Compounds and methods for exon 50 skipping in Duchenne muscular dystrophy

Abstract

This specification describes EEV-cargo conjugates and compositions thereof, comprising (a) an endosomal escape vehicle (EEV) containing a cyclic cell-permeable peptide (cCPP), and (b) a cargo containing an oligonucleotide complementary to a target sequence of the pre-mRNA transcript of the DMD gene, wherein the target sequence comprises at least a portion of the 5' adjacent intron of exon 50, at least a portion of exon 50, at least a portion of the 3' adjacent intron of exon 50, or a combination thereof. This specification also describes methods for treating DMD using the compounds described herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority based on the filing dates of U.S. Provisional Application No. 63 / 585,311 filed on 26 September 2023; U.S. Provisional Application No. 63 / 588,115 filed on 5 October 2023; U.S. Provisional Application No. 63 / 557,125 filed on 23 February 2024; and U.S. Provisional Application No. 63 / 640,506 filed on 30 April 2024, the disclosures of which are incorporated herein by reference in whole. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to mutations in the dystrophin protein. DMD is caused by genetic mutations in the gene encoding dystrophin (i.e., the DMD gene). These genetic modifications shift the reading frame of the DMD gene, producing a non-functional, truncated DMD protein. One way to treat DMD is to administer compounds that restore the reading frame of the DMD gene. Antisense oligonucleotides can restore the reading frame of the DMD gene by skipping internal exons associated with the shift in the DMD gene's reading frame that leads to the non-functional, truncated DMD protein. Exon skipping produces a functional dystrophin protein that would otherwise be lost in the disease.

[0003] DMD is caused by mutations in one or more exons, such as the region between exons 44 and 55. In 2016, the U.S. Food and Drug Administration (FDA) granted accelerated approval for Exondys 51 (also known as eteplirsene), the first phosphorodiamidate morpholino oligonucleotide (PMO) drug developed for DMD exon 51 skipping. Approximately 13% of DMD patients have mutations that are suitable for exon 51 skipping (Aoki, et al., Proc Natl Acad Sci USA. 109(34):13763-13768). On the other hand, there are currently no approved treatments for DMD patients with mutations that are suitable for exon 50 skipping. [Overview of the project]

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

[0005] This disclosure is an EEV-cargo conjugate, (a) an endosomal escape vehicle (EEV) containing a circular cell-permeable peptide ("cCPP"), (b) The EEV-cargo conjugate comprises a cargo containing an oligonucleotide complementary to a target sequence of the pre-mRNA transcript of the DMD gene, wherein the target sequence comprises at least a portion of the 5' adjacent intron of exon 50, at least a portion of exon 50, at least a portion of the 3' adjacent intron of exon 50, or a combination thereof.

[0006] In some embodiments, the EEV-cargo conjugate is (a) A cyclic cell-permeable peptide (cCPP), wherein the cCPP has the structure of formula (2): [ka] or having a protonated form thereof, During the ceremony, R1 , R 2 , and R 3 each can independently be H, or can be an amino acid residue having a side chain comprising an aryl group or a heteroaryl group, R 1 , R 2 , and R 3 at least two of are independently an aryl side chain or a heteroaryl side chain of an amino acid, R 4 and R 6 are independently H or an amino acid side chain, AA SC is an amino acid side chain, q is 1, 2, 3 or 4, m' and m'' are each independently an integer of 0 to 3, a cCPP, and (b) a linear exocyclic peptide (EP), the EP comprising 2 to 10 amino acid residues, and (c) a linker of formula (A'): [Chemical Formula] (In the formula, ** is the point of attachment to the linear exocyclic peptide (EP), * is the point of attachment to the cyclic cell-penetrating peptide (cCPP), L 1 and L 2 are independently a linker arm, ^ indicates L-stereochemistry or D-stereochemistry, y' is an integer of 1 to 5, M is a reactive handle comprising a functional group that reacts with a corresponding functional group on the cargo to form a linking group (M')), and (d) a cargo comprising an oligonucleotide that hybridizes to exon 50 in the pre-mRNA transcript of the human dystrophin gene (DMD) to cause exon skipping, wherein the oligonucleotide hybridizes to or comprises the nucleic acid sequence shown in Tables 11A to 11D, Tables 12A to 12D, or Table 13, and the cargo may be comprised.

[0007] In some embodiments, the EEV-cargo conjugate is (a) A cyclic cell-permeable peptide (cCPP), wherein the cCPP has the structure of formula (2): [ka] or having a protonated form thereof, During the ceremony, R 1 , R 2 , and R 3 Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least two of these are, independently, aryl side chains or heteroaryl side chains of amino acids. R 4 and R 6 These are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4. m' and m'' are independent integers between 0 and 3, cCPP and (b) A linear extracyclic peptide (EP) comprising 2 to 10 amino acid residues, (c) Linker having the structure of formula (B'): [ka] (In the formula, ** is a binding site to a linear extracyclic peptide (EP), * indicates a binding site to a cyclic cell-permeable peptide (cCPP), ^ indicates L-stereochemistry or D-stereochemistry. y' is an integer between 1 and 5. M is a reactive handle containing a functional group that reacts with the corresponding functional group on the cargo to form a bonding group (M'). x' is an integer between 1 and 12. z' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. (d) A cargo comprising an oligonucleotide that hybridizes to exon 50 in the pre-mRNA transcript of the human dystrophin gene (DMD) and causes exon skipping, wherein the oligonucleotide hybridizes to or comprises nucleic acid sequences shown in Tables 11A-11D, 12A-12D, or 13. It may include.

[0008] In some embodiments, the EEV-cargo conjugate is (a) A cyclic cell-permeable peptide (cCPP), wherein the cCPP has the structure of formula (2): [ka] or having a protonated form thereof, During the ceremony, R 1 , R 2 , and R 3 Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least two of these are, independently, aryl side chains or heteroaryl side chains of amino acids. R 4 and R 6 These are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4. m' and m'' are independent integers between 0 and 3, cCPP and (b) A linear extracyclic peptide (EP) containing 2 to 10 amino acid residues, (c) Linker having the structure of formula (C'): [ka] (In the formula, ** is a binding site to a linear extracyclic peptide (EP), * indicates a binding site to a cyclic cell-permeable peptide (cCPP), ^ indicates L-stereochemistry or D-stereochemistry. y' is an integer between 1 and 5. M is a reactive handle containing 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 between 1 and 12. z' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. (d) A cargo comprising an oligonucleotide that hybridizes to exon 50 in the pre-mRNA transcript of the human dystrophin gene (DMD) and causes exon skipping, wherein the oligonucleotide hybridizes to or comprises nucleic acid sequences shown in Tables 11A-11D, 12A-12D, or 13. It may include.

[0009] In some embodiments, the EEV-cargo conjugate is (a) A cyclic cell-permeable peptide (cCPP), wherein the cCPP has the structure of formula (2): [ka] or having a protonated form thereof, During the ceremony, R 1 , R 2 , and R3 Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least two of these are, independently, aryl side chains or heteroaryl side chains of amino acids. R 4 and R 6 These are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4. m' and m'' are independent integers between 0 and 3, cCPP and (b) A linear extracyclic peptide (EP) containing 2 to 10 amino acid residues, (c) Linker having the structure of formula (D'): [ka] (In the formula, ** is a binding site to a linear extracyclic peptide (EP), * indicates a binding site to a cyclic cell-permeable peptide (cCPP), ^ indicates L-stereochemistry or D-stereochemistry. y' is an integer between 1 and 5. M is a reactive handle containing 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 # These are D-lysine residues or L-lysine residues, x' is an integer between 1 and 12. z' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. (d) A cargo comprising an oligonucleotide that hybridizes to exon 50 in the pre-mRNA transcript of the human dystrophin gene (DMD) and causes exon skipping, wherein the oligonucleotide hybridizes to or comprises nucleic acid sequences shown in Tables 11A-11D, 12A-12D, or 13. It may include.

[0010] In several embodiments, the cargo is an oligonucleotide. In several embodiments, the oligonucleotide is a therapeutic oligonucleotide. In several embodiments, the cargo is an antisense oligonucleotide. In several embodiments, the cargo is an oligonucleotide comprising at least one modified nucleotide or nucleic acid. In several embodiments, the modified nucleotide or nucleic acid is a phosphorothioate (PS) nucleotide, a phosphorodiamidate morpholino oligonucleotide (PMO), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a nucleotide containing a 2'-O-methyl (2'-OMe) modified skeleton, a 2'-O-methoxyethyl (2'-MOE) nucleotide, a 2',4'-restricted ethyl (cEt) nucleotide, or a 2'-deoxy-2'-fluoro-beta-D-arabino nucleic acid (2'F-ANA). In several embodiments, the oligonucleotide comprises at least one phosphorodiamidate morpholino oligonucleotide (PMO). In several embodiments, each nucleotide in the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0011] In some embodiments, the oligonucleotide hybridizes to the exon 50 nucleic acid sequence shown in Tables 11A-11D, 12A-12D, or Table 13. In some embodiments, the oligonucleotide includes the reverse complementary sequence of the nucleic acid sequence in Tables 11A-11D, 12A-12D, or Table 13. In some embodiments, the oligonucleotide includes 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 Table 13.

[0012] In several embodiments, the oligonucleotide is complementary to a portion of exon 50. In several embodiments, the oligonucleotide is complementary to a portion of the 5' adjacent intron of exon 50 and a portion of exon 50. In several embodiments, the oligonucleotide is complementary to a portion of the 3' adjacent intron of exon 50 and a portion of exon 50.

[0013] In multiple embodiments, the first nucleotide of the oligonucleotide is -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 of exon 50 of the DMD gene of SEQ ID NO: 1 +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, It can hybridize to nucleotides located at positions +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 pharmaceutical compositions comprising the EEV-cargo conjugate described herein.

[0015] This disclosure relates to cells containing the EEV-cargo conjugate described herein.

[0016] This disclosure relates to a method for treating DMD, comprising administering the EEV-cargo conjugate described herein to a patient in need thereof. [Brief explanation of the drawing]

[0017] [Figure 1A] This paper presents the conjugation chemistry for conjugating oligonucleotides into endosomal escape vehicles (EEVs). [Figure 1B]This paper presents the conjugation chemistry for conjugating oligonucleotides into endosomal escape vehicles (EEVs). [Figure 2] Figures A and B show the conjugation chemistry for linking an EEV containing CPP and a linker to an antisense oligonucleotide. The antisense oligonucleotide is shown both without a component containing PEG2 (A) and with a component containing PEG2 (B). [Figure 3A] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 21-mer PMO. [Figure 3B] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 21-mer PMO. [Figure 4A] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 22-mer PMO. [Figure 4B] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 22-mer PMO. [Figure 5A] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 23-mer PMO. [Figure 5B] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 23-mer PMO. [Figure 6A] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 24-mer PMO. [Figure 6B] This shows the exon 50 skipping rate in CRL-2061™ cells treated with 5 μM 24-mer PMO. [Figure 7A] This shows the exon 50 skipping rates in CRL-2061® cells treated with 1 μM and 3 μM PMO. Table 14A shows the exon 50 skipping in CRL-2061® cells treated with 1 μM and 3 μM 21-mer PMO as listed. [Figure 7B] This shows the exon 50 skipping rates in CRL-2061® cells treated with 1 μM and 3 μM PMO. Table 14B shows the exon 50 skipping in CRL-2061® cells treated with 1 μM and 3 μM 22-mer PMO as listed. [Figure 7C] This shows the exon 50 skipping rates in CRL-2061® cells treated with 1 μM and 3 μM PMO. Table 14C shows exon skipping in CRL-2061® cells treated with 1 μM and 3 μM 23-mer PMO as listed. [Figure 7D] This shows the exon 50 skipping rates in CRL-2061® cells treated with 1 μM and 3 μM PMO. Table 14D shows exon skipping in CRL-2061® cells treated with 1 μM and 3 μM 24-mer PMO as listed. [Figure 8A] This shows the exon 50 skipping rate in gastrocnemius ("gastroc") tissue of hDMD mice treated with various 15mpk EEV-PMOs via IV injection. [Figure 8B] This shows the exon 50 skipping rate in the diaphragmatic tissue of hDMD mice treated with various 15mpk EEV-PMOs via IV injection. [Figure 8C] This shows the exon 50 skipping rate in cardiac tissue of hDMD mice treated with various 15mpk EEV-PMOs via IV injection. [Figure 9A] This shows the exon 50 skipping rate in gastrocnemius ("gastroc") tissue of hDMD mice treated with various 30 mpk EEV-PMOs via IV injection. [Figure 9B] This shows the exon 50 skipping rate in the diaphragmatic tissue of hDMD mice treated with various 30 mpk EEV-PMOs via IV injection. [Figure 9C] This shows the exon 50 skipping rate in cardiac tissue of hDMD mice treated with various 30 mpk EEV-PMOs via IV injection. [Figure 10] This table shows the in silico off-target analysis of EEV-PMO in primary and mature RNA. mm = mismatch. [Figure 11A] This shows the exon 50 skipping rate in the gastrocnemius muscle tissue of hDMD mice treated with 15mpk, 30mpk, and 60mpk EEV-PMO24mer-14 via IV injection. [Figure 11B] This shows the exon 50 skipping rate in triceps muscle tissue of hDMD mice treated with 15mpk, 30mpk, and 60mpk EEV-PMO24mer-14 via IV injection. [Figure 11C] This shows the exon 50 skipping rate in the tibialis anterior muscle tissue of hDMD mice treated with 15mpk, 30mpk, and 60mpk EEV-PMO24mer-14 via IV injection. [Figure 11D] This shows the exon 50 skipping rates in cardiac tissue of hDMD mice treated with 15mpk, 30mpk, and 60mpk EEV-PMO24mer-14 via IV injection. [Figure 11E] This shows the exon 50 skipping rates in the diaphragmatic tissue of hDMD mice treated with 15mpk, 30mpk, and 60mpk EEV-PMO24mer-14 via IV injection. [Figure 12A] This shows the exon 50 skipping rate in the gastrocnemius muscle one week after injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 12B] This shows the exon 50 skipping rate in the triceps muscle one week after injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 12C] This shows the exon 50 skipping rate in the tibialis anterior muscle one week after injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 12D] This shows the exon 50 skipping rate in the heart of hDMD mice treated with EEV-PMO24mer-14 via IV injection, one week after injection. [Figure 12E] This shows the exon 50 skipping rate in the diaphragm one week after injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 13A] This shows the exon 50 skipping rate in the gastrocnemius muscle 6 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 13B] This shows the exon 50 skipping rate in the triceps muscle 6 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 13C] This shows the exon 50 skipping rate in the tibialis anterior muscle 6 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 13D] This shows the exon 50 skipping rate in the heart of hDMD mice treated with EEV-PMO24mer-14 via IV injection, 6 weeks after injection. [Figure 13E] This shows the exon 50 skipping rate in the diaphragm 6 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection. [Figure 14A] This shows the exon 50 skipping rate in the triceps muscle at 1, 2, 4, 6, 8, and 12 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose duration of effect study. [Figure 14B] This shows the exon 50 skipping rate in the tibialis anterior muscle at 1, 2, 4, 6, 8, and 12 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose duration of effect study. [Figure 14C] This shows the exon 50 skipping rate in the gastrocnemius muscle at 1, 2, 4, 6, 8, and 12 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose duration of action study. [Figure 14D]This shows the exon 50 skipping rate in the diaphragm at 1, 2, 4, 6, 8, and 12 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose duration of effect study. [Figure 14E] This shows the exon 50 skipping rate in the heart at 1, 2, 4, 6, 8, and 12 weeks post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose duration of effect study. [Figure 15A] This shows the exon 50 skipping rates in the gastrocnemius muscle at 4, 8, 16, 24, 48, 96, and 168 hours post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose initial time-point study. [Figure 15B] This shows the exon 50 skipping rate in the triceps muscle at 4, 8, 16, 24, 48, 96, and 168 hours post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose initial time-point study. [Figure 15C] This shows the exon 50 skipping rate in the tibialis anterior muscle at 4, 8, 16, 24, 48, 96, and 168 hours post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose initial time-point study. [Figure 15D] This shows the exon 50 skipping rates in the diaphragm at 4, 8, 16, 24, 48, 96, and 168 hours post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose initial time-point study. [Figure 15E] This shows the exon 50 skipping rates in the heart at 4, 8, 16, 24, 48, 96, and 168 hours post-injection in hDMD mice treated with EEV-PMO24mer-14 via IV injection in a single-dose initial time-point study. [Figure 16] This is a plot of DMD exon 50 skipping rates in DMDΔ51 patient iPSC-derived cardiomyocytes (CMs) treated with 0, 0.1, 0.3, 1, 3, and 10 μM EEV-PMO24mer-14, as reported by ddPCR. [Figure 17] This plot shows the relative expression of DMD in DMDΔ51 patient iPSC-derived cardiomyocytes (CMs) treated with 0, 0.1, 0.3, 1, 3, and 10 μM EEV-PMO24mer-14, as reported by Simple Western Jess analysis. Dystrophin is shown as a percentage relative to untreated normal cells, normalized to total protein. [Figure 18] This is a plot of the mean dystrophin fluorescence signal intensity in DMDΔ51 patient iPSC-derived cardiomyocytes (CMs) treated with 0, 0.1, 0.3, 1, 3, and 10 μM EEV-PMO24mer-14, as measured by immunofluorescence. [Figure 19] This is a plot of dystrophin-positive cardiomyocyte area in DMDΔ51 patient iPSC-derived cardiomyocytes (CMs) treated with 0, 0.1, 0.3, 1, 3, and 10 μM EEV-PMO24mer-14, as measured by immunofluorescence. [Figure 20] Plots of DMD exon 50 skipping rates in DMDΔ51-55 inducible direct reprogramming myotubes (iDRMs) treated with 0, 1, 3, 10, and 30 μM EEV-PMO24mer-14, as reported by ddPCR. [Figure 21] This plot shows the relative expression of DMD in DMDΔ51-55-inducible direct reprogramming myotubes (iDRMs) treated with 0, 1, 3, 10, and 30 μM EEV-PMO24mer-14, analyzed by ddPCR, normalized for HPRT1 expression, and normalized for the control group. [Figure 22] This is a plot of dystrophin protein relative expression in DMDΔ51-55 inducible direct reprogramming myotubes (iDRMs) treated with 0, 1, 3, 10, and 30 μM EEV-PMO24mer-14, measured by Simple Western Jess analysis. [Figure 23]This is a plot of the mean dystrophin fluorescence signal intensity in DMDΔ51-55-inducible direct reprogramming myotubes (iDRMs) treated with 0, 1, 3, 10, and 30 μM EEV-PMO24mer-14, as measured by immunofluorescence. [Figure 24] This is a plot of dystrophin-positive myotube area in DMDΔ51-55-inducible direct reprogramming myotubes (iDRMs) treated with 0, 1, 3, 10, and 30 μM EEV-PMO24mer-14, measured by immunofluorescence. [Figure 25] This shows the exon 50 skipping rates in skeletal muscle tubes of DMDΔ51 derived from AdMyoD-induced patients treated with 0, 1, 3, 10, and 20 μM EEV-PMO24mer-14. [Figure 26] The ratios of dystrophin to total protein in DMDΔ51 skeletal muscle tubes from AdMyoD-induced patients treated with 0, 1, 3, 10, and 20 μM EEV-PMO24mer-14 are shown. [Figure 27] This is the structure of an EEV-cargo conjugate formed via amide chemistry by conjugating EEV having the sequence Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG12-OH to the 3' base of a phosphorodiamidate morpholino oligonucleotide (PMO). The symbol # represents the remaining part of the PMO. [Figure 28] This is the structure of an EEV-cargo conjugate formed via amide chemistry by conjugating EEV having the sequence Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG12-K(Ac-Bip)-O to the 3' base of a phosphorodiamidate morpholino oligonucleotide (PMO). The symbol # represents the remaining part of the PMO. [Figure 29]This is the structure of an EEV-cargo conjugate formed via amide chemistry by conjugating EEV having the sequence Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG12-Nal-OH to the 3' base of a phosphorodiamidate morpholino oligonucleotide (PMO). The symbol # represents the remaining part of the PMO. [Modes for carrying out the invention]

[0018] This specification discloses compounds for treating Duchenne muscular dystrophy (DMD) in patients with mutations correctable by exon skipping. In several embodiments, DMD is caused by mutations in the region of exons 44–55 of the DMD gene. In several embodiments, DMD is caused by mutations in exon 50. In several embodiments, the compound comprises an EEV designed to deliver an oligonucleotide cargo to a cell, the oligonucleotide cargo being complementary to a target sequence in the pre-mRNA transcript of the DMD gene, the target sequence comprising at least a portion of the 5' adjacent intron of exon 50, at least a portion of exon 50, at least a portion of the 3' adjacent intron of exon 50, or a combination thereof. In several embodiments, the EEV-cargo conjugate is delivered intracellularly to a target requiring it. In several embodiments, the EEV delivers an oligonucleotide cargo complementary to a target sequence including an intron-exon junction in exon 50 of the DMD gene. In some embodiments, the EEV delivers an oligonucleotide cargo complementary to a target sequence containing an intronic nucleotide sequence upstream (or 5') of exon 50 of the DMD gene. In some embodiments, the EEV delivers an oligonucleotide cargo complementary to a target sequence containing an intronic nucleotide sequence downstream (or 3') of exon 50 of the DMD gene.

