; j′′ is 0, 1 or 2, where j′′ is 0 when z′ is 0; X o ′ is a hydrophobic component; and K # It is a D-lysine or L-lysine residue.

19. The EEV-cargo conjugate as claimed in any of the preceding claims, wherein M′ comprises: -NH-, -C(O)-, -O-, ; ; ; or , where y′′ is an integer from 1 to 4, and t′ is an integer from 0 to 10.

20. The EEV-cargo conjugate of claim 19, wherein M comprises -C(O)-.

21. The EEV-cargo conjugate of claim 19, wherein M comprises or , where t′ is an integer from 0 to 10.

22. The EEV-cargo conjugate as claimed in any one of claims 2-4, 6-8 and 19-21, wherein z′ is 0, 2, 4, 8 or 12.

23. The EEV-cargo conjugate of claim 22, wherein z′ is 2 or 12.

24. The EEV-cargo conjugate of claim 22, wherein z′ is 2.

25. The EEV-cargo conjugate of claim 22, wherein z′ is 12.

26. The EEV-cargo conjugate as claimed in any one of claims 2-4, 6-8, 17 and 22-25, wherein x′ is 0, 2, 4, 8 or 12.

27. The EEV-cargo conjugate of claim 26, wherein x′ is 0 or 2.

28. The EEV-cargo conjugate of claim 26, wherein x′ is 0.

29. The EEV-cargo conjugate of claim 26, wherein x′ is 2.

30. The EEV-cargo conjugate as described in any one of claims 3-4, 7-8, 19-21, and 22-25, wherein X o ′ is selected from: L-2-naphthylalanine (L-Nal), D-2-naphthylalanine (d-nal), 3-(4′,4-biphenyl)-L-alanine (L-Bip) or 3-(4′,4-biphenyl)-D-alanine (d-bip).

31. The EEV-cargo conjugate of claim 30, wherein X o ′ represents L-2-naphthylalanine (L-Nal) or D-2-naphthylalanine (d-nal).

32. The EEV-cargo conjugate of claim 30, wherein X o ′ is 3-(4′,4-biphenyl)-L-alanine (L-Bip) or 3-(4′,4-biphenyl)-D-alanine (d-bip).

33. The EEV-cargo conjugate of claim 30, wherein X o ′ represents L-2-naphthylalanine (L-Nal).

34. The EEV-cargo conjugate of claim 30, wherein X o ′ represents D-2-naphthylalanine (d-nal).

35. The EEV-cargo conjugate of claim 30, wherein X o ′ is 3-(4′,4-biphenyl)-L-alanine (L-Bip).

36. The EEV-cargo conjugate of claim 30, wherein X o ′ is 3-(4′,4-biphenyl)-D-alanine (d-bip).

37. The EEV-cargo conjugate as claimed in any of the preceding claims, wherein the linker is covalently bound to the 5' end, the 3' end, or the backbone of the oligonucleotide cargo.

38. The EEV-cargo conjugate as claimed in any of the preceding claims, wherein the EP comprises 2 to 10 amino acid residues, wherein at least one amino acid residue comprises a side chain containing a guanidinium, a terminal amine, an imidazole, or a protonated form thereof.

39. The EEV-cargo conjugate of claim 38, wherein the EP comprises 2 to 8 amino acid residues.

40. The EEV-cargo conjugate of claim 38, wherein the EP comprises 2 to 6 amino acid residues.

41. The EEV-cargo conjugate according to any one of claims 38-40, wherein the EP comprises one, two, three or four arginine residues.

42. The EEV-cargo conjugate according to any one of claims 38-40, wherein the EP comprises one, two, three or four lysine residues.

43. The EEV-cargo conjugate according to any one of claims 38-40, wherein the EP comprises one or two uncharged hydrophobic amino acid residues.

44. The EEV-cargo conjugate of claim 43, wherein the uncharged hydrophobic amino acid residue is selected from valine, proline, β-alanine, glycine, or combinations thereof.

45. The EEV-cargo conjugate according to any one of claims 1-40, wherein the EP 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.

46. ​​The EEV-cargo conjugate according to any one of claims 1-40, wherein the EP has the following structure: Ac-PKKKRKV.

47. The EEV-cargo conjugate as claimed in claim 1, wherein the conjugate is selected from: 。 48. The EEV-cargo conjugate as claimed in claim 1, wherein the conjugate is selected from: 。 49. The EEV-cargo conjugate as claimed in claim 1, wherein the conjugate is selected from: 。 50. The EEV-cargo conjugate of claim 1, comprising: 。 51. The EEV-cargo conjugate as claimed in claim 1, wherein the conjugate is selected from: 。 52. The EEV-cargo conjugate as claimed in claim 1, wherein the conjugate is selected from: 。 53. The EEV-cargo conjugate as claimed in claim 1, having the following structure , where # is diaminophosphate morpholino oligonucleotide (PMO), and the 3' base is the 3' end nucleobase of the PMO.

54. The EEV-cargo conjugate as claimed in claim 1, having the following structure , where # is diaminophosphate morpholino oligonucleotide (PMO), and the 3' base is the 3' end nucleobase of the PMO.