[0019] In several embodiments, the oligonucleotide alters the splicing pattern of the target pre-mRNA with which the oligonucleotide hybridizes, resulting in the formation of a respliced ​​target protein. In several embodiments, the respliced ​​target protein exhibits enhanced function compared to the target protein produced by splicing of the target pre-mRNA in the absence of the oligonucleotide. In several embodiments, the respliced ​​target protein increases the function of the target protein by 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 ​​and ranges in between) compared to the function of the target protein produced by splicing in the absence of the oligonucleotide. In several embodiments, the respliced ​​target protein restores 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 ​​and ranges in between).

[0020] In several embodiments, the EEV-cargo conjugate comprises an endosomal escape vehicle (EEV) and a cargo. In several embodiments, the cargo is an oligonucleotide. In several embodiments, the oligonucleotide is a therapeutic oligonucleotide. In several embodiments, the oligonucleotide is an antisense oligonucleotide. In several embodiments, the EEV comprises a cyclic cell-permeable peptide (cCPP). In several embodiments, the EEV-cargo conjugate can cross the cell membrane and bind to a target pre-mRNA in vivo. In several embodiments, an EEV-cargo conjugate is provided comprising a) at least one endosomal escape vehicle (EEV) comprising a cyclic cell-permeable peptide (cCPP), and b) at least one cargo. As used herein, “conjugation” may mean a covalent or non-covalent bond between the EEV and the cargo, and also includes the chemical conjugation of the EEV and the cargo. Non-limiting examples of conjugation chemistry used for covalent bonding between EEV and cargo include, but are not limited to, conjugations using click chemistry or amide formation chemistry. Non-limiting examples of non-covalent bonding include those mediated by streptavidin / biotin interactions, in which case the EEV is bound to the cargo via a non-covalent bond between biotin and streptavidin.

[0021] In several embodiments, the cargo comprises an oligonucleotide, and the conjugation of the oligonucleotide cargo to the EEV can occur at any suitable site on the oligonucleotide. In several embodiments, the EEV can be conjugated to the 5' end, 3' end, or backbone of the oligonucleotide cargo.

[0022] Endosome escape vehicle (EEV) Provided herein are endosome escape vehicles (EEVs) that can be used to transport cargo molecules across the cell membrane, for example, to deliver cargo to the cytosol 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. Oligonucleotides may be antisense oligonucleotides. Oligonucleotides may be phosphorodiamidate morpholino oligonucleotides (PMOs). The EEV may include a cell-permeable peptide (CPP), e.g., a cyclic cell-permeable peptide (cCPP), which is conjugated to a linear extracyclic peptide (EP). The EEV may include one or more linkers. The EEV may include a hydrophobic component (X). One or more linker arms of the EEV or cCPP may include a hydrophobic component (X). In some embodiments, if the linker arms of the EEV include multiple hydrophobic components (X), the hydrophobic components are (e.g., X o ', X o This can be indicated using '', etc. In some embodiments, the EEV may comprise a hydrocarbon component, a polyethylene glycol (PEG) component, a hydrophobic component (X), an amino acid component (AA) comprising one or more amino acid residues, or a combination thereof. In some embodiments, EEVs are provided in which the stereochemistry of amino acids in the peptide sequence of the EEV is modified.

[0023] In several embodiments, the EEV is coupled to a cargo. The cargo can be coupled to a cCPP. The cargo can be coupled to an EP. The cargo can be coupled to a linker. The EP can be coupled to a cCPP. The EP can be coupled to both the cargo and the cCPP. The coupling between the EP, cargo, cCPP, or any combination thereof may be non-covalent or covalent. The EP, cargo, cCPP, or any combination thereof may be coupled via one or more linkers.

[0024] Cell-permeable peptides (CPPs) An endosome escape vehicle (EEV) comprising at least one cell-permeable peptide (CPP) is provided herein. The cell-permeable peptide may be a cyclic cell-permeable peptide (cCPP). In some embodiments, the cCPP can permeate the cell membrane. In some embodiments, the cCPP can deliver cargo to the cytosol of a cell. The cCPP can deliver cargo to a cellular location where a target gene, target transcript, and / or target protein is located. At least one bond or lone pair on the cCPP can be replaced to conjugate the cCPP to cargo, EP, and / or linker.

[0025] In several embodiments, the hydrophobic component (X) is attached to one or more amino acid residues of cCPP. In several embodiments, the hydrophobic component (X) is attached to the side chain of a lysine amino acid residue of cCPP.

[0026] X may be a D or L amino acid residue having a hydrophobic side chain. X may be a naturally occurring or unnatural amino acid residue having a hydrophobic side chain. X may be an amino acid residue having an aromatic side chain. X may be an amino acid residue having a heteroaromatic side chain. X may be selected from phenylalanine, 3-(4',4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or a combination thereof. X may be 2-naphthylalanine. X may be Naal. X may be d-Nal(nal). X may be 3-(4',4-biphenyl)-L-alanine. X may be Bip. X may be D-Bip(bip). X may be a C4-C8 alkyl hydrocarbon. X may be a C6 alkyl hydrocarbon.

[0027] The total number of amino acid residues in a cCPP is 6 to 20 amino acid residues, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, encompassing all ranges and partial ranges between these. A cCPP may contain 6 to 13 amino acid residues. A cCPP may contain 6 to 10 amino acids. For example, a cCPP containing 6 to 10 amino acid residues is given by formulas IA to IE:

[0028] [ka] It may have a structure according to any of the following, where AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and AA 10 This is an amino acid residue.

[0029] cCPP may contain 6 to 8 amino acids. cCPP may contain 8 amino acids.

[0030] Each amino acid in cCPP may be a natural or non-natural amino acid. Table 1 shows the abbreviations used herein for some natural and non-natural amino acids.

[0031] As used herein, the term "amino acid" refers to a compound having 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 characteristic substituent bonded to the α-carbon of an amino acid.

[0032] An "α-amino acid" is an amino acid in which the amino group is bonded to the first carbon (alpha carbon) adjacent to the carboxylic acid group, and therefore the carbon atom of the carbonyl group and the nitrogen atom of the amino group are separated 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 bonded to the second carbon (beta carbon) instead of the alpha carbon, and therefore the carbon atom of the carbonyl group and the nitrogen atom of the amino group are separated by two carbon atoms. Examples of β-amino acids include, but are not limited to, β-alanine and β-homophenylalanine.

[0033] "Uncharged" amino acids are those that have no charge at physiological pH (e.g., 6.5–8.0 or 6.8–7.6). Note that histidine can exist in a neutral or positively charged form at physiological pH.

[0034] Side chains that do not contain an aryl group or a heteroaryl group may be referred to herein as “non-aryl” side chains. In some embodiments, side chains that do not contain an aryl group or a heteroaryl group may be uncharged and may be referred herein as uncharged non-aryl side chains. Amino acids having uncharged non-arylamino 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.

[0035] The term "non-natural amino acid" refers to organic compounds that are congeners of natural amino acids, in that they have a structure similar to natural amino acids, mimicking the structure and reactivity of natural amino acids. Non-natural amino acids may be modified amino acids and / or amino acid analogs that are not one of the 20 common natural amino acids or the rare natural amino acids selenocysteine ​​or pyrrolicin. Non-natural amino acids may also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives, or combinations thereof. [Table 1-1] [Table 1-2]

[0036] In several embodiments, cCPP may comprise two consecutive amino acids having hydrophobic side chains. In several embodiments, cCPP may comprise three consecutive amino acids having hydrophobic side chains. In several embodiments, the amino acids having hydrophobic side chains are selected from phenylalanine and naphthylalanine. In several embodiments, consecutive amino acids having hydrophobic side chains may have D-stereochemistry or L-stereochemistry. In several embodiments, consecutive amino acids having hydrophobic side chains may have the same stereochemistry. In several embodiments, consecutive amino acids having hydrophobic side chains may have alternating stereochemistry. In several embodiments, consecutive amino acids having hydrophobic side chains may all have L-stereochemistry. In several embodiments, consecutive amino acids having hydrophobic side chains may all have D-stereochemistry.

[0037] In some embodiments, one or two amino acids in the cCPP may not have a side chain. In some embodiments, all amino acids in the cCPP have a side chain. As used herein, when a side chain is absent, the amino acid has two hydrogen atoms on the carbon atom(s) linking the amine and carboxylic acid of the amino acid residue (e.g., -CH2). An amino acid without a side chain may be glycine. An amino acid without a side chain may be β-alanine.

[0038] In several embodiments, cCPP may comprise two consecutive amino acids, one having a hydrophobic side chain, one without a side chain, or a combination thereof. In several embodiments, cCPP may comprise three consecutive amino acids, one having a hydrophobic side chain, one without a side chain, or a combination thereof. In several embodiments, consecutive amino acids, one having a hydrophobic side chain, one without a side chain, or a combination thereof, may have alternating stereochemistry. In several embodiments, consecutive amino acids, one having a hydrophobic side chain, one without a side chain, or a combination thereof, may all have L-stereochemistry. In several embodiments, consecutive amino acids, one having a hydrophobic side chain, one without a side chain, or a combination thereof, may all have D-stereochemistry.

[0039] cCPP may contain 6 to 20, 6 to 10, or 6 to 8 amino acid residues, where (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 an aryl group or a heteroaryl group; and (iii) at least two amino acids may independently have side chains containing a guanidine group or a protonated form thereof. In some embodiments, (i) 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 an aryl group or a heteroaryl group; and (iii) two amino acids may independently have side chains containing a guanidine group or a protonated form thereof.

[0040] In some embodiments, one amino acid of cCPP may be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid. In some embodiments, two amino acids may independently be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid. In some embodiments, three amino acids may be glycine, β-alanine, serine, histidine, citrulline, or 4-aminobutyric acid.

[0041] In several embodiments, one amino acid of cCPP may have a side chain containing an aryl group or a heteroaryl group. In several embodiments, two amino acids of cCPP may have side chains containing an aryl group or a heteroaryl group. In several embodiments, three amino acids of cCPP may have side chains containing an aryl group or a heteroaryl group. In several embodiments, the amino acids having side chains containing an aryl group or a heteroaryl group are not consecutive. In several embodiments, two amino acids having side chains containing an aryl group or a heteroaryl group may be consecutive. In several embodiments, two consecutive amino acids may have opposite stereochemistry. In several embodiments, two consecutive amino acids may have the same stereochemistry. In several embodiments, three amino acids having side chains containing an aryl group or a heteroaryl group may be consecutive. In several embodiments, three consecutive amino acids may have the same stereochemistry. In several embodiments, three consecutive amino acids may have alternating stereochemistry. Each amino acid residue having a side chain containing an aryl group or a heteroaryl group may independently be a residue of phenylalanine, naphthylalanine, or β-homophenylalanine, each of which may optionally be substituted with one or more substituents. At least one amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a phenylalanine residue. One amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a phenylalanine residue. At least two amino acid residues having side chains containing an aryl group or a heteroaryl group may be phenylalanine residues. Two amino acid residues having side chains containing an aryl group or a heteroaryl group may be phenylalanine residues. Each amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a phenylalanine residue. At least one amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a naphthylalanine residue. One amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a naphthylalanine residue.At least one amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a residue of β-homophenylalanine. One amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a residue of β-homophenylalanine.

[0042] In several embodiments, one amino acid of cCPP may have a side chain that does not contain an aryl or heteroaryl group, and is referred to herein as a “non-aryl” side chain. In several embodiments, a side chain that does not contain an aryl or heteroaryl group may be uncharged, and is referred to herein as an uncharged non-aryl side chain. In several embodiments, two amino acids of cCPP may have uncharged non-aryl side chains. In several embodiments, three amino acids of cCPP may have uncharged non-aryl side chains. Amino acids having uncharged non-arylamino 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.

[0043] In several embodiments, one amino acid of cCPP has a side chain containing a guanidine group or its protonated form. In several embodiments, two amino acids of cCPP may have side chains containing a guanidine group or its protonated form. In several embodiments, three amino acids of cCPP may have side chains containing a guanidine group or its protonated form. In several embodiments, four amino acids of cCPP may have side chains containing a guanidine group or its protonated form. In several embodiments, the amino acids of cCPP having side chains containing a guanidine group or its protonated form are consecutive. In several embodiments, the amino acids of cCPP having side chains containing a guanidine group or its protonated form are not consecutive. In several embodiments, the amino acid containing a guanidine group or its protonated form is arginine. In several embodiments, the amino acids of cCPP having side chains containing a guanidine group or its protonated form have D-stereochemistry or L-stereochemistry. In several embodiments, the amino acids of cCPP having a side chain containing a guanidine group or its protonated form have alternating D-stereochemistry and L-stereochemistry. In several embodiments, the amino acids of cCPP having a side chain containing a guanidine group or its protonated form have the same stereochemistry. In several embodiments, the amino acids of cCPP having a side chain containing a guanidine group or its protonated form have D-stereochemistry. In several embodiments, the amino acids of cCPP having a side chain containing a guanidine group or its protonated form have L-stereochemistry.

[0044] In several embodiments, cCPP may comprise 6 to 10 amino acid residues, where (i) at least two amino acids may independently be glycine, β-alanine, or 4-aminobutyric acid residues; (ii) at least two amino acids may have side chains comprising an aryl group or a heteroaryl group; and (iii) at least two amino acids may independently have side chains comprising a guanidine group or a protonated form thereof.

[0045] In several embodiments, cCPP may comprise 6 to 10 amino acid residues, where (i) two amino acids may independently be glycine, β-alanine, or 4-aminobutyric acid residues; (ii) two or three amino acids may have side chains containing an aryl group or a heteroaryl group; and (iii) two amino acids may independently have side chains containing a guanidine group or a protonated form thereof.

[0046] In several embodiments, cCPP may comprise 6 to 10 amino acid residues, where (i) at least two amino acids may independently be glycine, β-alanine, or 4-aminobutyric acid residues; (ii) at least three amino acids may have side chains comprising an aryl group or a heteroaryl group; and (iii) at least two amino acids may comprise arginine.

[0047] Glycine and related amino acid residues In multiple embodiments, cCPP may contain 1, 2, 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. In multiple embodiments, cCPP may contain 2 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. In multiple embodiments, cCPP may contain 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. In multiple embodiments, cCPP may contain 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. In multiple embodiments, cCPP may contain 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. In multiple embodiments, cCPP may contain 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. In multiple embodiments, cCPP may contain 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. In some embodiments, cCPP may comprise three, four, or five glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof.

[0048] In multiple embodiments, cCPP may contain 1, 2, 3, 4, 5, or 6 glycine residues. cCPP may contain 2 glycine residues. In multiple embodiments, cCPP may contain 3 glycine residues. In multiple embodiments, cCPP may contain 4 glycine residues. In multiple embodiments, cCPP may contain 5 glycine residues. In multiple embodiments, cCPP may contain 6 glycine residues. In multiple embodiments, cCPP may contain 3, 4, or 5 glycine residues. In multiple embodiments, cCPP may contain 3 or 4 glycine residues. In multiple embodiments, cCPP may contain 2 or 3 glycine residues. In multiple embodiments, cCPP may contain 1 or 2 glycine residues.

[0049] In some embodiments, cCPP may contain at least three glycine residues. In some embodiments, cCPP may contain three, four, five, or six glycine residues. In some embodiments, cCPP may contain three glycine residues. In some embodiments, cCPP may contain four glycine residues. In some embodiments, cCPP may contain five glycine residues. In some embodiments, cCPP may contain six glycine residues. In some embodiments, cCPP may contain three, four, or five glycine residues. cCPP may contain three or four glycine residues.

[0050] In several embodiments, the glycine, β-alanine, or 4-aminobutyric acid residues in cCPP are not consecutive. In several embodiments, two or three glycine, β-alanine, or 4-aminobutyric acid residues may be consecutive. In several embodiments, two glycine, β-alanine, or 4-aminobutyric acid residues may be consecutive.

[0051] In several embodiments, the glycine residues in cCPP are not consecutive. For example, each glycine residue in cCPP may be separated by a non-glycine amino acid residue.

[0052] In several embodiments, two or more glycine residues in cCPP are consecutive. In several embodiments, two or three glycine residues may be consecutive. In several embodiments, two glycine residues are consecutive.

[0053] Amino acid side chains having an aryl group or a heteroaryl group In several embodiments, cCPP may comprise 2, 3, 4, 5, or 6 amino acid residues, each independently having a side chain containing an aryl group or a heteroaryl group. In several embodiments, cCPP may comprise 2 amino acid residues, each independently having a side chain containing an aryl group or a heteroaryl group. In several embodiments, cCPP may comprise 3 amino acid residues, each independently having a side chain containing an aryl group or a heteroaryl group. In several embodiments, cCPP may comprise 2, 3, or 4 amino acid residues, each independently having a side chain containing an aryl group or a heteroaryl group. In several embodiments, cCPP may comprise 2 or 3 amino acid residues, each independently having a side chain containing an aryl group or a heteroaryl group.

[0054] The aryl group may be a 6- to 14-membered aryl. The aryl may be phenyl, naphthyl, or anthracenyl, each of which may be optionally substituted. The heteroaryl group may be a 6- to 14-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S. The heteroaryl may be pyridyl, quinolyl, or isoquinolyl.

[0055] Each amino acid residue having a side chain containing an aryl group or a heteroaryl group can independently be a residue of phenylalanine, naphthylalanine, phenylglycine, β-homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan, or tyrosine, each of which may be optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aryl group or a heteroaryl group can independently be tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butylphenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 3-(9-anthryl)-alanine. The amino acid residues having a side chain containing an aryl or heteroaryl group may independently be 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalene-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, or 3-(3-benzothienyl)-alanine. The amino acid residues having a side chain containing an aryl or heteroaryl group may independently be phenylalanine, 1-naphthylalanine, 2-naphthylalanine, phenylglycine, β-homophenylalanine, or homonaphthylalanine, each of which may optionally be substituted with one or more substituents. Each amino acid residue having a side chain containing an aryl group or a heteroaryl group can independently be a residue of phenylalanine, 2-naphthylalanine, or β-homophenylalanine, each of which may be optionally substituted with one or more substituents.Each amino acid residue having a side chain containing an aryl group or a heteroaryl group may independently be a phenylalanine or 2-naphthylalanine residue, each of which may optionally be substituted with one or more substituents. At least one amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a phenylalanine residue. At least two amino acid residues having side chains containing an aryl group or a heteroaryl group may be phenylalanine residues. Each amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a phenylalanine residue. One amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a 2-naphthylalanine residue. One amino acid residue having a side chain containing an aryl group or a heteroaryl group may be a β-homophenylalanine residue.

[0056] In several embodiments, amino acids having side chains containing an aryl group or a heteroaryl group are not consecutive. In several embodiments, two amino acids having side chains containing an aryl group or a heteroaryl group may be consecutive. In several embodiments, two consecutive amino acids may have opposite stereochemistry. In several embodiments, two consecutive amino acids may have the same stereochemistry. In several embodiments, three amino acids having side chains containing an aryl group or a heteroaryl group may be consecutive. In several embodiments, three consecutive amino acids having side chains containing an aryl group or a heteroaryl group have the same stereochemistry. In several embodiments, three consecutive amino acids may have alternating stereochemistry.

[0057] Amino acid residues containing an aryl group or a heteroaryl group may be L-amino acids. Amino acid residues containing an aryl group or a heteroaryl group may be D-amino acids. Amino acid residues containing an aryl group or a heteroaryl group may be a mixture of D-amino acids and L-amino acids.

[0058] The hydrophobicity of amino acid residues can be measured and / or calculated using various techniques. In several embodiments, the hydrophobicity of an amino acid residue can be determined by calculating its consensus value based 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.). Hydrophobic amino acids are amino acids that have hydrophobic side chains.

[0059] Amino acid residues having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. When used herein, guanidine has the following structure: [ka] It refers to.

[0060] When used herein, the protonated form of guanidine is structural: [ka] It refers to.