55. The EEV-cargo conjugate as claimed in claim 1, having the following structure , where # is diaminophosphate morpholino oligonucleotide (PMO), and the 3' base is the 3' end nucleobase of the PMO.

56. The EEV-cargo conjugate according to any one of claims 1-55, wherein the cargo is a therapeutic oligonucleotide.

57. The EEV-cargo conjugate according to any one of claims 1-56, wherein the oligonucleotide is an antisense oligonucleotide.

58. The EEV-cargo conjugate according to any one of claims 1-57, wherein the oligonucleotide comprises at least one modified nucleotide or nucleic acid selected from phosphate thioester (PS) nucleotides, diaminophosphate morpholino oligonucleotides (PMO), locked nucleic acids (LNA), peptide nucleic acids (PNA), nucleotides comprising a 2'-O-methyl (2'-OMe) modified backbone, 2'-O-methoxy-ethyl (2'-MOE) nucleotides, 2',4' restricted ethyl (cEt) nucleotides, and 2'-deoxy-2'-fluoro-β-D-arabinose (2'F-ANA).

59. The EEV-cargo conjugate according to any one of claims 1-58, wherein the oligonucleotide comprises at least one diaminophosphate morpholino oligonucleotide (PMO).

60. The EEV-cargo conjugate according to any one of claims 1-59, wherein the oligonucleotide comprises diaminophosphate morpholino oligonucleotide (PMO).

61. The EEV-cargo conjugate according to any one of claims 1-60, wherein the oligonucleotide comprises 15 to 30 nucleotides.

62. The EEV-cargo conjugate according to any one of claims 1-61, wherein the oligonucleotide comprises 20 to 30 nucleotides.

63. The EEV-cargo conjugate according to any one of claims 1-62, wherein the oligonucleotide comprises 20 to 25 nucleotides.

64. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide is bound to or comprises the nucleic acid sequence shown in Tables 11A-11D, 12A-12D or 13.

65. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide is bound to or comprises the nucleic acid sequences shown in Tables 11A-11D.

66. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide is bound to or comprises the nucleic acid sequences shown in Tables 12A-12D.

67. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide is bound to or comprises the nucleic acid sequence shown in Table 13.

68. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide comprises a sequence containing 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X = U or T), its reverse complementary sequence, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

69. The EEV-cargo conjugate as claimed in any one of claims 1-63, wherein, The oligonucleotide comprises a sequence of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleic acids containing a base sequence, wherein the base sequence comprises: 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3', where X = U or T.

70. The EEV-cargo conjugate according to any one of claims 1-63, wherein the oligonucleotide comprises a sequence selected from: 5'-AGTGGTCAGTCCAGGAGCTAGGTC-3', 5'-GTGGTCAGTCCAGGAGCTAGG-3', 5'-GGTCAGTCCAGGAGCTAGGTCA-3', 5'-TAGTGGTCAGTCCAGGAGCTAGGT-3', 5'-GCTCCAATAGTGGTCAGTCCAG-3', 5'-ACCGCCTTCCACTCAGAGCTCAGA-3', 5'-TTACCGCCTTCCACTCAGAGCTCA,-3' 5'-GGAGCTAGGTCAGGCTGCTTTG-3', 5'-GTGGTCAGTCCAGGAGCTAGGTC-3', and 5'-AGGAGCTAGGTCAGGCTGCTTT-3'.

71. The EEV-cargo conjugate according to any one of claims 1-63, wherein the oligonucleotide comprises: 5'-AGTGGTCAGTCCAGGAGCTAGGTC-3'.

72. The EEV-cargo conjugate according to any one of claims 1-63, wherein the oligonucleotide comprises: 5'-GTGGTCAGTCCAGGAGCTAGG-3'.

73. The EEV-cargo conjugate according to any one of claims 1-63, wherein the oligonucleotide comprises: 5'-GGTCAGTCCAGGAGCTAGGTCA-3'.

74. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide comprises: 5'-TAGTGGTCAGTCCAGGAGCTAGGT-3'.

75. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide comprises: 5'-GCTCCAATAGTGGTCAGTCCAG-3'.

76. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide comprises: 5'-ACCGCCTTCCACTCAGAGCTCAGA-3'.

77. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide comprises: 5'-TTACCGCCTTCCACTCAGAGCTCA,-3'.

78. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide comprises: 5'-GGAGCTAGGTCAGGCTGCTTTG-3'.

79. The EEV-cargo conjugate according to any one of claims 1-63, wherein the oligonucleotide comprises: 5'-GTGGTCAGTCCAGGAGCTAGGTC-3'.

80. The EEV-cargo conjugate of any one of claims 1-63, wherein the oligonucleotide comprises: 5'-AGGAGCTAGGTCAGGCTGCTTT-3'.

81. A pharmaceutical composition comprising the EEV-cargo conjugate of any one of claims 1-80 and a pharmaceutically acceptable carrier.

82. A method of treating DMD, comprising administering the pharmaceutical composition of claim 81 to a patient in need.

83. The method of claim 82, wherein administration includes parenteral administration.

84. The method of claim 83, wherein parenteral administration is selected from: subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal, and intrathecal administration.

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