[0061] A guanidine substituent refers to a functional group on the side chain of an amino acid that is positively charged above physiological pH, or a functional group that can replicate the hydrogen bond donating and accepting activity of a guanidinium group.

[0062] While we do not wish to be bound by theory, guanidine substituents are thought to facilitate the cellular penetration and delivery of therapeutic agents while reducing the toxicity associated with the guanidine group or its protonated forms. A cCPP may contain at least one amino acid having a side chain containing a guanidine or guanidinium substituent. A cCPP may contain two amino acids having a side chain containing a guanidine or guanidinium substituent. A cCPP may contain three amino acids having a side chain containing a guanidine or guanidinium substituent. A cCPP may contain four amino acids having a side chain containing a guanidine or guanidinium substituent. A cCPP may contain five amino acids having a side chain containing a guanidine or guanidinium substituent. A cCPP may contain six amino acids having a side chain containing a guanidine or guanidinium substituent.

[0063] The guanidine or guanidinium group may be an isostea of ​​guanidine or guanidinium. The guanidine or guanidinium substituent may be less basic than guanidine.

[0064] As used herein, guanidine substituents are [ka] Or it refers to its protonated form.

[0065] In several embodiments, a cCPP is provided comprising 6 to 10 amino acid residues, where (i) at least two amino acids have side chains comprising a guanidine group or its protonated form; (ii) at least one amino acid residue has no side chain or has a side chain comprising a guanidine substituent or its protonated form; and (iii) at least two amino acid residues independently have side chains comprising an aryl group or a heteroaryl group. In several embodiments, two amino acids independently have side chains comprising an aryl group or a heteroaryl group. In several embodiments, three amino acids independently have side chains comprising an aryl group or a heteroaryl group. In several embodiments, at least two amino acids have no side chain or have side chains comprising a guanidine substituent or its protonated form. As used herein, if a side chain is absent, the amino acid has two hydrogen atoms on the carbon atom(s) linking the amine and carboxylic acid (e.g., -CH2). In several embodiments, the amino acid without a side chain may be glycine.

[0066] In some embodiments, cCPP may comprise 2, 3, 4, 5, or 6 amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In some embodiments, cCPP may comprise 2 amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In some embodiments, cCPP may comprise 3 amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In some embodiments, cCPP may comprise 4 amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In some embodiments, cCPP may comprise 5 amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In several embodiments, cCPP may comprise six amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In several embodiments, cCPP may comprise two, three, four, or five amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In several embodiments, cCPP may comprise two, three, or four amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof. In several embodiments, cCPP may comprise two or three amino acid residues, each independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof.

[0067] In several embodiments, an amino acid residue independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be an L-amino acid. In several embodiments, an amino acid residue independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be a D-amino acid. In several embodiments, an amino acid residue independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be a mixture of L-amino acids and D-amino acids.

[0068] In several embodiments, each amino acid residue having a side chain containing a guanidine group or its protonated form may independently be arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid, or a protonated form thereof. In several embodiments, each amino acid residue having a side chain containing a guanidine group or its protonated form may independently be arginine or a protonated form thereof. In several embodiments, an amino acid residue having a side chain containing a guanidine substituent or its protonated form may independently be a citrulline residue.

[0069] In several embodiments, cCPP may contain residues of asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine. In several embodiments, cCPP may contain residues of asparagine. In several embodiments, cCPP may contain residues of glutamine.

[0070] Chirality While we do not wish to be bound by theory, it is thought that the chirality of amino acids in cCPP may affect cytosolic uptake efficiency. In several embodiments, cCPP may contain at least one D amino acid. In several embodiments, cCPP may contain 1 to 20 D amino acids. In several embodiments, cCPP may contain 1 to 15 D amino acids. In several embodiments, cCPP may contain 1 to 10 D amino acids. In several embodiments, cCPP may contain 1 to 8 D amino acids. In several embodiments, cCPP may contain 1, 2, 3, 4, 5, 6, 7, or 8 D amino acids. In several embodiments, cCPP may contain all D amino acids. In several embodiments, cCPP may contain at least one L amino acid. In several embodiments, cCPP may contain 1 to 20 L amino acids. In several embodiments, cCPP may contain 1 to 15 L amino acids. In several embodiments, cCPP may contain 1 to 10 L amino acids. In several embodiments, cCPP may contain 1 to 8 L amino acids. In several embodiments, cCPP may contain 1, 2, 3, 4, 5, 6, 7, or 8 L amino acids. In several embodiments, cCPP may contain all L amino acids. In several embodiments, cCPP may contain 2, 3, 4, 5, 6, 7, or 8 consecutive amino acids having alternating D and L chiralities. In several embodiments, cCPP may contain 3 consecutive amino acids having the same chirality. In several embodiments, cCPP may contain 2 consecutive amino acids having the same chirality. In several embodiments, at least two amino acids may have opposite chiralities. In several embodiments, at least two amino acids having opposite chiralities may be adjacent to each other. In several embodiments, at least three amino acids may have alternating stereochemistry with respect to each other. In several embodiments, at least three amino acids having alternating chiralities with respect to each other may be adjacent to each other. In some embodiments, at least four amino acids have alternating stereochemistry with respect to each other. In some embodiments, at least four amino acids having alternating chiralities with respect to each other may be adjacent to each other.In several embodiments, at least two amino acids may have the same chirality. In several embodiments, at least two amino acids having the same chirality may be adjacent to each other. In several embodiments, at least two amino acids may have the same chirality and at least two amino acids may have opposite chirality. In several embodiments, at least two amino acids having opposite chirality may be adjacent to at least two amino acids having the same chirality. Therefore, adjacent amino acids in a cCPP may have any of the following sequences: DL;LD;DLD, LDL, DLLD;LDDL;LDLLD;DLDDL;DLLDL; or LDDLD. In several embodiments, all amino acid residues forming a cCPP may be L-amino acids. In several embodiments, all amino acid residues forming a cCPP may be D-amino acids.

[0071] In some embodiments, one or more amino acid residues forming a cCPP may be achiral. In some embodiments, a cCPP may comprise three, four, or five amino acid motifs, where two amino acids having the same chirality may be separated by an achiral amino acid. A cCPP may comprise the following sequences: D / LXD / L;D / LXD / LX;D / LXD / LXD / L;DXD;DXDX;DXDXD;LXL;LXLX; or LXLXL, where D / L indicates that the amino acid may be a D amino acid or an L amino acid, and X is an achiral amino acid. The achiral amino acid may be glycine.

[0072] In several embodiments, an amino acid having a side chain containing a guanidine substituent or its protonated form may be adjacent to an amino acid having a side chain containing an aryl group or a heteroaryl group. In several embodiments, an amino acid having a side chain containing a guanidine substituent or its protonated form may be adjacent to at least one amino acid having a side chain containing guanidine or its protonated form. In several embodiments, an amino acid having a side chain containing guanidine or its protonated form may be adjacent to an amino acid having a side chain containing an aryl group or a heteroaryl group. In several embodiments, two amino acids having side chains containing a guanidine substituent or its protonated form may be adjacent to each other. In several embodiments, two amino acids having side chains containing guanidine or its protonated form are adjacent to each other. In several embodiments, cCPP may comprise at least two consecutive amino acids having side chains that may contain an aryl group or a heteroaryl group, and at least two non-adjacent amino acids having side chains containing a guanidine substituent or its protonated form. In several embodiments, cCPP comprises at least two consecutive amino acids having side chains containing an aryl group or a heteroaryl group, [ka] or may include at least two non-adjacent amino acids having side chains containing the protonated form thereof. In some embodiments, adjacent amino acids may have the same chirality. In some 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 of the sequences described in the preceding paragraph.

[0073] In several embodiments, at least two amino acids having a side chain containing a guanidine substituent or a protonated form thereof are alternating with at least two amino acids having a side chain containing a guanidine group or a protonated form thereof.

[0074] In several embodiments, cCPP has the structure of formula (1): [ka] or may include its protonated form, During the ceremony, R 1 , R 2 , R 3 、 R 4 , R 5 , R 6 , and R 7 These are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4.

[0075] The cCPP of formula (1) may have any configuration and / or amino acid side chains, but is not limited to PCT Publication WO2015 / 179691, titled “CELL PENETRATING PEPTIDES AND METHODS OF MAKING AND USING THEREOF,” filed on 21 May 2015; PCT Publication WO2021 / 127650, titled “COMPOSITIONS FOR DELIVERY OF ANTISENSE COMPOUNDS,” filed on 21 December 2020; PCT Publication WO2022 / 213118, titled “CYCLIC CELL PENETRATING PEPTIDES,” filed on 31 March 2022; and “COMPOSITIONS AND METHODS FOR MODULATING TISSUE DISTRIBUTION OF INTRACELLULAR This includes the information contained in PCT Publication WO2022 / 241408, entitled “THERAPEUTICS”, U.S. Patent Publication 2023 / 0312653, entitled “CYCLIC CELL PENETRATING PEPTIDES,” filed March 30, 2023, and U.S. Patent No. 11,225,506, entitled “CELL PENETRATING PEPTIDES AND METHODS OF MAKING AND USING THEREOF,” filed April 20, 2020, the disclosures of which are incorporated herein by reference in their entirety.

[0076] In several embodiments, cCPP is represented by formula (1) or its protonated form, where, R 1 , R 2 , and R 3 Each of these is independently either H, or an aryl side chain or heteroaryl side chain of an amino acid. R 1 , R 2 , and R 3 At least two of these are aryl or heteroaryl side chains of amino acids. R 4 , R5 , R 6 , R 7 These are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4.

[0077] In several embodiments, cCPP is represented by formula (1) or its protonated form, where, R 1 , R 2 , and R 3 Each of these is independently either H, or an aryl side chain or heteroaryl side chain of an amino acid. R 1 , R 2 , and R 3 At least two of these are aryl or heteroaryl side chains of amino acids. R 4 , R 5 , R 6 , R 7 These are independently H or amino acid side chains. R 4 , R 5 , R 6 , R 7 At least two of these are independently arginine side chains, AA SC These are amino acid side chains, q is 1, 2, 3, or 4.

[0078] In the embodiment of formula (1), R 1 , R 2 , and R 3 Each of these is independently either H or a side chain containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least two of these are phenylalanine side chains, R 4 , R 5 , R 6 , and R7 is, independently, H or an amino acid side chain, AA SC is an amino acid side chain, q is 1, 2, 3, or 4.

[0079] In an embodiment of Formula (1), R 1 , R 2 , and R 3 one of is H, R 1 , R 2 , and R 3 two of are CH2Ph, and Ph is a phenyl group, R 4 , R 5 , R 6 , and R 7 are, independently, H or an amino acid side chain.

[0080] In an embodiment of Formula (1), R 1 , R 2 , and R 3 one of is H, R 1 , R 2 , and R 3 two of are CH2Ph, R 4 , R 5 , R 6 , and R 7 are, independently, H or the amino acid side chain of arginine.

[0081] In an embodiment of Formula (1), R 1 , R 2 , and R 3 are -CH2Ph, R 4 , R 5 , R 6 , and R 7 are, independently, H or an amino acid side chain.

[0082] In an embodiment of Formula (1), R 1, R 2 , and R 3 Each of these is a side chain containing an aryl group or a heteroaryl group, R 1 , R 2 , and R 3 At least two of these are phenylalanine side chains, R 4 , R 5 , R 6 , and R 7 These are independently H or amino acid side chains. q is 1, 2, 3, or 4.

[0083] In the embodiment of formula (1), R 1 , R 2 , and R 3 These are, independently, H or an amino acid side chain. R 1 , R 2 , and R 3 At least one of these is an aryl side chain or heteroaryl side chain of an amino acid. R 1 , R 2 , and R 3 At least one of them is an arginine side chain, R 4 , R 5 , R 6 , and R 7 These are independently H or amino acid side chains. q is 1, 2, 3, or 4.

[0084] In the embodiment of formula (1), R 1 , R 2 , and R 3 These are, independently, H or an amino acid side chain. R 1 , R 2 , and R 3 At least two of these are arginine side chains, R 4 , R 5 , R 6, and R 7 These are independently H or amino acid side chains. q is 1, 2, 3, or 4.

[0085] In the embodiment of formula (I), R 1 , R 2 , and R 3 Each of these is a side chain containing an aryl group or a 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 These are independently H or amino acid side chains. q is 1, 2, 3, or 4.

[0086] In the embodiment of formula (I), R 1 , R 2 , and R 3 Each of these is a side chain containing an aryl group or a heteroaryl group, R 1 , R 2 , and R 3 At least two of these are side chains of naphthylalanine, R 4 , R 5 , R 6 , and R 7 These are independently H or amino acid side chains. q is 1, 2, 3, or 4.

[0087] In the embodiment of formula (I), R 1 , R 2 , and R 3 Each of these is a side chain containing an aryl group or a heteroaryl group, R 1 , R 2 , and R 3At least two of these are phenylalanine side chains, 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 These are independently H or amino acid side chains. q is 1, 2, 3, or 4.

[0088] AAsc can be a side chain or terminal 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 some embodiments, AA SC teeth, [ka] This is possible, and in the formula, t can be an integer from 0 to 5. AA SC teeth, [ka] This is possible, where t can be 0 or an integer from 1 to 5. In some embodiments, t can be 1 to 5. In some embodiments, t is 2 or 3. In some embodiments, t can be 2. In some embodiments, t can be 3. In some embodiments, AA SC It can be conjugated to a linker. In several embodiments, AAsc is a side chain of a glutamine residue. In several embodiments, AAsc is a side chain of a glutamic acid residue. In several embodiments, AA SC If it is a glutamine side chain, the carboxamide nitrogen of the glutamine side chain is -(CH2) y’ -Forms a bond with a group.

[0089] In some embodiments, cCPP is represented by formula (1), where R4 , R 5 , R 6 , R 7 At least one of these is independently an uncharged, non-aromatic side chain of an amino acid. In some embodiments, R 4 , R 5 , R 6 , and R 7 At least one of these is independently H, or a side chain of serine, histidine, or citrulline. In some embodiments, R 4 , R 5 , R 6 , and R 7 One of these is independently H, or a side chain of serine, histidine, or citrulline. In some embodiments, R 4 , R 5 , R 6 , and R 7 At least two of these are independently H or a side chain of serine, histidine, or citrulline. In some embodiments, R 4 , R 5 , R 6 , and R 7 Two of these are independently either H or a side chain of serine, histidine, or citrulline.

[0090] In several embodiments, an EEV is provided comprising a cCPP having 6 to 10 amino acids, where at least two of the amino acids in the cCPP are charged amino acids, at least two of the amino acids in the cCPP are aromatic hydrophobic amino acids, and at least two of the amino acids in the cCPP are uncharged non-aromatic amino acids. In several embodiments, at least two of the charged amino acids in the cCPP are arginine. In several embodiments, two of the charged amino acids in the cCPP are arginine. In several embodiments, at least two of the aromatic hydrophobic amino acids in the cCPP are independently phenylalanine, 2-naphthylalanine, or a combination thereof. In several embodiments, two or three of the aromatic hydrophobic amino acids in the cCPP are independently phenylalanine, 2-naphthylalanine, or a combination thereof. In several embodiments, at least two of the uncharged non-aromatic amino acids in the cyclic peptide are citrulline, histidine, serine, glycine, or a combination thereof. In some embodiments, the two amino acids of cCPP are citrulline, histidine, serine, glycine, or a combination thereof. In some embodiments, cCPP has 6 to 10 amino acids, where two amino acids of cCPP are arginine, two or three amino acids are aromatic hydrophobic amino acids independently selected from phenylalanine, 2-naphthylalanine, and β-homophenylalanine, and two amino acids are uncharged non-aromatic amino acids independently selected from citrulline, serine, histidine, and glycine.

[0091] In some embodiments, cCPP is represented by formula (1), where R 4 , R 5 , R 6 , and R 7 At least two of these are positively charged side chains of amino acid residues. In several embodiments, R 4 , R 5 , R 6 , and R 7 Two of these are the positively charged side chains of amino acid residues. In several embodiments, R 4 , R 5 , R 6, and R 7 At least three of these are positively charged side chains of amino acid residues. In several embodiments, R 4 , R 5 , R 6 , R 7 Three of these are the positively charged side chains of amino acid residues. In several embodiments, R 4 , R 5 , R 6 , and R 7 This is the positively charged side chain of an amino acid residue.

[0092] cCPP has the structure of equation (2): [ka] or may include its protonated form, During the ceremony, R 1 , R 2 , and R 3 Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least two of these are aryl or heteroaryl side chains of amino acids. R 4 and R 6 These are independently H or amino acid side chains. AA SC These are amino acid side chains, m' is an integer between 0 and 3. m'' is an integer between 0 and 3. q is 1, 2, 3, or 4.

[0093] In some embodiments, cCPP is represented by formula (1) or (2), where R 1 , R 2 , and R 3 Each of these can independently be an amino acid side chain containing H, an alkylene-aryl group, an alkylene-heteroaryl group, a guanidine group, a guanidine substituent, or a protonated form thereof.1 , R 2 , and R 3 These are H and -C, respectively, independently. 1-3 Alkylene-aryl, -C 1-3 It may be an alkylene heteroaryl group or an amino acid side chain containing a guanidine group or its protonated form. In several embodiments, cCPP is represented by formula (1) or (2), where R 1 , R 2 , and R 3 Each of these can independently be H, -alkylene-aryl, or -alkylene-heteroaryl. 1 , R 2 , and R 3 These are H and -C, respectively, independently. 1-3 Alkylene-aryl, or -C 1-3 It may be an alkylene-heteroaryl. 1 , R 2 , and R 3 Each of these can independently be H or an alkylene-aryl. 1 , R 2 , and R 3 These are, independently, H or -C 1-3 It can be alkylene-aryl. 1-3 Alkylene can be methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl can be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl can be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. The amino acid side chain containing a guanidine group can be arginine. R 1 , R 2 , and R 3 These are H and -C, respectively, independently. 1-3 Alkylene-Ph or -C 1-3 It may be alkylene-naphthyl. 1 , R 2 , and R 3These can each be independently H, -CH2Ph, or -CH2-naphthyl. 1 , R 2 , and R 3 These can each be independently H or -CH2Ph. 1 , R 2 , and R 3 One of them could be arginine.

[0094] In some embodiments, cCPP is represented by formula (1) or (2), where R 1 , R 2 , and R 3 Each of these can independently be a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butylphenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthuryl)-alanine, or arginine. 1 , R 2 , and R 3 These can each be independently a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, arginine, or tryptophan.

[0095] In some embodiments, cCPP is represented by formula (1) or (2), where R 1 This could be a tyrosine side chain. 1 This could be a side chain of phenylalanine. 1 This could be a side chain of 1-naphthylalanine. 1 This could be a side chain of 2-naphthylalanine. 1 This could be a side chain of tryptophan. 1 This could be a side chain of 3-benzothienylalanine. 1This could be a side chain of 4-phenylphenylalanine. 1 This could be a side chain of 3,4-difluorophenylalanine. 1 This could be a side chain of 4-trifluoromethylphenylalanine. 1 This could be a side chain of 2,3,4,5,6-pentafluorophenylalanine. 1 This could be a side chain of homophenylalanine. 1 This could be a side chain of β-homophenylalanine. 1 This could be a side chain of 4-tert-butyl-phenylalanine. 1 This could be a side chain of 4-pyridinylalanine. 1 This could be a side chain of 3-pyridinylalanine. 1 This could be a side chain of 4-methylphenylalanine. 1 This could be a side chain of 4-fluorophenylalanine. 1 This could be a side chain of 4-chlorophenylalanine. 1 This could be a side chain of 3-(9-anthuryl)-alanine. 1 This can be H. 1 This could be a side chain of arginine.

[0096] In some embodiments, cCPP is represented by formula (1) or (2), where R 2 This could be a tyrosine side chain. 2 This could be a side chain of phenylalanine. 2 This could be a side chain of 1-naphthylalanine. 2 This could be a side chain of 2-naphthylalanine. 2 This could be a side chain of tryptophan. 2 This could be a side chain of 3-benzothienylalanine. 2 This could be a side chain of 4-phenylphenylalanine. 2 This could be a side chain of 3,4-difluorophenylalanine. 2 This could be a side chain of 4-trifluoromethylphenylalanine. 2This could be a side chain of 2,3,4,5,6-pentafluorophenylalanine. 2 This could be a side chain of homophenylalanine. 2 This could be a side chain of β-homophenylalanine. 2 This could be a side chain of 4-tert-butyl-phenylalanine. 2 This could be a side chain of 4-pyridinylalanine. 2 This could be a side chain of 3-pyridinylalanine. 2 This could be a side chain of 4-methylphenylalanine. 2 This could be a side chain of 4-fluorophenylalanine. 2 This could be a side chain of 4-chlorophenylalanine. 2 This could be a side chain of 3-(9-anthuryl)-alanine. 2 This can be H. 2 This could be a side chain of arginine.

[0097] In some embodiments, cCPP is represented by formula (1) or (2), where R 3 This could be a tyrosine side chain. 3 This could be a side chain of phenylalanine. 3 This could be a side chain of 1-naphthylalanine. 3 This could be a side chain of 2-naphthylalanine. 3 This could be a side chain of tryptophan. 3 This could be a side chain of 3-benzothienylalanine. 3 This could be a side chain of 4-phenylphenylalanine. 3 This could be a side chain of 3,4-difluorophenylalanine. 3 This could be a side chain of 4-trifluoromethylphenylalanine. 3 This could be a side chain of 2,3,4,5,6-pentafluorophenylalanine. 3 This could be a side chain of homophenylalanine. 3 This could be a side chain of β-homophenylalanine. 3 This could be a side chain of 4-tert-butyl-phenylalanine.3 This could be a side chain of 4-pyridinylalanine. 3 This could be a side chain of 3-pyridinylalanine. 3 This could be a side chain of 4-methylphenylalanine. 3 This could be a side chain of 4-fluorophenylalanine. 3 This could be a side chain of 4-chlorophenylalanine. 3 This could be a side chain of 3-(9-anthuryl)-alanine. 3 This can be H. 3 This could be a side chain of arginine.

[0098] In some embodiments, cCPP is represented by formula (1) or (2), where R 4 R may be H, or it may be a side chain of arginine, citrulline, serine, or histidine. 4 R can be a side chain of H or arginine. 4 This can be H. 4 This could be a side chain of arginine. 4 This could be a side chain of citrulline. 4 This could be a serine side chain. 4 This could be a histidine side chain. 4 This could be a side chain of valine. 4 This could be a side chain of leucine.

[0099] In some embodiments, cCPP is represented by formula (1), where R 5 R may be H, or it may be a side chain of arginine, citrulline, serine, or histidine. 5 R can be a side chain of H or arginine. 5 This can be H. 5 This could be a side chain of arginine. 5 This could be a side chain of citrulline. 5 This could be a serine side chain. 5 This could be a histidine side chain.

[0100] In some embodiments, cCPP is represented by formula (1) or (2), where R 6 R may be H, or it may be a side chain of arginine, citrulline, serine, or histidine. 6 R can be a side chain of H or arginine. 6 This can be H. 6 This could be a side chain of arginine. 6 This could be a side chain of citrulline. 6 This could be a serine side chain. 6 This could be a histidine side chain. 6 This could be a side chain of valine. 6 This could be a side chain of leucine.

[0101] In some embodiments, cCPP is represented by formula (2), where R 7 R may be H, or it may be a side chain of arginine, citrulline, serine, or histidine. 7 R can be a side chain of H or arginine. 7 This can be H. 7 This could be a side chain of arginine. 7 This could be a side chain of citrulline. 7 This could be a serine side chain. 7 This could be a histidine side chain.

[0102] In some embodiments, cCPP is represented by formula (1) or (2), where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 One, two, or three of these 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. 1 , R 2 , R 3, R 4 , R 5 , R 6 , and R 7 One of them could be H. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 Two of them could be H. 1 , R 2 , R 3 , R 5 , R 6 , and R 7 Three of them could be H. 1 , R 2 , and R 3 One of them could be H. 4 , R 5 , R 6 , and R 7 At least one of them can be H. 4 , R 5 , R 6 , and R 7 One of them could be H. 4 , R 5 , R 6 , and R 7 Two of them could be H. 4 , R 5 , R 6 , and R 7 Three of them could be H. 4 , R 5 , R 6 , and R 7 Four of them could be H.

[0103] In some embodiments, cCPP is represented by formula (1) or (2), where R 4 , R 5 , R 6 , and R 7 At least one of these may be H, or may be a side chain of arginine, citrulline, serine, or histidine. 4 , R 5 , R 6 , and R 7At least one of them is H. 4 , R 5 , R 6 , and R 7 At least one of these may be an arginine side chain. 4 , R 5 , R 6 , and R 7 At least one of these may be a citrulline side chain. 4 , R 5 , R 6 , and R 7 At least one of these may be a serine side chain. 4 , R 5 , R 6 , and R 7 At least one of these may be a histidine side chain. 4 , R 5 , R 6 , and R 7 One of these may be H, or it may be a side chain of arginine, citrulline, serine, or histidine. 4 , R 5 , R 6 , and R 7 One of them is H. 4 , R 5 , R 6 , and R 7 One of them could be the side chain of arginine. 4 , R 5 , R 6 , and R 7 One of them could be the side chain of citrulline. 4 , R 5 , R 6 , and R 7 One of them could be a serine side chain. 4 , R 5 , R 6 , and R 7 One of these could be a histidine side chain.

[0104] In some embodiments, cCPP is represented by formula (1) or (2), where R 4 , R 5 , R 6, and R 7 Two of these may be H, or they may be side chains of arginine, citrulline, serine, or histidine. 4 , R 5 , R 6 , and R 7 Two of them could be H. 4 , R 5 , R 6 , and R 7 Two of these could be arginine side chains. 4 , R 5 , R 6 , and R 7 Two of these could be citrulline side chains. 4 , R 5 , R 6 , and R 7 Two of these could be serine side chains. 4 , R 5 , R 6 , and R 7 Two of these could be histidine side chains.

[0105] In some embodiments, cCPP is represented by formula (1) or (2), where R 4 , R 5 , R 6 , and R 7 Three of these may be H, or may be the side chain of arginine, citrulline, serine, or histidine. 4 , R 5 , R 6 , and R 7 Three of them could be H. 4 , R 5 , R 6 , and R 7 Three of these could be arginine side chains. 4 , R 5 , R 6 , and R 7 Three of these could be citrulline side chains. 4 , R 5 , R 6 , and R 7 Three of these could be serine side chains. 4, R 5 , R 6 , and R 7 Three of these could be histidine side chains.

[0106] In some embodiments, cCPP is represented by formula (1) or (2), where AA SC This can be a side chain of asparagine, glutamine, or homoglutamine residues. AA SC This can be a side chain of a glutamine residue. In several embodiments, AA SC teeth, [ka] This is possible, and in the formula, t can be an integer from 0 to 5. AA SC teeth, [ka] This can be the case, where t can be 0 or an integer from 1 to 5. In some embodiments, t can be from 1 to 5. In some embodiments, t is 2 or 3. In some embodiments, t can be 2. In some embodiments, t can be 3. In some embodiments, AAsc is a side chain of a glutamine residue. In some embodiments, AAsc is a side chain of a glutamic acid residue. In some embodiments, AA SC If it is a glutamine side chain, the carboxamide nitrogen of the glutamine side chain is -(CH2) y’ -Forms a bond with a group.

[0107] In several embodiments, cCPP is represented by formula (1) or (2), where q may be 1, 2, or 3. q may be 1 or 2. q may be 1. q may be 2. q may be 3. q may be 4.

[0108] In several embodiments, cCPP is represented by 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.

[0109] In several embodiments, cCPP is represented by 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.

[0110] In several embodiments, cCPP comprises the structure of formula (2) or a protonated form thereof, where, R 1 , R 2 , and R 3 One of them is H, R 1 , R 2 , and R 3 Two of them are -CH2Ph, R 4 and R 6 These are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4. m' is an integer between 0 and 3. m'' is an integer between 0 and 3.

[0111] In several embodiments, cCPP comprises the structure of formula (2) or a protonated form thereof, where, R 1 , R 2 , and R 3 It is -CH2Ph, R 4 and R 6 These are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4. m' is an integer between 0 and 3. m'' is an integer between 0 and 3.

[0112] In several embodiments, cCPP comprises the structure of formula (2) or a protonated form thereof, where, R 1, R 2 , and R 3 These are, independently, side chains of amino acids containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least one of them is a side chain of naphthylalanine, AA SC These are amino acid side chains, R 4 and R 6 It is independently either H, or an amino acid side chain of arginine, serine, histidine, or citrulline. q is 1, 2, 3, or 4. m' is an integer between 0 and 3. m'' is an integer between 0 and 3.

[0113] In some embodiments, cCPP comprises the structure of formula (2) or its protonated form, R 4 and R 6 Independently, is either H or a side chain of arginine, histidine, serine, or citrulline. In some embodiments, R 4 and R 6 R is independently either H or a side chain of arginine, histidine, or serine. In some embodiments, R 4 H is H. In some embodiments, R 4 R is the side chain of arginine. In some embodiments, R 4 R is a side chain of histidine. In some embodiments, R 4 R is a serine side chain. In some embodiments, R 4 R is the side chain of citrulline. In some embodiments, R 6 H is H. In some embodiments, R 6 R is the side chain of arginine. In some embodiments, R 6 R is a side chain of histidine. In some embodiments, R 6 R is a serine side chain. In some embodiments, R 6 This is the side chain of citrulline.

[0114] The cCPP in equation (2) has the structure of equation (2-a) or equation (II-b): [ka] or may include its protonated form, where AA SC , R 1 , R 2 , R 3 , R 4 , R 6 m', and m'' are as defined herein with respect to formula (2).

[0115] The cCPP in equation (2) has the structure of equations (2-a1), (2-b1), (2-a2), (2-b2), (2-a3), or (2-b3): [ka] [ka] or may include its protonated form, where AA SC m', and m'' are as defined herein with respect to formula (2).

[0116] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: FGFGRGR;GfFGrGr;FfFGRGR;FGFGRRR; or FGFRRRR.

[0117] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: FGFGHGH or FGFSHSH.

[0118] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: FfFSRSR or FGFSRSR.

[0119] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: Ff-Nal-RrRr;Ff-Nal-GrGr;Ff-Nal-GRGR;Ff-Nal-HrHr; or Ff-Nal-SrSr.

[0120] In some embodiments, 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.

[0121] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: FGFGRGRQ;GfFGrGrQ;FfFGRGRQ;FGFGRRRQ; or FGFRRRRQ.

[0122] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: FGFGHGHQ or FGFSHSHQ.

[0123] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: FfFSRSRQ or FGRSRSRQ.

[0124] In some embodiments, the cCPP of formula (1) or (2) may include having one of the following sequences: Ff-Nal-RrRrQ;Ff-Nal-GrGrQ;Ff-Nal-GRGRQ;Ff-Nal-HrHrQ; or Ff-Nal-SrSrQ.

[0125] In some embodiments, the cCPP of formula (1) or (2) may include 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. [Table 2]

[0126] In several embodiments, the cCPP in formula (1) or (2) may be selected from Ff-Nal-GrGrQ;FfFGRGRQ;FGFGRGRQ;GfFGrGrQ;FfFGRGRQ;FGFGRRRQ; and FGFRRRRQ.

[0127] In several embodiments, the cCPP of formula (1) or (2) may be selected from fNalRrRrQ, Ff-Nal-Cit-r-Cit-rQ, and Ff-Nal-GrGrQ.

[0128] In some embodiments, the cCPP of formula (1) or (2) has an array of FfFGRGRQ.

[0129] In some embodiments, the cCPP of formula (1) or (2) has an array of FGFGRGRQ or GfFGrGrQ.

[0130] In several embodiments, cCPP in formula (1) or (2) may be FAFARARQ.

[0131] In several embodiments, cCPP in formula (1) or (2) may be Ff-Nal-GrGrQ.

[0132] In several embodiments, the cCPP in formula (1) or (2) may be selected from FfFGRGRQ, FfFRRRRQ, FfFRrRrQ, fffrrrrQ, or FfFRrRrQ.

[0133] In several embodiments, the cCPP in formula (1) or (2) may be FG-Nal-GRGRQ.

[0134] In several embodiments, the cCPP of formula (1) or (2) may be selected from FGFGRGRQ, fGfGrGrQ, fGfGrGrQ, FGFGRGRQ, FGFGRQ, fGfrrrrQ, FGFRRRRQ, or FGFRRRRQ.

[0135] In some embodiments, the cCPP in formula (I) or (II) may be selected from FFFRRRRQ, FFFGRRRQ, FFFGRRQ; FFFRRGRQ, FFFRRRGQ, GFFRRRRQ, or FFFRRRRQ.

[0136] In some embodiments, cCPP in formula (I) or (II) may be FFFRRRRQ.

[0137] In some embodiments, cCPP in formula (I) or (II) may be FFFGRRRQ.

[0138] In some embodiments, cCPP in formula (I) or (II) may be FFFRGRRQ.

[0139] In some embodiments, cCPP in formula (I) or (II) may be FFFRRGRQ.

[0140] In some embodiments, cCPP in formula (I) or (II) may be FFFRRRGQ.

[0141] In some embodiments, cCPP in formula (I) or (II) may be GFFRRRRQ.

[0142] In some embodiments, cCPP in formula (I) or (II) may be FFFRRRRQ.

[0143] In several embodiments, the cCPP in formula (I) or (II) may be selected from Nal-G-Nal-GRGRQ;FGFKRKRQ;FGFRRRRQ;FfGFRRRRQ; or FGFRRGRRQ.

[0144] In some embodiments, cCPP in formula (I) or (II) may be Nal-G-Nal-GRGRQ.

[0145] In some embodiments, cCPP in formula (I) or (II) may be FGFKRKRQ.

[0146] In some embodiments, cCPP in formula (I) or (II) may be FGFRRRRQ.

[0147] In some embodiments, cCPP in formula (I) or (II) may be FfGFRRRRQ.

[0148] In some embodiments, cCPP in formula (I) or (II) may be FGFRRGRRQ.

[0149] In several embodiments, the cCPP in formula (I) or (II) may be selected from RGRGRGRQ;FFFGRGRQ;FGFRRRRQ;FGFRGRGQ;FRGRGRGQ;FRFGRGRQ;FRFRFRFQ;FGFLRLRQ; or FGFVRVRQ.

[0150] In some embodiments, cCPP in formula (I) or (II) may be RGRGRGRQ.

[0151] In some embodiments, cCPP in formula (I) or (II) may be FFFGRGRQ.

[0152] In some embodiments, cCPP in formula (I) or (II) may be FGFRRRRQ.

[0153] In some embodiments, cCPP in formula (I) or (II) may be FGFRGRGQ.

[0154] In some embodiments, cCPP in formula (I) or (II) may be FRGRGRGQ.

[0155] In some embodiments, cCPP in formula (I) or (II) may be FRFGRGRQ.

[0156] In some embodiments, cCPP in formula (I) or (II) may be FRFRFRFQ.

[0157] In some embodiments, cCPP in formula (I) or (II) may be FGFLRLRQ.

[0158] In some embodiments, the cCPP of formula (I) or (II) may be FGFVRVRQ. In some embodiments, the cCPP of formula (1) or (2) may be FGWRGRQ.

[0159] In some embodiments, cCPP in formula (1) or (2) may be fFRGRQ.

[0160] The cCPP in equation (2) has the structure of equation (II-c): [ka] or it may have a protonated form thereof, In the formula, R 4 , R 6 q, m', m'', and AAsc are as defined herein.

[0161] The cCPP in equation (2) has the structure of equation (2-d): [ka] or it may have a protonated form thereof, In the formula, R 4 , R 6 q, m', m'', and AAsc are as defined herein.

[0162] The cCPP in equation (2) has the structure of equation (2-e): [ka] or it may have a protonated form thereof, In the formula, R 2 , R 4 , R 6 m', m'', and AAsc are as defined herein. In some embodiments, R2 is H.

[0163] In several embodiments, cCPP is given by formula (3): [ka] It can be represented as, or in its protonated form, During the ceremony, R 1 , R 2 , and R 3 Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least two of these are aryl or heteroaryl side chains of amino acids. R 4 , R 5 , R 6 , and R 7 These are independently H or amino acid side chains. R 4 , R 5 , R 6 , and R 7 At least two of these are independently arginine side chains, AA SC These are amino acid side chains, n X is 0 or 1, q is 1, 2, 3, or 4.

[0164] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , or R 3 At least one of them is H. In some embodiments, the amino acid residue having a side chain containing an aryl group or a heteroaryl group is phenylalanine, homophenylalanine, or 2-naphthylalanine. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7At least two of these are, independently, amino acid residues having a side chain containing a charged group. In some embodiments, the amino acid residue having a side chain containing a charged group is arginine. In some embodiments, cCPP is represented by formula (3), where q is 1.

[0165] In several embodiments, cCPP is represented by formula (3), where n X is 1, and R 5 and R 7 is the side chain of arginine. In some embodiments, cCPP is represented by formula (3), where n X is 1, and the aryl group or heteroaryl group is phenylalanine, beta-homophenylalanine, or 2-naphthylalanine, R 4 , R 5 , R 6 and R 7 At least two of these are arginine side chains. In several embodiments, cCPP is represented by formula (3), where n X is 1, and R 4 , R 5 , R 6 and R 7 is the side chain of arginine. In some embodiments, cCPP is represented by formula (3), where n X is 1, and R 5 and R 7 R is the side chain of arginine. 4 and R 6 H is H.

[0166] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least one of these is a serine or histidine amino acid side chain. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7At least two of these are, independently, serine, citrulline, or histidine amino acid side chains. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least three of these are independently serine or histidine amino acid side chains. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least four of these are, independently, serine or histidine amino acid side chains.

[0167] In several embodiments of the cCPP of formula (3), R 1 , R 2 , and R 3 At least two of these are independently side chains of phenylalanine or 2-naphthylalanine. R 4 , R 5 , R 6 , or R 7 At least two of these are independently arginine side chains, R 4 , R 5 , R 6 , or R 7 At least two of these are independently H, or side chains of arginine, serine, citrulline, or histidine. AA SC These are amino acid side chains, n X is 0 or 1, q is 1.

[0168] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 Two of these are serine side chains. In several embodiments, cCPP is represented by formula (3), where R 4 , R5 , R 6 , or R 7 Two of these are histidine side chains. In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 Two of these are citrulline side chains. In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 Two of them are independently H.

[0169] In some embodiments, cCPP is represented by formula (3), where, R 1 , R 2 , and R 3 At least two of these are side chains of phenylalanine or naphthylalanine. R 4 , R 5 , R 6 , or R 7 At least two of these are arginine side chains, R 4 , R 5 , R 6 , or R 7 At least two of these are independently H or side chains of uncharged nonaryl amino acids 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 These are amino acid side chains, n X is 0 or 1, q is 1.

[0170] In some embodiments, cCPP is represented by formula (3), where, R 1 , R 2, and R 3 At least two of these are independently side chains of phenylalanine or naphthylalanine. R 4 , R 5 , R 6 , or R 7 At least two of these are arginine side chains, R 4 , R 5 , R 6 , or R 7 At least two of these are independently serine, citrulline, or histidine side chains. AA SC These are amino acid side chains, n X is 0 or 1, q is 1.

[0171] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , or R 3 At least one of them is H. In some embodiments, cCPP is represented by formula (3), where R 1 , R 2 , or R 3 At least one of these is a side chain of phenylalanine. In some embodiments, cCPP is represented by formula (3), where R 1 , R 2 , or R 3 At least two of these are side chains of phenylalanine. In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , or R 3 At least one of them is a side chain of 2-naphthylalanine.

[0172] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least two of these are, independently, serine, citrulline, or histidine side chains.

[0173] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least one of these is independently an uncharged, non-aryl side chain of an amino acid. In some embodiments, R 4 , R 5 , R 6 , or R 7 At least two of these are independently side chains of uncharged nonaryl 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 some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least two of these are, independently, side chains of uncharged nonaryl amino acids 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 some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least two of these are, independently, side chains of uncharged, non-aryl amino acids selected from histidine, citrulline, and serine.

[0174] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least one of them is independently H. In some embodiments, cCPP is represented by formula (3), where R 4 , R5 , R 6 , or R 7 Two of them are independently H.

[0175] In some embodiments, cCPP may be represented by formula (3), where, R 1 , R 2 , and R 3 Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R 1 , R 2 , and R 3 At least two of them are phenylalanine, R 4 , R 5 , R 6 , R 7 These are independently H or amino acid side chains. R 4 , R 5 , R 6 , R 7 Two of them are independently arginine side chains. AA SC These are amino acid side chains, n X is 0 or 1, q is 1, 2, 3, or 4.

[0176] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , or R 3 At least one of them is independently H. In some embodiments, cCPP is represented by formula (3), where R 1 , R 2 , and R 3At least one of these is an amino acid residue having a side chain containing an aryl group or a heteroaryl group. In some embodiments, the amino acid residue having a side chain containing an aryl group or a heteroaryl group is phenylalanine, β-homophenylalanine, or 2-naphthylalanine. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , and R 7 At least two of these are, independently, amino acid residues having a side chain containing a charged group. In some embodiments, the amino acid residue having a side chain containing a charged group is arginine. In some embodiments, cCPP is represented by formula (3), where q is 1.

[0177] In several embodiments, cCPP is represented by formula (3), where n X is 1, and R 5 and R 7 is the side chain of arginine. In some embodiments, cCPP is represented by formula (3), where n X is 1, and the aryl group or heteroaryl group is phenylalanine, β-homophenylalanine, or 2-naphthylalanine, R 4 , R 5 , R 6 and R 7 At least two of these are arginine side chains. In several embodiments, cCPP is represented by formula (3), where n X is 1, and R 4 , R 5 , R 6 and R 7 is the side chain of arginine. In some embodiments, cCPP is represented by formula (3), where n X is 1, and in the formula, R 5 and R 7 R is the side chain of arginine. 4 and R 6 is H. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6, or R 7 At least one of (if present) is H, or an amino acid side chain of serine, citrulline, or histidine. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , or R 7 At least two of (if present) are independently H or serine or histidine amino acid side chains. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least three of these are independently H, or the amino acid side chains of serine, citrulline, or histidine. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least four of these are independently either H or the amino acid side chains of serine, citrulline, or histidine.

[0178] In several embodiments of the cCPP in equation (3), R 1 , R 2 , and R 3 At least two of these are independently the side chains of phenylalanine, β-homophenylalanine, or 2-naphthylalanine, and R 4 , R 5 , R 6 , or R 7 At least two of these are independently arginine side chains, and R 4 , R 5 , R 6 , or R 7 At least two of these are independently H, or side chains of arginine, serine, citrulline, or histidine, and AA SC n is an amino acid side chain, X is 0 or 1, and q is 1. 1If is the side chain of β-homophenylalanine, then n X It is understood that this is 1.

[0179] In several embodiments of the cCPP in equation (3), R 1 , R 2 , and R 3 At least two of these are independently side chains of phenylalanine or 2-naphthylalanine, and R 4 and R 6 Each is independently either H, or a side chain of arginine, serine, citrulline, or histidine, and R 5 and R 7 is the side chain of arginine, AA SC n is an amino acid side chain, X is either 0 or 1, and q is 1.

[0180] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , or R 7 Two of (if present) are independently serine side chains. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , or R 7 Two of (if present) are independently histidine side chains. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , or R 7 Two of (if any) are independently H.

[0181] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , and R 3 At least two of these are independently phenylalanine beta-homophenylalanine, or naphthylalanine side chains, and R 4 , R5 , R 6 , or R 7 At least two of these are independently arginine side chains, and R 4 , R 5 , R 6 , or R 7 At least two of these are independently H or side chains of uncharged nonaryl amino acids 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, and AA SC n is an amino acid side chain, X is 0 or 1, and q is 1. 1 If is the side chain of β-homophenylalanine, then n X It is understood that this is 1.

[0182] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , and R 3 At least two of these are independently the side chains of phenylalanine, 2-naphthylalanine, or β-homophenylalanine, and R 4 and R 6 R is independently H, or a side chain of an uncharged nonaryl amino acid 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. 5 and R 7 is the side chain of arginine, AA SC n is an amino acid side chain, X is 0 or 1, and q is 1. 1 If is the side chain of β-homophenylalanine, then n X It is understood that this is 1.

[0183] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , and R 3 At least two of these are independently the side chains of phenylalanine, β-homophenylalanine, or 2-naphthylalanine, and R 4 , R 5 , R 6 , or R 7 At least two of these are independently arginine side chains, and R 4 , R 5 , R 6 , or R 7 At least two of these are independently serine, citrulline, or histidine side chains, and AA SC n is an amino acid side chain, X is 0 or 1, and q is 1. 1 If is the side chain of β-homophenylalanine, then n X It is understood that this is 1.

[0184] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , and R 3 At least two of these are independently side chains of phenylalanine, beta-homophenylalanine, or naphthylalanine, and R 4 and R 6 R is independently a side chain of serine, citrulline, or histidine. 5 and R 7 is the side chain of arginine, AA SC n is an amino acid side chain, X is 0 or 1, and q is 1. 1 If is the side chain of β-homophenylalanine, then n X It is understood that this is 1.

[0185] In several embodiments, cCPP is represented by formula (3), where R 1 , R 2 , and R 3at least one of is H. In some embodiments, cCPP is represented by Formula (3), wherein R 1 , R 2 , and R 3 at least one of is a side chain of phenylalanine. In some embodiments, CPP is represented by Formula (3), wherein R 1 , R 2 , and R 3 at least two of are side chains of phenylalanine. In some embodiments, CPP is represented by Formula (3), wherein R 1 , R 2 , and R 3 two of are side chains of phenylalanine. In some embodiments, CPP is represented by Formula (3), wherein R 1 , R 2 , and R 3 three of are side chains of phenylalanine. In some embodiments, cCPP is represented by General Formula (3), wherein R 1 , R 2 , and R 3 one of is a side chain of naphthylalanine.

[0186] In some embodiments, cCPP is represented by Formula (3), wherein R 4 , R 5 , R 6 , or R 7 at least two of are independently a side chain of serine or histidine. In some embodiments, cCPP is represented by General Formula (3), wherein R 4 and R 6 are independently H, or a side chain of serine, citrulline, or histidine. In some embodiments, cCPP is represented by Formula (3), wherein R 4 and R 6 are side chains of serine. In some embodiments, cCPP is represented by Formula (3), wherein R 4 and R 6 are side chains of histidine. In some embodiments, cCPP is represented by Formula (3), wherein R 4 and R 6is the side chain of citrulline. In some embodiments, cCPP is represented by formula (3), where R 4 and R 6 is H. In some embodiments, cCPP is represented by formula (3), where R 5 and R 7 This is the side chain of arginine.

[0187] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , R 7 At least one of (if present) is independently an uncharged, non-aryl side chain of an amino acid. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , R 7 At least two of (if present) are independently side chains of uncharged nonaryl 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 some embodiments, cCPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , R 7 At least two of (if present) are side chains of uncharged nonaryl amino acids 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 some embodiments, CPP is represented by formula (3), where R 4 , R 5 (if present), R 6 , R 7At least two of (if present) are side chains of uncharged non-aryl amino acids independently selected from histidine and serine. In some embodiments, cCPP is represented by formula (3), where R 5 and R 7 This is the side chain of arginine.

[0188] In several embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 At least one of them is independently H. In some embodiments, cCPP is represented by formula (3), where R 4 , R 5 , R 6 , or R 7 Two of these are independently H. In several embodiments, cCPP is represented by formula (3), where R 4 or R 6 At least one of them is independently H. In some embodiments, cCPP is represented by formula (3), where R 4 and R 6 is H. In some embodiments, cCPP is represented by formula (3), where R 5 and R 7 This is the side chain of arginine.

[0189] In several embodiments, an EEV is provided comprising a cCPP having 6 to 10 amino acids, where at least two of the amino acids in the cCPP are charged amino acids, at least two of the amino acids in the cCPP are aryl or heteroaryl hydrophobic amino acids, and at least two of the amino acids in the cCPP are uncharged nonaryl amino acids. In several embodiments, the at least two charged amino acids in the cCPP are arginine. In several embodiments, the at least two aryl or heteroaryl hydrophobic amino acids in the cCPP are phenylalanine, naphthylalanine (3-naphthi-2-yl-alanine), β-homophenylalanine, or a combination thereof. In several embodiments, the at least two uncharged nonaryl amino acids in the cCPP are glycine. In several embodiments, two of the uncharged amino acids are serine, citrulline, histidine, or a combination thereof.

[0190] In several embodiments, the cCPP of formula (3) may include 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 (bhF: β-homophenylalanine; hFf: homophenylalanine).

[0191] In several embodiments, the cCPP of formula (3) may include 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 (bhF: β-homophenylalanine; hFf: homophenylalanine).

[0192] In several embodiments, the cCPP of formula (3) may include 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 (bhF: beta-homophenylalanine; hFf: homophenylalanine).

[0193] In several embodiments, the cCPP of formula (3) may include one of the following sequences: hFf-Nal-GrGrQ;βhF-F-Nal-SRSRQ;βhF-F-Nal-GRGRQ;βhF-F-Nal-HRHRQ;βhF-f-Nal-GrGrQ;βhF-f-Nal-SRSRQ;βhF-f-Nal-SrSrQ; or βhFf-Nal-HrHrQ.

[0194] Linker An EEV may include one or more linker arms. A linker can link a cargo to a cCPP. A linker can link an EP to a cCPP. A linker can be attached to the side chains of amino acids in a cCPP, and a cargo can be attached to a suitable position on the linker.

[0195] The linker can be any suitable part that can conjugate cCPP to one or more further parts, e.g., an extracyclic peptide (EP) and / or a cargo. Before conjugation (e.g., before conjugation to cCPP and / or one or more further parts), the linker has two or more functional groups, each of which can independently form a covalent bond (e.g., a covalent bond to cCPP and / or one or more further parts). If the cargo is an oligonucleotide, the linker can be covalently attached to the 5' end of the cargo or the 3' end of the oligonucleotide cargo. The linker can be covalently attached to the 5' end of the oligonucleotide cargo. The linker can be covalently attached to the 3' end of the oligonucleotide cargo. If the cargo is a peptide, 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 (e.g., somewhere in the middle, rather than at the ends). The linker may be any suitable part that conjugates the cCPP described herein to a therapeutic moiety such as an oligonucleotide, peptide, or small molecule.

[0196] The linker can be covalently attached to the cargo at any suitable position on the cargo. The linker can be covalently attached to the 3' end or 5' end of an oligonucleotide cargo. The linker can be covalently attached to the N-terminus or C-terminus of a peptide cargo. The linker can be covalently attached to the backbone of an oligonucleotide or peptide cargo. The linker can be covalently attached to the cargo via a binding group M'.

[0197] The linker is the side chain of the amino acid on the cCPP (AA SCFor example, it can be covalently bonded to aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or to a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker can be bonded to the lysine side chain on the cCPP.

[0198] In some embodiments, the linker 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 represents a -(CH2CH2O)- group and may be repeated any number of times.

[0199] While we do not wish to be bound by theory, it is believed that linkers can affect the efficacy and tolerability of EEV-cargo conjugates (e.g., increase efficacy). In several embodiments, efficacy increases without decreasing tolerability. In several embodiments, one or more hydrophobic components (X) are added to the EEV linker. In several embodiments, the linker length can vary. The linker length can affect efficacy and tolerability. In some cases, as the linker length decreases, efficacy increases and tolerability decreases, and as the linker length increases, efficacy decreases and tolerability increases. In several embodiments, efficacy can be increased by increasing the hydrophobicity of the linker.

[0200] The hydrophobic component X can be added to one or more positions on the linker. For example, X may be present in the linker between CPP and EP; or in the linker between CPP and cargo. In some embodiments, X may be a binding group between CPP and EP. In some embodiments, X is not a binding group between CPP and EP. In some embodiments, X may be a binding group between CPP and cargo. In some embodiments, X is not a binding group between CPP and cargo. X may form part of the backbone of the linker between CPP and EP. X may form part of the backbone of the linker between CPP and cargo. X can be added to the linker. X can be added to the side chain of a lysine residue in the linker. X can be added to the linker between CPP and EP. X can be added to the linker between CPP and cargo.

[0201] In several embodiments, the hydrophobic component (X) in the linker may be aliphatic, alkene, alkyne, aromatic (e.g., carbocyclic or heteroaromatic), or a combination of aliphatic and aromatic.

[0202] X may be a D or L amino acid residue having a hydrophobic side chain. X may be a naturally occurring or unnatural amino acid residue having a hydrophobic side chain. X may be an amino acid residue having an aromatic side chain. X may be an amino acid residue having a heteroaromatic side chain. X may be selected from phenylalanine, 3-(4',4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or a combination thereof. X may be 2-naphthylalanine. X may be Naal. X may be d-Nal(nal). X may be 3-(4',4-biphenyl)-L-alanine. X may be Bip. X may be D-Bip(bip). X may be a C4-C8 alkyl hydrocarbon. X may be a C6 alkyl hydrocarbon.

[0203] In a plurality of embodiments, the hydrophobic component X comprises an optionally substituted alkyl group. The alkyl group may comprise a branched alkyl group. In a plurality of embodiments, the alkyl group comprises a double bond. In a plurality of embodiments, the alkyl group comprises an ethyl group or a propyl group. In an embodiment, the alkyl group is a butyl group, a pentyl group, or a hexyl group. In an 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) n CH2O-, -OCH2(CH2) n CH2S-, NH2(CH2) n CH2-, SH(CH2) n CH2-, or N3-C(O)CH2(CH2) n CH2- group (wherein "n" is 4 to 34, inclusive).

[0204] In several 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, and the like. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0205] In several embodiments, the 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 naturally occurring hydrophobic amino acids or unnaturally occurring hydrophobic amino acids. In several embodiments, the hydrophobic amino acid residues may comprise substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, or aralkyl side chains, where the alkyl, alkenyl, and alkynyl side chains comprise up to one heteroatom for every six carbon atoms. X may comprise a D or L amino acid residue having a hydrophobic side chain. X may comprise a naturally occurring or unnaturally occurring amino acid residue having a hydrophobic side chain. X may comprise an amino acid residue having an aromatic side chain. X may comprise an amino acid residue having a heteroaromatic side chain. The hydrophobic component X may comprise an amino acid residue having a hydrophobic side chain that can be selected from valine, proline, alanine, leucine, isoleucine, phenylalanine, cysteine, glycine, histidine, and methionine and tryptophan. The amino acid residue having a hydrophobic side chain may be selected from the group consisting of glycine, phenylglycine, alanine, valine, leucine, isoleucine, norleucine, phenylalanine, tryptophan, naphthylalanine, proline, and combinations thereof, where the aromatic side chain on phenylglycine, phenylalanine, tryptophan, or naphthylalanine is optionally substituted. The amino acid residue having a hydrophobic side chain may be naphthylalanine, lysine-naphthylalanine, biphenylalanine, or lysine-biphenylalanine. X may be an aliphatic hydrocarbon. X is C4-C 12It may be an aliphatic hydrocarbon. X may be a C4-C8 aliphatic hydrocarbon. X may be a C6 aliphatic hydrocarbon. X may be selected from phenylalanine, 3-(4',4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or a combination thereof. X may be an amino acid selected from tryptophan, tyrosine, isoleucine, leucine, histidine, phenylalanine, or a combination thereof.

[0206] X can be 2-naphthylalanine. X can be Naal. 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).

[0207] In several embodiments, the hydrophobic component X may include a modified amino acid subunit. For example, an amino-terminal or carboxy-terminal amino acid subunit may be modified. Such modification includes capping the amino-terminal or carboxy-terminal with a group that makes the amino acid subunit more hydrophobic. For example, the amino-terminal may be capped with an acyl group (e.g., acetyl, benzoyl, or stearoyl moiety). For example, the amino-terminal of a modified amino acid subunit may be shown as follows: [ka]

[0208] In some embodiments, X is lysine or SH(CH2) n CH2-, or N3-C(O)CH2(CH2) n It can be added to the linker's framework via CH2-.

[0209] In several embodiments, the size of the aromatic or heteroaromatic groups present in the hydrophobic component X may be selected to improve the cytosol delivery efficiency of the cargo. While we do not wish to be constrained by theory, it is believed that the presence of hydrophobic component X in the linker of an EEV can improve the cytosol delivery efficiency of the cargo compared to an EEV that is identical except for the absence of hydrophobic component X.

[0210] The linker may include hydrocarbon linkers.

[0211] The linker may contain cleavage sites. These cleavage sites may be disulfide or caspase cleavage sites (e.g., Val-Cit-p-aminobenzyloxycarbonyl, also known as Val-Cit-PABC).

[0212] The linker consists of (i) one or more D amino acids or L amino acids (each of which may be optionally substituted), and (ii) one or more -(U 1 -JU 2 )i'-subunit(in the formula, U 1 and U 2 Each of these is independently selected from alkylene in each case, and each J is independently selected from C, NU 3 ,-NU 3 C(O)-, S, and O, where U 3 i' can independently be selected from H, alkyl, alkenyl, alkynyl, carbocykryl, and heterocyclyl, each of which may be substituted by arbitrary choice, and i' is an integer from 1 to 50.

[0213] The linker consists of one or more D-amino acids or L-amino acids and / or -(OCH2CH2) i -(i is an integer from 0 to 60); or may include combinations 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) iThis can also be called polyethylene glycol (PEG).

[0214] The linker is one or more PEG components - (OCH2CH2) i - may be included, where i is an integer from 0 to 12. PEG components can be expressed as (PEG)x', (PEG)z', and / or (PEG)z''. The linker contains one or more hydrophobic components (X o ') and (X o It may contain ''). Hydrophobic components can be added to the side chains of amino acids in the linker. Hydrophobic component (X o ') can be added to the side chain of lysine in the linker (K(X o The linker may contain a bonding group (M).

[0215] The linker consists of (i) β-alanine residues and lysine residues; (ii)-(JU 1 ) i’ ; or (iii) a combination thereof. Each U 1 J can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can independently be C, NU 3 ,-NU 3 C(O)-, S, or O, where U 3 z'' is H, alkyl, alkenyl, alkynyl, carbocykyl, or heterocyclyl, each of which can be substituted by choice, and z'' can be an integer from 1 to 50. 1 can be alkylene, and each J can be O.

[0216] The linker is (i) a residue of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid or a combination thereof; and (ii)-(U 1- J) i’ or -(JU 1 ) i’ May include. Each U 1 J can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can independently be C, NU3 ,-NU 3 C(O)-, S, or O, where U 3 is H, alkyl, alkenyl, alkynyl, carbocykyl, or heterocyclyl, each of which can be substituted by any choice, and i' can be an integer from 1 to 50. 1 is an alkylene, and each J can be O. The linker may contain glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof.

[0217] The linker is also a disulfide [NH2-(CH2O) n -SS-(CH2O) n The cleavage site can be incorporated, including -COOH, or a caspase cleavage site (Val-Cit-PABC).

[0218] The linker may contain glycine or β-alanine residues.

[0219] The linker is divalent and can link cCPP to the cargo. The linker is divalent and can link cCPP to the extracyclic peptide (EP).

[0220] The linker is trivalent and can connect cCPP to cargo and EP.

[0221] The linker is divalent or trivalent C1-C 50It can be an alkylene, where 1 to 25 methylene groups can be optionally and independently replaced with -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl, or cycloalkenyl. The linker is divalent or trivalent C1-C 50 The resulting material is an alkylene, where 1 to 25 methylene groups can be optionally and independently replaced with -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.

[0222] The cargo can be bonded to the glutamic acid of the cyclic peptide, thereby converting the glutamic acid to glutamine. The linker (L) can couple the cargo to the glutamine / glutamic acid of the cyclic peptide. In several embodiments, the linker (L) is covalently bonded to the cargo skeleton.

[0223] The linker has the structure shown in equation (A'): [ka] It may be a trivalent linker containing During the ceremony, ** is a binding site to a linear extracyclic peptide (EP) as defined herein, * indicates a binding site to a cell-permeable peptide (CPP) as defined herein. L 1 and L 2 It is independently a linker arm, ^ indicates L-stereochemistry or D-stereochemistry. y' is an integer between 1 and 5. M includes the reactive handle.

[0224] In some embodiments, L 1 and L 2 Each of these independently comprises either a hydrocarbon component, a polyethylene glycol (PEG) component, a hydrophobic component (X), an amino acid (AA) component comprising one or more amino acid residues, or a combination thereof. In some embodiments, L 1 It contains polyethylene glycol (PEG) x' component, where x' is an integer from 1 to 12. In some embodiments, L 2 It contains polyethylene glycol (PEG) z' component, where z' is an integer from 1 to 12. In some embodiments, L 2 This is the hydrophobic component (X o Includes ').

[0225] In some embodiments, L 1 , L 2 Alternatively, cCPP contains one or more hydrophobic components (X).

[0226] In some embodiments, L 1 It contains PEG components, L 2 This includes a PEG component, a hydrophobic component (X), or a combination thereof.

[0227] In some embodiments, L 1 is absent. In some embodiments, when x' is 0, L 1 L is absent. In some embodiments, L 1 In the absence of lysine, the lysine linked to the cyclic peptide is acylated (Ac). In several embodiments, L 1 L is absent. 2 This consists of the PEG component (PEG)z' and the hydrophobic component (X o '), or combinations thereof.

[0228] In some embodiments, L 2 (PEG)z', hydrophobic component (X o '), or a combination thereof. In some embodiments, L 2It contains the PEG component (PEG)z'. In some embodiments, L 2 This is the hydrophobic component (X o Includes '). In some embodiments, L 2 (PEG)z' and the hydrophobic component (X o ') is included. In some embodiments, the hydrophobic component (X o ') can be added to the side chain of a lysine amino acid residue.

[0229] M comprises a functional group (referred to herein as a reactive handle) that can react with a corresponding functional group on the cargo to form an EEV-cargo conjugate or a linker-cargo conjugate. After conjugation, the EEV-cargo or linker-cargo conjugate contains a bonding group M' which is the reaction product of the two functional groups.

[0230] In some embodiments, M can be -OH (for example, as part of the C-terminus of an amino acid). In some embodiments, M can be -OH, [ka] The expression includes the following, where y'' is an integer between 1 and 4. y'' can be 1. y'' can be 2. y'' can be 3. y'' can be 4.

[0231] In some embodiments, linker formula (A') is formula (B'), (C'), or (D'): [ka] It may have an expression selected from, x' is an integer between 1 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. X o ' is a hydrophobic component, K #This is a D-lysine residue or an L-lysine residue.

[0232] With the exception of glycine (G), which has H as a side chain, each amino acid has an asymmetric or chiral α-carbon atom and exists in two enantiomer forms, denoted as "D-" and "L-". The "L-" isomer is the most common in naturally occurring proteins. While we do not wish to be bound by theory, it is thought that the chirality of amino acids in cell-permeable peptides (CPPs) or endosomal escape vehicles (EEVs) may affect cytosolic delivery efficiency and toxicity. This specification discloses EEVs in which the stereochemistry of amino acids within the EEV peptide sequence is modified to affect cytosolic delivery efficiency and / or reduce toxicity.

[0233] In several embodiments, the linker comprises a structure selected from formula (B'), (C'), or (D'), where EP comprises all D-amino acids, cCPP comprises a D-amino acid, an achiral amino acid, and AAsc, where AAsc is an amino acid side chain, and AA SC This conjugates cCPP to the linker.

[0234] In multiple embodiments, cCPP comprises at least two D-amino acid residues having hydrophobic side chains. In multiple embodiments, cCPP comprises two D-amino acid residues having hydrophobic side chains. In multiple embodiments, cCPP comprises three D-amino acid residues having hydrophobic side chains. In multiple embodiments, cCPP comprises at least two D-arginine amino acid residues. In multiple embodiments, cCPP comprises two D-arginine amino acid residues. In multiple embodiments, cCPP comprises three D-arginine amino acid residues. In multiple embodiments, cCPP comprises four D-arginine amino acid residues. In multiple embodiments, cCPP comprises five D-arginine amino acid residues. In multiple embodiments, cCPP comprises six D-arginine amino acid residues. In multiple embodiments, cCPP comprises at least two D-phenylalanine residues. In multiple embodiments, cCPP comprises two D-phenylalanine residues. In multiple embodiments, cCPP comprises three D-phenylalanine residues. In multiple embodiments, cCPP comprises at least two glycine residues. In multiple embodiments, cCPP comprises two glycine residues. In multiple embodiments, cCPP comprises three glycine residues. In multiple embodiments, cCPP comprises four glycine residues. In multiple embodiments, cCPP comprises five glycine residues. In multiple embodiments, cCPP comprises six glycine residues. In multiple embodiments, cCPP comprises at least two D-amino acid residues and at least two D-arginine residues having hydrophobic side chains. In multiple embodiments, cCPP comprises at least two D-phenylalanine residues and at least two D-arginine residues. In multiple embodiments, AA SC This is the side chain of L-glutamine.

[0235] The linker is given by equation (B'): [ka] It can be expressed as, where **, *, x', j', ^, y', z', j'', and M are as defined herein.

[0236] The linker is given by equation (C'): [ka] It can be expressed as, in the formula, **, *, x', j', ^, y', z', j'', X o ', and M are as defined herein.

[0237] The linker is given by equation (D'): [ka] It can be expressed as, in the formula, **, *, x', j', ^, y', z', j'', X o ', K # , and M are as defined herein.

[0238] In some embodiments, M is -OH, [ka] The expression includes the following, where y'' is an integer between 1 and 4. y'' can be 1. y'' can be 2. y'' can be 3. y'' can be 4.

[0239] 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.

[0240] j' can be 0, 1, or 2. j' can be 1. j' can be 2. If x' is 0, then j' is understood to be 0.

[0241] ^ may indicate L-stereochemistry. ^ may indicate D-stereochemistry.

[0242] y' can be an integer between 1 and 5, for example, 1, 2, 3, 4, or 5, including all ranges and subranges between these. y' can be an integer between 2 and 5. y' can be an integer between 3 and 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.

[0243] 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.

[0244] j'' can be 0, 1, or 2. j'' can be 0. j'' can be 1. j'' can be 2. If z' is 0, then j'' is understood to be 0.

[0245] K # This indicates an L-lysine residue. # This indicates a D-lysine residue.

[0246] In some embodiments, ^ represents L-stereochemistry. In some embodiments, ^ represents D-stereochemistry.

[0247] In multiple embodiments, M' is -NH-, -C(O)-, -O-, [ka] The formula includes, where y'' is an integer from 1 to 4 and t' is an integer from 0 to 10. In some embodiments, M' is -C(O)-, [ka] This includes, where y'' is an integer from 1 to 4. In some embodiments, M' is [ka] The formula includes, where t' is an integer from 0 to 10. In the embodiment, M' is [ka] Includes.

[0248] AAsc can be a side chain or terminal 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 some embodiments, AAsc is a side chain of a glutamine residue. In some embodiments, AAsc is a side chain of a glutamic acid residue. In some embodiments, AA SC If it is a glutamine side chain, the carboxamide nitrogen of the glutamine side chain is -(CH2) y’ -Forms a bond with a group.

[0249] y'' can be an integer between 0 and 10, for example, 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.

[0250] X o ' can be an aliphatic hydrocarbon. o ' is C4-C 12 It can be an aliphatic hydrocarbon. o' can be a C4-C8 aliphatic hydrocarbon. o ' can be a C6 aliphatic hydrocarbon. o ' may be selected from phenylalanine, 3-(4',4-biphenyl)-L-alanine, tryptophan, tyrosine, valine, isoleucine, leucine, or histidine, or any combination thereof. o ' may be an amino acid residue selected from tryptophan, tyrosine, isoleucine, leucine, histidine, phenylalanine, or any combination thereof.

[0251] X o ' may be a D-amino acid residue or an L-amino acid residue having a hydrophobic side chain. o ' may be a naturally occurring or unnatural amino acid residue having a hydrophobic side chain. o ' can be an amino acid residue having an aromatic side chain. o ' may be an amino acid residue having a heteroaromatic side chain. o ' may 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. o ' may be 2-naphthylalanine (Nal or nal). o ' may be L-2-naphthylalanine (L-Nal or Nal). o ' may be D-2-naphthylalanine (d-nal or nal). o ' may be 3-(4',4-biphenyl)-L-alanine. o ' may be 3-(4',4-biphenyl)-L-alanine (L-Bip or Bip). o ' may be 3-(4',4-biphenyl)-D-alanine (d-bip or bip). o ' may be a C4-C8 alkyl or dialkyl hydrocarbon. o' may be a C6 alkyl or dialkyl hydrocarbon.

[0252] Endosome escape vehicle (EEV) An EEV comprising a cyclic cell-permeable peptide (cCPP) and one or more linkers is provided. In some embodiments, the EEV comprises an extracyclic peptide (EP). In some embodiments, the EP is absent.

[0253] EEV has the structure of equation (A): [ka] It may include, in the formula, EP is a linear extracyclic peptide, cCPP is a cell-permeable peptide, L 1 and L 2 It is independently a linker arm, ^ indicates L-stereochemistry or D-stereochemistry. y' is an integer between 1 and 5. M includes the reactive handle.

[0254] In some embodiments, the cCPP is transmitted via the AAsc of the cCPP to (CH2) y’ -Connected to the base. In some embodiments, the cCPP is connected to the AAsc of the cCPP via (CH2) y’ -It is connected to the base, and that connection is structure [ka] It is expressed as, where n is an integer from 1 to 5. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, AAsc is the side chain of a glutamine residue. In some embodiments, when n is 1, AAsc is the side chain of a glutamic acid residue. In some embodiments, AA SCIf it is a glutamine side chain, the carboxamide nitrogen of the glutamine side chain is -(CH2) y’ -Forms a bond with a group.

[0255] In some embodiments, the EEV of formula (A) is formula (B), (C), or (D): [ka] Includes a structure selected from, in the formula, EP is a linear extracyclic peptide, cCPP is a cyclic cell-permeable peptide, x' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. ^ indicates L-stereochemistry or D-stereochemistry. y' is an integer between 1 and 5. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. X o ' contains hydrophobic components, K # These are D-lysine residues or L-lysine residues, M includes the reactive handle.

[0256] EEV has the structure of equation (A): [ka] It may include, in the formula, EP is a linear extracyclic peptide, cCPP is a cell-permeable peptide, L 1 and L 2 It is independently a linker arm, ^ indicates D-stereochemistry. y' is an integer between 1 and 5. M includes a reactive handle, Here, EP contains all D-amino acids, cCPP contains D-amino acids, achiral amino acids and AAsc, AAsc is an amino acid side chain, and AAsc conjugates cCPP to a linker.

[0257] In several embodiments, the EEV of formula (A) comprises a structure selected from formulas (B), (C), or (D), where EP comprises all D-amino acids, cCPP comprises a D-amino acid, an achiral amino acid, and AAsc, where AAsc is an amino acid side chain, and AAsc conjugates cCPP to a linker.

[0258] X may be a D or L amino acid residue having a hydrophobic side chain. X may be a naturally occurring or unnatural amino acid residue having a hydrophobic side chain. X may be an amino acid residue having an aromatic side chain. X may be an amino acid residue having a heteroaromatic side chain. X may be 2-naphthylalanine. X may be Naal. X may be d-Nal(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 a combination thereof.

[0259] In some embodiments, L 1 and L 2 If present, each independently acts as a hydrocarbon linker (e.g., NR h H-(CH2)n-COOH), PEG components (e.g., NR h H-(CH2O) n -COOH, in the formula, R h The linker is a hydrophobic component (which is H, methyl, or ethyl), one or more amino acid residues, or a combination thereof.

[0260] In several embodiments, AAsc is a glutamine side chain. In several embodiments, AAsc is an L-glutamine side chain. In several embodiments, AAsc is a D-glutamine side chain.

[0261] In some embodiments, M is -OH or [ka] The expression includes , where y'' is an integer between 1 and 4.

[0262] In several embodiments, EEV is given by formula (B): [ka] The formula includes, where EP, cCPP, x', j', ^, y', z', j'', and M are as defined herein.

[0263] In some embodiments, the EEV is given by formula (C): [ka] The formula includes EP, cCPP, x', j', ^, y', z', j'', X o ', and M are as defined herein.

[0264] In several embodiments, the EEV is given by formula (D): [ka] The formula includes EP, cCPP, x', j', ^, y', z', j'', X o ', K # , and M are as defined herein.

[0265] In several embodiments, EEV is given by formula (A-2): [ka] It may include, in the formula, EP, L 1 , L 2M, y', ^, n, q, R1, R2, R3, R4, R5, R6, and R7 are as defined herein.

[0266] In some embodiments, the EEV of formula (A-2) may include the linker of formula (B'). In some embodiments, the EEV of formula (A-2) may include the linker of formula (C'). In some embodiments, the EEV of formula (A-2) may include the linker of formula (D').

[0267] In several embodiments, the EEV is given by formula (A-2a): [ka] It may include, in the formula, EP, L 1 , L 2 ,M,y',^,n,m',m'',R 1 , R 2 , R 3 , R 4 , and R 6 This is defined herein.

[0268] In some embodiments, the EEV of formula (A-2a) may include the linker of formula (B'). In some embodiments, the EEV of formula (A-2a) may include the linker of formula (C'). In some embodiments, the EEV of formula (A-2a) may include the linker of formula (D').

[0269] In several embodiments, the EEV may have the structure shown in formula (B), where x' is 0 and z' is 12. In several embodiments, the EEV may have the arrangement shown in Table 2a. [Table 3]

[0270] In several embodiments, the EEV may have the structure shown in formula (B), where x' is 2 and z' is 12. In several embodiments, the EEV may have the arrangement shown in Table 2b. [Table 4]

[0271] In several embodiments, the EEV may have the structure shown in formula (B), where x' is 2 and z' is 2. In several embodiments, the EEV may have the arrangement shown in Table 2c. [Table 5]

[0272] In several embodiments, the EEV may have the structure shown in formula (B), where x' is 2 and z' is 12. In several embodiments, the EEV may have the arrangement shown in Table 3a. [Table 6]

[0273] In several embodiments, the EEV may have the structure shown in formula (D), where x' is 2 and z' is 12. In several embodiments, the EEV may have the arrangement shown in Table 3b. [Table 7]

[0274] In several embodiments, the EEV may have the structure shown in formula (C), where x' is 2 and z' is 12. In several embodiments, the EEV may have the arrangement shown in Table 3c. [Table 8]

[0275] In multiple embodiments, the EEV can be selected from Table 4, where cCPP has the formula:FGFGRGRQ. [Table 9]

[0276] In several embodiments, the EEV can be selected from Table 5, where cCPP has the formula: GfFGrGrQ. [Table 10]

[0277] In multiple embodiments, the EEV construct can be selected from Table 6, where cCPP has the formula: FfFGRGRQ. [Table 11]

[0278] In several embodiments, the EEV can be selected from Table 7, where cCPP has the formula: Ff-Nal-GrGrQ. [Table 12]

[0279] In several embodiments, the EEV can be selected from Table 8A, where the linker contains a hydrophobic group. [Table 13]

[0280] In several embodiments, EEV can be selected from Table 8B, where cCPP includes citrulline (Cit=L-citrulline, cit=D-citrulline). [Table 14]

[0281] In the EEV provided herein, the cCPP can be coupled to the linker via AAsc. In some embodiments, the cCPP is coupled to the linker via AAsc-(CH2) y’ - is bonded to the linker via a group. In several embodiments, cCPP is bonded to the linker via AAsc-(CH2) y’- The linker is connected by the base, thereby the connection is structure [ka] It is represented as , where n is an integer from 1 to 5. In several embodiments, cCPP may be conjugated to the lysine component of the linker via AAsc. In several embodiments, when AAsc is glutamic acid, n is 1. In the EEVs shown in Tables 2a-2c and 3a-3c, cCPP is conjugated to the lysine component of the linker via AAsc, where AAsc is glutamic acid (E) and n before conjugation is 1. After conjugation to the linker, AAsc is represented as glutamine (Q).

[0282] In the provided EEV sequences, lysine may contain a protecting group (not shown). In several embodiments, the protecting group is a trifluoroacetyl (Tfa) group. In several embodiments, the protecting group is an acetyl (Ac) group. Other protecting groups may also be used, and it is understood that the protecting group can be removed after the EEV is conjugated to the cargo. In several embodiments, the EEV is deprotected after conjugation to the PMO.

[0283] Extracyclic peptide An extracyclic peptide (EP) may contain 2 to 10 amino acid residues, for example, 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 some embodiments, the EP contains 2 amino acid residues. In some embodiments, the EP contains 3 amino acid residues. In some embodiments, the EP contains 4 amino acid residues. In some embodiments, the EP contains 5 amino acid residues. In some embodiments, the EP contains 6 amino acid residues. In some embodiments, the EP contains 7 amino acid residues. In some embodiments, the EP contains 8 amino acid residues.

[0284] The amino acids in EP may have D-stereochemistry or L-stereochemistry. All amino acid residues in EP may be D-amino acids. All amino acid residues in EP may be L-amino acids. Amino acid residues in EP may be a combination of D-amino acids and L-amino acids.

[0285] The extracyclic peptide may be acylated at its N-terminus (Ac-EP). It is understood that EP and Ac-EP are interchangeable throughout this application. For example, EP may include Ac-PKKKRKV.

[0286] Each amino acid in an extracyclic peptide may be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a homolog of a natural amino acid in that it has a structure similar to a natural amino acid in order to mimic the structure and reactivity of a natural amino acid. A non-natural amino acid may be a modified amino acid and / or amino acid analog that is not one of the 20 common natural amino acids or the rare natural amino acids selenocysteine ​​or pyrrolicin. A non-natural amino acid may also be a D-isomer of a natural amino acid. Amino acids and / or amino acid residues may be referred to using their full name, a conventional three-letter abbreviation, or a conventional single-letter abbreviation. When a single-letter abbreviation is used, uppercase letters indicate L amino acids or residues, and lowercase letters indicate D amino acids or residues. For example, arginine may be referred to as Arg, R, or r.

[0287] The EP may comprise at least one amino acid residue that is positively charged at physiologically relevant pH values. In some embodiments, the EP comprises at least one amino acid residue comprising a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. In some embodiments, the EP comprises at least one amino acid residue comprising a guanidine group or a protonated form thereof. In some embodiments, the EP comprises at least one amino acid residue comprising a terminal amine or a protonated form thereof. In some embodiments, the EP comprises at least one amino acid residue comprising an imidazole or a protonated form thereof. Throughout this disclosure, the protonated form may mean a salt thereof.

[0288] EP may contain 1 to 5 amino acid residues, including a side chain containing a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. EP may contain 1 to 4 amino acid residues, including a side chain containing a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. EP may contain 1 to 3 amino acid residues, including a side chain containing a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. EP may contain 1 or 2 amino acid residues, including a side chain containing a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. EP may contain 2 or 3 amino acid residues, including a side chain containing a guanidine group, a terminal amine, an imidazole, or a protonated form thereof. EP may contain 2 to 4 amino acid residues, including a side chain containing a guanidine group, a terminal amine, an imidazole, or a protonated form thereof.

[0289] In several embodiments, the amino acid containing a guanidine group is arginine. In several embodiments, the amino acid containing a terminal amine is lysine. In several embodiments, the amino acid containing an imidazole is histidine.

[0290] EP may contain 1, 2, 3, 4, or 5 amino acid residues, each containing a side chain with a guanidine group or its protonated form. EP may contain 1 amino acid residue, each containing a side chain with a guanidine group or its protonated form. EP may contain multiple amino acid residues, each containing a side chain with a guanidine group or its protonated form. EP may contain 3 amino acid residues, each containing a side chain with a guanidine group or its protonated form. EP may contain 4 amino acid residues, each containing a side chain with a guanidine group or its protonated form. EP may contain 5 amino acid residues, each containing a side chain with a guanidine group or its protonated form. The amino acid residue containing a side chain with a guanidine group may be an arginine residue. EP may contain 1, 2, 3, 4, or 5 arginine residues. EP may contain 1 arginine residue. EP may contain 2 arginine residues. EP may contain 3 arginine residues. EP may contain four arginine residues. EP may contain five arginine residues.

[0291] EP may contain 1, 2, 3, 4, or 5 amino acid residues, including a terminal amine or its protonated form. EP may contain 1 amino acid residue, including a terminal amine or its protonated form. EP may contain 2 amino acid residues, including a terminal amine or its protonated form. EP may contain 3 amino acid residues, including a terminal amine or its protonated form. EP may contain 4 amino acid residues, including a terminal amine or its protonated form. EP may contain 5 amino acid residues, including a terminal amine or its protonated form. The amino acid having a terminal amine may be lysine. EP may contain 1, 2, 3, 4, or 5 lysine residues. EP may contain 1 lysine residue. EP may contain 2 lysine residues. EP may contain 3 lysine residues. EP may contain 4 lysine residues. EP may contain 5 lysine residues. The amino group on the side chain of each lysine residue can be substituted with a protecting group, for example, trifluoroacetyl (-COCF3), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), or (4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene-3)-methylbutyl (ivDde). The amino group on the side chain of each lysine residue can be substituted with a trifluoroacetyl (-COCF3) group. The inclusion of a protecting group may enable amide conjugation. The protecting group can be removed after conjugating EP to cCPP.

[0292] EP may contain 1, 2, 3, 4, or 5 amino acid residues, including imidazole or its protonated form. EP may contain 1 amino acid residue, including imidazole or its protonated form. EP may contain 2 amino acid residues, including imidazole or its protonated form. EP may contain 3 amino acid residues, including imidazole or its protonated form. EP may contain 4 amino acid residues, including imidazole or its protonated form. EP may contain 5 amino acid residues, including imidazole or its protonated form. The amino acid containing imidazole may be histidine. EP may contain 1, 2, 3, 4, or 5 histidine residues. EP may contain 1 histidine residue. EP may contain 2 histidine residues. EP may contain 3 histidine residues. EP may contain 4 histidine residues. EP may contain five histidine residues.

[0293] EP may contain 1, 2, 3, or 4 amino acid residues having uncharged side chains. The uncharged side chains may include hydrophobic side chains. EP may contain 1, 2, 3, or 4 amino acid residues having hydrophobic side chains. EP may contain 1, 2, 3, or 4 amino acid residues having uncharged hydrophobic side chains. EP may contain 1 amino acid residue having an uncharged hydrophobic side chain. EP may contain 2 amino acid residues having uncharged hydrophobic side chains. EP may contain 3 amino acid residues having uncharged hydrophobic side chains. EP may contain 4 amino acid residues having uncharged hydrophobic side chains. The amino acid residue having an uncharged hydrophobic side chain may be selected from valine, proline, β-alanine, and glycine. The amino acid residue having a hydrophobic side chain may be valine or proline. The amino acid residue having a hydrophobic side chain may be valine. The amino acid residue with a hydrophobic side chain may be proline. The amino acid residue with a hydrophobic side chain may be β-alanine. The amino acid residue with a hydrophobic side chain may be glycine.

[0294] EP may contain at least one positively charged amino acid residue and at least one uncharged hydrophobic residue. EP may contain two positively charged amino acid residues and one uncharged hydrophobic residue. EP may contain three positively charged amino acid residues and one uncharged hydrophobic residue. EP may contain four positively charged amino acid residues and one uncharged hydrophobic residue. EP may contain two positively charged amino acid residues and two uncharged hydrophobic residues. EP may contain three positively charged amino acid residues and two uncharged hydrophobic residues. EP may contain four positively charged amino acid residues and two uncharged hydrophobic residues.

[0295] EP may contain at least one lysine residue and at least one arginine residue. EP may contain two, three, four, or five lysine residues and / or arginine residues. EP may contain two lysine residues and / or arginine residues. EP may contain three lysine residues and / or arginine residues. EP may contain four lysine residues and / or arginine residues. EP may contain five lysine residues and / or arginine residues.

[0296] 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) residues. EP may contain three lysine (K) residues. EP may contain four lysine (K) residues. EP may contain one or more arginine (R) residues. EP may contain 1 arginine(R). EP may contain 2 arginine(R). EP may contain 3 arginine(R). EP may contain 4 arginine(R). EP may contain 5 arginine(R). EP may contain 6 arginine(R). EP may contain 1 or more histidine(H). EP may contain 1 histidine(H). EP may contain 2 histidine(H). EP may contain 3 histidine(H). EP may contain 4 histidine(H). EP may contain 5 histidine(H). EP may contain 6 histidine(H). EP may contain 1 or more β-alanine(B). EP may contain 2 β-alanine(B). EP may contain two β-alanine (B) groups. The amino acid residues in EP may have D-stereochemistry or L-stereochemistry.

[0297] EP is 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, KHK H, 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, May include KRKKK, KKKRK, KKKKR, KKFRK, KBKBK, KRKIL, RBFBR, RBRBR, RBHBR, RFRRF, RIRRI, RKKKKG, KRKKKG, KKKRKG, 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 beta-alanine. EP may include PKKKRKV. EP may contain RR, RRR, RHR, RBR, RBRBR, RBRRBR, RBGRBR, RBRGBR, RBRRBG, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D stereochemistry or L stereochemistry.

[0298] EPは、kk、kr、rr、rk、rf、hh、hk、hr、kkk、kk、kfk、kgk、kbk、kbr、krk、krr、rrk、rrk rrv、frr、rbr、kkkh、khk、hkk hrr、hrh、hhr、hbh hhhhhhhh pkkkk、khkk kkkkhk kh、khkhkh、hkhk、kkkk、kkrk、krkk、krrk、krkf、rkkr、rrrr、rbrb、brbr、hrhr、rhrh、hb hb、bhbh、kgkk、kkgk、hbhbh、rbhbh、brbrb、hbrbh、bhrhr、brhrh、hbkbh、rrrrr、kkkkk kkkrk、rkkkk、krkkk、kkrkkk、kkkkr、kkfrk、kbkbk、krkil、rbfbr、rbrbr、rbhbr、rfr rf、rirri、rkkkkg、krkkkg、kkrkkkg、kkkkrg、rkkkkb、krkkkb、kkrkkkb、kkkkrb、kkkrkv rrrrrr、hhhhhhh、rhrrhr、krkkkp、rkkkrk、krkrkr、rkrkrk、rbrbrb、rbrrbr、 kbkbkb、pkkkrkv、pgkkrkv、pkggkrkv、pkkgrkv、pkkkgkv、pkkkrgv

[0299] EP is 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, KHK H, 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, It can consist of KRKKK, KRKKK, KKKKR, KKFRK, KBKBK, KRKIL, RBFBR, RBRBR, RBHBR, RFRRF, RIRRI, RKKKKG, KRKKKG, KRKRKKG, 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 beta-alanine. EP can consist of PKKKRKV. EP may consist of RR, RRR, RHR, RBR, RBRBR, RBRRBR, RBGRBR, RBRGBR, RBRRBG, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D stereochemistry or L stereochemistry.

[0300] EPは、kk、kr、rr、rk、rf、hh、hk、hr、rh、kkkk、kfk、kgk、kbk、kbr、krk、krr、rkk、rrrrrrr rrv、frr、rfr、rbr、kkh、khk、hkk、hrr、hrh、hhr、hbh、hhh、hhhh、pkkk、khkk、kkhk、kk kh、khkh、hkhk、kkkk、kkrk、krkk、krrk、krkf、rkkr、rrrr、rbrb、brbr、hrhr、rhrh、hb hb、bhbh、kgkk、kkgk、hbhbh、rbhbh、brbrb、hbrbh、bhrhr、brhrh、hbkbh、rrrrr、kkkkk 、kkkrk、rkkkk、krkkk、kkrkk、kkkkr、kkfrk、kbkbk、krkil、rbfbr、rbrbr、rbhbr、rfr rf、rirri、rkkkkg、krkkkg、kkrkkg、kkkkrg、rkkkkb、krkkkb、kkrkkb、kkkkrb、kkkrkv 、rrrrrr、hhhhhh、rhrhrh、hrhrhr、rkkkp、pkkkrk、krkrkr、rkrkrk、rbrbrb、rbrrbr、k bkbkb、pkkkrkv、pgkkrkv、pkgkrkv、pkkgrkv、pkkkgkv、pkkkrgv、またはpkkkrkgからな

[0301] EP、KK、KR、RR、RK、RF、KKK、KGK、KFK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK、KKRK、KRKKKK RRK、KRKF、RKKR、RRRR、KGKK、KKGK、KKKKK、KKKRK、KBKBK、KKFRK、KRKIL、RFRRF、RIRRI、 KKKRKV、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV、またはPKKKRKG、 EP、PKKKRKV、RR、RRR、RHR、RBR、RBRBR、RB HBR, HBRBH, B, B, and HBRBH.

[0302] EP may include 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 may include pkkkrkv, rr, rrr, rhr, rbr, rbrbr, rbhbr, or hbrbh.

[0303] EP may consist of 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 may consist of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine.

[0304] EP can consist of 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.

[0305] EP may contain one of the following sequences: HBH, HBHBH, or HRBH. EP may contain HHRBFBR. EP may contain one of the following sequences: KBK, KBKBK, or KBR. EP may contain one of the following sequences: KGK or KGKK. EP may contain one of the following sequences: KKGK, KKKK, KKKKR, KKKRK, KKKRKG, or KKRK. EP may contain one of the following sequences: KR, KRK, or KRKKK. EP may contain PGKKRKV. EP may contain one of the following sequences: PKGKRKV, PKK, PKKGRKV, PKKK, PKKKGKV, PKKKRGV, PKKKRKG, PKKRGV, PKKRKG, or PKKRKV. 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 beta-alanine.

[0306] EP may include an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. EP may consist of an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. EP may include an NLS containing the amino acid sequence PKKKRKV. EP may consist of an NLS containing the amino acid sequence PKKKRKV.

[0307] An EP contains 2 to 10, 6 to 9, or 4 to 8 consecutive amino acid residues. While an EP may contain 2 to 10, 6 to 9, or 4 to 8 amino acids, not all amino acids are consecutive. For example, the amino acid residues of an EP may be separated by one or more components of an EEV, such as a linker, cCPP, or a combination thereof, thereby forming a "split" EP.

[0308] EEV Cargo Conjugate In several embodiments, the EEV is conjugated to the cargo, resulting in the structure of equation (A-1): [ka] It is possible to form an EEV-cargo conjugate that includes, During the ceremony, EP is a linear extracyclic peptide as defined herein, cCPP is a cyclic cell-permeable peptide as defined herein, L 1 and L 2 It is independently a linker arm, ^ indicates D-stereochemistry or L-stereochemistry. y' is an integer between 1 and 5. M' is a bonding group as defined herein, Cargo can be peptides, oligonucleotides, small molecules, or any combination thereof.

[0309] In several embodiments, the EEV-cargo conjugate formula (A-1) is (B-1), (C-1), and (D-1): [ka] It may have an expression selected from, In the formula, EP, cCPP, x', j', ^, y', z', j'', X o ', K # M', and Cargo are as defined herein.

[0310] In several embodiments, the EEV-cargo conjugate has the structure of formula (B-1), formula (C-1), or formula (D-1), where EP comprises all D-amino acids, cCPP comprises D-amino acids, achiral amino acids, and AAsc, where AAsc is an amino acid side chain, and AAsc conjugates cCPP to a linker.

[0311] In several embodiments, the EEV-cargo conjugate has the structure of formula (A-3): [ka] It has, During the ceremony, EP is a linear extracyclic peptide, L 1 and L 2 This is a linker arm, M' is a bonding group, y' is an integer between 1 and 5. ^ indicates D-stereochemistry or L-stereochemistry. R 1 , R 2 , and R 3 Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R 4 , R 5 , R 6 and R 7 These are independently H or amino acid side chains. q is an integer between 1 and 4. n is an integer between 1 and 4. Cargo consists of peptides, oligonucleotides, small molecules, or combinations thereof.

[0312] In several embodiments, the EEV-cargo conjugate of formula (A-3) has a linker structure of formula (B'). In several embodiments, the EEV-cargo conjugate of formula (A-3) has a linker structure of formula (C'). In several embodiments, the EEV-cargo conjugate of formula (A-3) has a linker structure of formula (D').

[0313] In several embodiments, the EEV-cargo conjugate has the structure of formula (A-3a): [ka] It has, In the formula, EP, M', L 1 , L 2 ,^,y',n,R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 A cargo is defined herein as a peptide, oligonucleotide, small molecule, or a combination thereof.

[0314] In several embodiments, the EEV-cargo conjugate of formula (A-3a) has a linker structure of formula (B'). In several embodiments, the EEV-cargo conjugate of formula (A-3a) has a linker structure of formula (C'). In several embodiments, the EEV-cargo conjugate of formula (A-3a) has a linker structure of formula (D').

[0315] In several embodiments, the EEV-cargo conjugate has the structure of formula (A-3b): [ka] It has, In the formula, EP, L 1 , L 2 , M', ^, y', n, m', m'', q, R 1 , R 2 , R 3 , R 4 , and R6 A cargo is defined herein as a peptide, oligonucleotide, small molecule, or a combination thereof.

[0316] In several embodiments, the EEV-cargo conjugate of formula (A-3b) has a linker structure of formula (B'). In several embodiments, the EEV-cargo conjugate of formula (A-3b) has a linker structure of formula (C'). In several embodiments, the EEV-cargo conjugate of formula (A-3b) has a linker structure of formula (D').

[0317] In several embodiments, the EEV-cargo conjugate has the structure of formula (A-3c): [ka] It has, In the formula, EP, L 1 , L 2 ,M',^,y',n,m',m'',R 1 , R 2 , R 3 , R 4 , and R 6 A cargo is defined herein as a peptide, oligonucleotide, small molecule, or a combination thereof.

[0318] In several embodiments, the EEV-cargo conjugate of formula (A-3c) has a linker structure of formula (B'). In several embodiments, the EEV-cargo conjugate of formula (A-3c) has a linker structure of formula (C'). In several embodiments, the EEV-cargo conjugate of formula (A-3c) has a linker structure of formula (D').

[0319] In several embodiments, the cargo is an oligonucleotide. In several embodiments, the oligonucleotide is a therapeutic oligonucleotide. In several embodiments, the oligonucleotide is an antisense oligonucleotide. In several embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0320] M' is -NH-, -C(O)-, -O-, [ka] The expression may include , where y'' is an integer between 1 and 4, and t' is between 0 and 10.

[0321] M' is -NH-, -C(O)-, -O-, [ka] The expression may include , where y'' is an integer between 1 and 4.

[0322] In some embodiments, M' is [ka] The formula includes , where t' is between 0 and 10.

[0323] M' may contain -NH-. M' may contain -C(O)-. M' may contain -O-.

[0324] M' is [ka] The formula may include a structure selected from, where R is alkyl, alkenyl, alkynyl, carbocykyl, or heterocyclyl.

[0325] M' is [ka] Includes a structure selected from. M' is [ka] It may include M', [ka] It may include M', [ka] It may include.

[0326] M' may contain -C(O)-. M' may contain -NH-. M' may contain -O-.

[0327] M' is [ka] It may include M', [ka] It may include, where t' is between 0 and 10. M' is, [ka] It may include. M' can be -O-. M' can be -NH-. M' may include -C(O)-.

[0328] This specification provides an EEV-cargo conjugate comprising EEV conjugated to a cargo via a binding group (M'), where M' is -NH-, -C(O)-, -O-, [ka] It may include.

[0329] The linker can be covalently bonded to the cargo at any suitable position on the cargo. The linker can be covalently bonded to the 3' or 5' end of the oligonucleotide cargo. The linker can be covalently bonded to the cargo backbone.

[0330] M' forms a bond to the free secondary amine on the morpholino ring of the terminal nucleotide of the PMO cargo. In some embodiments, M' forms a bond to the free secondary amine on the morpholino ring of the 3' terminal nucleotide of the PMO. In some embodiments, M' forms a bond to the free hydroxyl on the 5' terminal nucleotide of the PMO.

[0331] In several embodiments, the EEV-cargo conjugate has the structure shown in formula (B-1), where x' is 0 and z' is 12. In several embodiments, the EEV-cargo conjugate may have the arrangement shown in Table 9a. [Table 15]

[0332] In several embodiments, the EEV-cargo conjugate has the structure shown in formula (B-1), where x' is 2 and z' is 12. In several embodiments, the EEV-cargo conjugate may have the arrangement shown in Table 9b. [Table 16]

[0333] In several embodiments, the EEV-cargo conjugate has the structure shown in formula (B-1), where x' is 2 and z' is 2. In several embodiments, the EEV-cargo conjugate may have the arrangement shown in Table 9c. [Table 17]

[0334] In several embodiments, the EEV-cargo conjugate has the structure shown in formula (B-1), where x' is 2 and z' is 12. In several embodiments, the EEV-cargo conjugate may have the arrangement shown in Table 9d. [Table 18]

[0335] In several embodiments, the EEV-cargo conjugate has the structure shown in formula (D-1), where x' is 2 and z' is 12. In several embodiments, the EEV-cargo conjugate may have the arrangement shown in Table 10a. [Table 19]

[0336] In several embodiments, the EEV-cargo conjugate has the structure shown in formula (C-1), where x' is 2 and z' is 12. In several embodiments, the EEV-cargo conjugate may have the arrangement shown in Table 10b. [Table 20]

[0337] The linker can be covalently bonded to the cargo at any suitable position on the cargo. The linker can be covalently bonded to the 3' or 5' end of the oligonucleotide cargo. The linker can be covalently bonded to the cargo backbone.

[0338] In embodiments where the cargo is PMO, M' forms a bond to the free secondary amine on the morpholino ring of the terminal nucleotide of the PMO cargo. In some embodiments, M' forms a bond to the free secondary amine on the morpholino ring of the 3' terminal nucleotide of the PMO. In some embodiments, M' forms a bond to the free hydroxyl on the 5' terminal nucleotide of the PMO.

[0339] The structure of the EEV-cargo conjugate is shown in Figures 27-29.

[0340] cargo In several embodiments, an EEV is provided comprising a cyclic peptide conjugated to a cargo, such as a cyclic cell-permeable peptide (cCPP). In several embodiments, the cargo is an oligonucleotide. In several embodiments, the oligonucleotide is a therapeutic oligonucleotide. In several embodiments, the oligonucleotide is an antisense oligonucleotide. In several embodiments, the oligonucleotide is directed to a target polynucleotide. In several embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO). In several embodiments, the oligonucleotide includes siRNA, RNAi, microRNA, antagonists, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, supermilli, miRNA mimetics, miRNA inhibitors, or combinations thereof. For example, see Chery, J., “RNA therapeutics: 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, 23 July 2022, 12(644) (doi.org / 10.1038 / s41419-022-05075-2).

[0341] The term "antisense oligonucleotide" or simply "antisense" refers to an oligonucleotide that is complementary to a target polynucleotide sequence. An antisense nucleotide is a single strand of DNA or RNA that is complementary to a selected sequence, such as the target gene mRNA.

[0342] Antisense oligonucleotides can modulate one or more aspects of protein transcription, translation, and expression. In several embodiments, antisense oligonucleotides may be directed to a target sequence within a target pre-mRNA, modulating one or more aspects of pre-mRNA splicing. As used herein, modification of splicing refers to altering the processing of a pre-mRNA transcript such that the spliced ​​mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, deletion of one or more exons, or deletion or addition of a sequence not normally found in the spliced ​​mRNA (e.g., an intron sequence). In several embodiments, hybridization of an antisense oligonucleotide to a target sequence in a pre-mRNA molecule restores the original splicing of the mutated pre-mRNA sequence. In several embodiments, hybridization of an antisense oligonucleotide results in alternative splicing of the target pre-mRNA. In several embodiments, hybridization of an antisense oligonucleotide results in exon inclusion or exon skipping of one or more exons. In several embodiments, the skipped exon sequence includes a frameshift mutation, a nonsense mutation, or a missense mutation. In several embodiments, the skipped exon sequence includes a deletion, substitution, or insertion of nucleic acid. In several embodiments, the skipped exon itself does not contain a sequence mutation, but the adjacent intron contains a mutation that results in a frameshift mutation or a nonsense mutation. In several embodiments, hybridization of an antisense oligonucleotide against a target sequence in a target pre-mRNA prevents the incorporation of the exon sequence into a mature mRNA molecule. In several embodiments, hybridization of an antisense oligonucleotide against a target sequence in a target pre-mRNA results in the preferential expression of the wild-type target protein isomer.In several embodiments, hybridization of an antisense oligonucleotide against a target sequence within a target pre-mRNA results in the expression of a respliced ​​target protein containing an active fragment of the wild-type target protein.

[0343] The mechanism of action of antisense oligonucleotides is through hybridization between the antisense oligonucleotide and the target nucleic acid. In several embodiments, antisense oligonucleotides hybridizing to the target sequence suppress the expression of the target protein. In several embodiments, hybridization of an antisense oligonucleotide to its target sequence suppresses the expression of one or more wild-type target protein isomers. In several embodiments, hybridization of an antisense oligonucleotide to its target sequence upregulates the expression of the target protein. In several embodiments, hybridization of an antisense oligonucleotide to its target sequence increases the expression of one or more wild-type target protein isomers.

[0344] Methods for generating antisense oligonucleotides are known in the art and can be readily applied to the generation of antisense oligonucleotides targeting any polynucleotide sequence. The selection of antisense oligonucleotide sequences specific to a particular target sequence is based on the analysis of the selected target sequence, as well as the determination of its secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides may be selected based on their reluctance to form dimers, hairpins, or other secondary structures that reduce or inhibit specific binding to the target mRNA within the host cell. Target regions of mRNA include regions on or near the AUG translation start codon, and sequences substantially complementary to the 5' region of the mRNA. These secondary structure analyses and selection of target sites can be performed, for example, using OLIGO primer analysis software (Molecular Biology Insights) version 4 and / or BLASTN 2.0.5 algorithm software (Altschul et ai, Nucleic Acids Res. 1997, 25(17):3389-402).

[0345] In several embodiments, oligonucleotides, such as antisense oligonucleotides, can modify one or more aspects of splicing, translation, or expression of a target gene, for example, by altering the splicing of a target pre-mRNA in a eukaryote. The oligonucleotides contain nucleic acid sequences complementary to sequences found within the target pre-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 genes causing specific diseases. The principle of antisense technology is that antisense oligonucleotides hybridizing to a target nucleic acid modulate gene expression activities, such as splicing or translation, through one of several antisense mechanisms of action. Due to the sequence specificity of oligonucleotides, this technology is highly attractive as a therapeutic method for selectively modulating the splicing of pre-mRNAs involved in the pathogenesis of any one of a variety of diseases. Antisense technology is an effective means of altering the expression of one or more specific gene products and can therefore be shown to be useful in a variety of therapeutic, diagnostic, and research applications.

[0346] Oligonucleotides may contain one or more chiral centers, thus resulting in enantiomers, diastereomers, and other stereoisomer configurations, which can be defined from the viewpoint of absolute stereochemistry as (R) or (S), α or β, or (D) or (L). Oligonucleotides may include all such possible isomers, as well as their racemic and optically pure forms.

[0347] Oligonucleotide hybridization sites The mechanism of action of antisense depends on the hybridization of the antisense oligonucleotide to the target nucleic acid. In several embodiments, antisense oligonucleotides complementary to the target nucleic acid are provided. In several embodiments, the target nucleic acid sequence is located in the pre-mRNA molecule. In several embodiments, the target nucleic acid sequence is located in the exon of the pre-mRNA molecule. In several embodiments, the target nucleic acid sequence is located in the intron of the pre-mRNA molecule.

[0348] Pre-mRNA molecules are generated in the nucleus and undergo processing before or during transport to the cytoplasm for translation. Processing of pre-mRNA involves the addition of a 5' methylated cap and the addition of a poly(A) tail of approximately 200-250 bases to the 3' end of the transcript. The next step in mRNA processing is splicing of pre-mRNA, which occurs in the maturation of 90-95% of mammalian mRNA. Introns (or intervening sequences) are regions of the primary transcript (or the DNA that codes for it) that are not included in the coding sequence of mature mRNA. Exons are regions of the primary transcript that remain when mature mRNA reaches the cytoplasm. Exons are spliced ​​together to form the mature mRNA sequence. Splice junctions, also called splice sites, are often referred to as the "5' splice site" or "splice donor site" and the "3' splice site" or "splice acceptor site," with the 5' end of the junction being called the "5' splice site" or "splice donor site." In splicing, the 3' end of an upstream exon joins the 5' end of a downstream exon. Therefore, unspliced ​​RNA (or pre-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 intron removal, in mature mRNA, exons are contiguous, sometimes called exon / exon junctions or boundaries. Hidden splice sites are less common but may be used when normal splice sites are blocked or unavailable. Alternative splicing is defined as splicing together different combinations of exons, often resulting in the production of multiple mRNA transcripts from a single gene.

[0349] In multiple embodiments, oligonucleotides can hybridize with sequences of splice sites. In multiple embodiments, oligonucleotides can hybridize with sequences containing a portion of a splice site. In multiple embodiments, oligonucleotides can hybridize with sequences containing a portion or all of a splice site. In multiple embodiments, oligonucleotides can hybridize with sequences containing a portion or all of a splice donor site. In multiple embodiments, oligonucleotides can hybridize with sequences containing a portion or all of a splice acceptor site. In multiple embodiments, oligonucleotides can hybridize with sequences containing a portion or all of a hidden splice site. In multiple embodiments, oligonucleotides can hybridize with sequences containing an exon / intron junction.

[0350] Pre-mRNA splicing involves two consecutive biochemical reactions. Both reactions involve transesterification between RNA nucleotides by spliceosomes. In the first reaction, the 2'-OH group of a specific branching point nucleotide within an intron, defined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site, forming a lariat intermediate. In the second reaction, the 3'-OH group of the released 5' exon performs a nucleophilic attack on the last nucleotide of the intron at the 3' splice site, resulting in exon binding and the release of the intron lariat. Pre-mRNA splicing is regulated by the intron silencer sequence (ISS) and the terminal stem-loop (TSL) sequence. As used herein, the terms “intron silencer sequence (ISS)” and “terminal stem-loop (TSL)” refer to sequence elements within introns and exons, respectively, which regulate alternative splicing through the binding of transactive protein factors within pre-mRNA, thereby resulting in the differential use of splice sites. Typically, intron silencer sequences are 8–16 nucleotides long and less conserved than the splice sites of exon-intron junctions. Terminal stem-loop sequences are typically 12–24 nucleotides long and form secondary loop structures through complementarity, i.e., binding, within 12–24 nucleotide sequences.

[0351] In several embodiments, the oligonucleotide hybridizes with a sequence containing some or all of the intron silencer sequence. In several embodiments, the oligonucleotide hybridizes with a sequence containing some or all of the terminal stem loop.

[0352] Up to 50% of human genetic disorders caused by point mutations are due to abnormal splicing. Such point mutations can disrupt existing splice sites or create new ones, resulting in mRNA transcripts composed of different exon combinations or missing exons. Point mutations can also cause activation of hidden splice sites or disruption of regulatory cis elements (i.e., splicing enhancers or silencers).

[0353] In several embodiments, the oligonucleotide hybridizes with a sequence containing some or all of the abnormal splice sites resulting from a mutation in the target gene. In several embodiments, the oligonucleotide hybridizes with a sequence containing some or all of the regulatory elements. Antisense oligonucleotides targeting cis-regulatory elements are also provided. In several embodiments, the regulatory elements are located within an exon. In several embodiments, the regulatory elements are located within an intron.

[0354] In several embodiments, oligonucleotides may be specifically hybridized 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 several embodiments, oligonucleotides may hybridize with some or all of the pre-mRNA splice sites, exon-exon junctions, or intron-exon junctions. In several embodiments, oligonucleotides may hybridize with aberrant fusion junctions resulting from rearrangement or deletion. In several embodiments, oligonucleotides may hybridize with specific exons within alternative splicing mRNA.

[0355] In several embodiments, the oligonucleotide can hybridize with sequences of 5 to 50 nucleotides in length, and this length is also called the oligonucleotide length. In several embodiments, the oligonucleotide is 5 to 50, 10 to 40, 15 to 30, 20 to 30, or 20 to 25 nucleotides long. In several embodiments, the oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In several embodiments, the oligonucleotide is at least 15, 16, 17, 18, 19, or 20 nucleotides long and up to 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In several embodiments, the oligonucleotide is 10 nucleotides long. In several embodiments, the oligonucleotide is 15 nucleotides long. In several embodiments, the oligonucleotide is 16 nucleotides long. In several embodiments, the oligonucleotide is 17 nucleotides long. In multiple embodiments, the oligonucleotide is 18 nucleotides long. In multiple embodiments, the oligonucleotide is 19 nucleotides long. In multiple embodiments, the oligonucleotide is 20 nucleotides long. In multiple embodiments, the oligonucleotide is 21 nucleotides long. In multiple embodiments, the oligonucleotide is 22 nucleotides long. In multiple embodiments, the oligonucleotide is 23 nucleotides long. In multiple embodiments, the oligonucleotide is 24 nucleotides long. In multiple embodiments, the oligonucleotide is 25 nucleotides long. In multiple embodiments, the oligonucleotide is 26 nucleotides long. In multiple embodiments, the oligonucleotide is 27 nucleotides long. In multiple embodiments, the oligonucleotide is 28 nucleotides long. In multiple embodiments, the oligonucleotide is 29 nucleotides long. In multiple embodiments, the oligonucleotide is 30 nucleotides long.

[0356] In several embodiments, the oligonucleotide may be less than 100% complementary to the target nucleic acid sequence. As used herein, the term “percentage of complementarity” refers to the number of nucleic acid bases of an oligonucleotide that are nucleic acid base complementary to the corresponding nucleic acid base of the oligonucleotide or nucleic acid, divided by the total length of the oligonucleotide (number of nucleic acid bases). Those skilled in the art will recognize that it is possible to include mismatches without impairing the activity of the antisense oligonucleotide. In several embodiments, the oligonucleotide may contain up to 20% nucleotides that disrupt the base pairing between the oligonucleotide and the target nucleic acid. In several embodiments, the oligonucleotide may contain 15% or less, 10% or less, 5% or less mismatches, or no mismatches. In several embodiments, the oligonucleotide may contain 1, 2, 3, 4, or 5 or fewer mismatches. In several embodiments, the oligonucleotide is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% complementary to the target nucleic acid. The complementarity percentage of an oligonucleotide is calculated by dividing the number of complementary nucleic acid bases by the total number of nucleic acid bases in the oligonucleotide. The complementarity percentage within an oligonucleotide region is calculated by dividing the number of complementary nucleic acid bases within that region by the total number of nucleic acid base regions.

[0357] In several embodiments, incorporating nucleotide affinity modifications allows for a greater tolerance of mismatches compared to unmodified oligonucleotides. Similarly, certain 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 oligonucleotides and target nucleic acids, for example, by determining the melting temperature (Tm). Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443), those skilled in the art can evaluate the ability of nucleotide modifications to increase the melting temperature of RNA:DNA double helix.

[0358] Mechanism of action of antisense Oligonucleotides can be antisense oligonucleotides that modulate one or more aspects of protein transcription, translation, and expression. In several embodiments, oligonucleotides that hybridize to a target sequence within a target pre-mRNA can modulate one or more aspects of pre-mRNA splicing. As used herein, modification of splicing refers to altering the processing of a pre-mRNA transcript such that the spliced ​​mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, deletion of one or more exons, or deletion or addition of a sequence not normally found in the spliced ​​mRNA (e.g., an intron sequence). In several embodiments, oligonucleotide hybridization to a target sequence within a pre-mRNA molecule restores the original splicing of the mutated pre-mRNA sequence. In several embodiments, oligonucleotide hybridization results in alternative splicing of the target pre-mRNA. In several embodiments, oligonucleotide hybridization results in exon inclusion or exon skipping of one or more exons. In several embodiments, the skipped exon sequence includes a frameshift mutation, a nonsense mutation, or a missense mutation. In several embodiments, the skipped exon sequence includes a deletion, substitution, or insertion of nucleic acid. In several embodiments, the skipped exon itself does not contain a sequence mutation, but the adjacent intron contains a mutation that results in a frameshift mutation or a nonsense mutation. In several embodiments, the deletion of an exon that does not contain a sequence mutation restores the reading frame of the mature mRNA. In several embodiments, hybridization of an oligonucleotide to a target sequence in the target pre-mRNA results in the preferential expression of the wild-type target protein isomer. In several embodiments, hybridization of an oligonucleotide to a target sequence in the target pre-mRNA results in the expression of a respliced ​​target protein containing an active fragment of the wild-type target protein.

[0359] The mechanism of action of antisense oligonucleotides involves hybridization of the antisense oligonucleotide with a target nucleic acid. In several embodiments, the oligonucleotide hybridizing to the target sequence suppresses the expression of the target protein. In several embodiments, the oligonucleotide hybridizing to the target sequence suppresses the expression of one or more wild-type target protein isomers. In several embodiments, the oligonucleotide hybridizing to the target sequence upregulates the expression of the target protein. In several embodiments, the oligonucleotide hybridizing to the target sequence increases the expression of one or more wild-type target protein isomers.

[0360] The efficacy of an oligonucleotide may be evaluated by assessing the antisense activity induced by its administration. As used herein, the term “antisense activity” refers to any detectable and / or measurable activity that can be attributed to the hybridization of an antisense oligonucleotide with its target nucleic acid. Such detection and / or measurement may be direct or indirect. In some embodiments, antisense activity is evaluated by detecting and / or measuring the amount of the target protein. In some embodiments, antisense activity is evaluated by detecting and / or measuring the amount of the respliced ​​target protein. In some embodiments, antisense activity is evaluated 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.

[0361] Antisense oligonucleotide design The design of oligonucleotides may depend on the target sequence. Targeting an oligonucleotide to a specific target nucleic acid molecule can be a multi-step process. This process typically begins with identifying the target nucleic acid whose expression is to be regulated. As used herein, the terms “target nucleic acid” and “nucleic acid encoding a target gene” include DNA encoding a selected target gene, RNA transcribed from such DNA (including pre-mRNA and mRNA), and cDNA derived from such RNA. In embodiments, the target nucleic acid may be a cellular gene (or mRNA transcribed from such gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule derived from a pathogen.

[0362] Those skilled in the art can design, synthesize, and screen antisense oligonucleotides of different nucleic acid base sequences to identify sequences that yield antisense activity. For example, antisense oligonucleotides can be designed to alter the splicing of a target pre-mRNA or to inhibit the expression of a target protein. Methods for designing, synthesizing, and screening antisense oligonucleotides that have antisense activity against pre-selected target nucleic acids can be found, for example, in “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida, which is incorporated by reference in its entirety for all purposes.

[0363] In several embodiments, the antisense oligonucleotide comprises a modified nucleoside, a modified nucleoside linkage, and / or a conjugate group.

[0364] In several embodiments, the antisense oligonucleotide is “tricyclo-DNA (tc-DNA),” which refers to a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring, limiting the conformational flexibility of the skeleton and optimizing the geometric shape of the skeleton with a twist angle γ. tc-DNA containing homonucleotides adenine and thymine forms highly stable AT base pairs with complementary RNA.

[0365] Nucleoside In several embodiments, antisense oligonucleotides comprising linked nucleosides are provided. In several embodiments, some or all of the nucleosides are modified nucleosides. In several embodiments, one or more nucleosides comprise modified nucleic acid bases. In several embodiments, one or more nucleosides comprise modified sugars. Chemically modified nucleosides are routinely incorporated into antisense oligonucleotides to improve one or more properties such as nuclease resistance, pharmacokinetics, or affinity for target RNA.

[0366] Generally, a nucleic acid base is any group containing one or more atoms or groups of atoms that can hydrogen bond to a base of another nucleic acid. In addition to “unmodified” or “natural” nucleic acid bases such as the purine nucleic acid bases adenine (A) and guanine (G), and the pyrimidine nucleic acid bases thymine (T), cytosine (C), and uracil (U), many modified nucleic acid bases or nucleic acid base mimetics known to those skilled in the art can also be used in the oligonucleotides described herein. The terms modified nucleic acid base and nucleic acid base mimetic may overlap, but generally, a modified nucleic acid base refers to a nucleic acid base that is structurally quite similar to the parent nucleic acid base, such as 7-deazapurine, 5-methylcytosine, or G-clamp, while a nucleic acid base mimetic involves more complex structures, such as the tricyclic phenoxazine nucleic acid base mimetic. Methods for preparing the above-mentioned modified nucleic acid bases are well known to those skilled in the art.

[0367] In several embodiments, the oligonucleotides provided herein comprise one or more nucleosides having a modified sugar moiety. In several embodiments, the furanosyl sugar ring of a natural nucleoside may be substituted, but not limited to, two non-geminal ring atoms may be bridged to form a bicyclic nucleic acid (BNA), and the 4' ring oxygen may be -S-, -N(U)-, or -C(U) 1 )(U 2 )(In the formula, U, U 1 , and U 2 Each of these can be modified in various ways, including substitution with an atom or group such as H or any preferred substituent. Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of antisense oligonucleotides for their targets and / or to increase nuclease resistance. A representative list of modified sugars includes, but is not limited to, non-bicyclic substituted sugars, in particular non-bicyclic 2'-substituted sugars having 2'-F, 2'-OCH3 or 2'-O(CH2)2-OCH3 substituents; and 4'-thio-modified sugars. Sugars can also be replaced with sugar mimetic groups, among other things. In several embodiments, the sugar is replaced with a six-membered morpholine ring. Methods for preparing modified sugars are known to those skilled in the art.

[0368] In several embodiments, the nucleoside includes bicyclic modified sugars (BNAs), such as LNA (4'-(CH2)-O-2' bridged), 2'-thio-LNA (4'-(CH2)-S-2' bridged), 2'-amino-LNA (4'-(CH2)-NR-2' bridged), ENA (4'-(CH2)2-O-2' bridged), 4'-(CH2)3-2' bridged BNA, 4'-(CH2CH(CH3))-2' bridged BNA''cEt(4'-(CH(CH3)-O-2' bridged), and cMOE BNA (4'-(CH(CH2OCH3)-O-2' bridged).

[0369] Furthermore, this specification also provides locked nucleic acids (LNAs) in which the 2'-hydroxyl group of a ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, forming a 2'-C,4'-C-oxymethylene linkage to form a bicyclic sugar moiety. The linkage may also be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, in which case the term LNA is used for the bicyclic moiety. In the case of an ethylene group at this position, the term ENA(trademark) is used (Singh et al., Chem.Commun., 1998, 4, 455-456; ENA(trademark): Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogs exhibit extremely high double-strand thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against degradation by 3'-exonucleases, and excellent solubility. Potent and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0370] Furthermore, among the LNA isomers being studied, alpha-L-LNA has been shown to have improved stability against 3'-exonucleases. Alpha-L-LNA has been incorporated into antisense gapmers and chimeras that exhibit potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0371] 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, are described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNA and its preparation are also described in WO98 / 39352 and WO99 / 14226.

[0372] Phosphothioate LNA and 2'-thio-LNA, analogs of LNA, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide double helixes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO99 / 14226). The synthesis of 2'-amino-LNA, a novel high-affinity oligonucleotide analog with restricted higher-order structure, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-LNA have also been prepared, and the thermal stability of their double helix structures with complementary RNA and DNA strands has been previously reported.

[0373] Nucleoside linkage This specification describes internucleoside linking groups that link nucleosides or other modified monomer units together to form antisense oligonucleotides. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linking groups include, but are not limited to, phosphodiesters, phosphotriesters, methylmethylphosphonates, phosphoramidates (including phosphorodiamidates), and phosphorothioates. Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Antisense oligonucleotides having non-phosphorus nucleoside linking groups are called oligonucleosides. Modified nucleoside linkages can typically be used to alter and increase the nuclease resistance of antisense oligonucleotides compared to natural phosphodiester linkages. Nucleoside linkages with chiral atoms can be prepared as racemates, chiral, or mixtures. Typical chiral nucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0374] In several embodiments, the phosphate group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In oligonucleotide formation, the phosphate group covalently links adjacent nucleosides to each other, forming linear polymer oligonucleotides. Within oligonucleotides, the phosphate group is generally referred to as forming the internucleoside skeleton of that oligonucleotide. The usual linkage or skeleton of RNA and DNA is the 3'→5' phosphodiester linkage.

[0375] conjugate group In several embodiments, oligonucleotides are modified by covalent bonding of one or more conjugate groups. Generally, conjugate groups can alter one or more properties of oligonucleotides, for example, but not limited to, pharmacodynamic properties, pharmacokinetic properties, binding properties, absorption properties, cell distribution properties, cell uptake properties, charge properties, and clearance properties. Conjugate groups are commonly used in the chemical art and are conjugated directly to oligonucleotides or via optionally selected linking moieties or linking groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In several embodiments, the conjugate group is polyethylene glycol (PEG), which is conjugated to either oligonucleotides or cyclic peptides.

[0376] The conjugate group consists of lipid moieties, e.g., cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NYA 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, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923), etc.

[0377] Linking groups or difunctional linking moieties, such as those known in the art, may be included in the oligonucleotides provided herein. Linking groups are useful for attaching chemical functional groups, conjugate groups, reporter groups, and other groups to selective sites within oligonucleotides, such as antisense oligonucleotides. In some embodiments, the difunctional linking moiety includes a hydrocarbyl moiety having two functional groups. Any linker described herein may be used. In some embodiments, the linker includes a chain structure or oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups used in the difunctional linking moiety include, but are not limited to, electrophiles that react with nucleophiles and electrophiles that react with nucleophiles. In some embodiments, the difunctional linking moiety includes amino, hydroxyl, carboxylic acid, thiol, and unsaturated bonds (e.g., double or triple bonds). Some non-exclusive examples of difunctional linkages include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other linkage groups include, but are not limited to, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Alkynnyl is included, where an unrestricted list of substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0378] In several embodiments, oligonucleotides can be ligated to 10 arginine-serine dipeptide repeats. Oligonucleotides ligated to 10 arginine-serine dipeptide repeats for artificially recruiting splicing enhancer factors have been applied in vitro to induce insertion of mutant BRCA1 and SMN2 exons that would otherwise be skipped. See Cartegni and Krainer 2003, which are incorporated herein by reference.

[0379] In several embodiments, the cargo is an oligonucleotide. In several embodiments, the oligonucleotide is a therapeutic oligonucleotide. In several embodiments, the oligonucleotide is an antisense oligonucleotide. In several embodiments, the cargo is an oligonucleotide that can hybridize to the nucleic acid sequence of the human DMD gene encoding dystrophin. In several embodiments, the oligonucleotide can hybridize to at least a portion of exon 50 of the DMD gene. In several embodiments, the oligonucleotide that hybridizes to at least a portion of exon 50 of the DMD gene may also hybridize to at least a portion of the 5' adjacent intron of the exon, or to at least a portion of the 3' adjacent intron of exon 50. In several embodiments, the oligonucleotides that hybridize to at least a portion of exon 50 of the DMD gene are 5–50, 10–40, 15–35, or 20–30 nucleotides long, for example, at least 15, 16, 17, 18, 19, or 20 nucleotides long and up to 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides long. In several embodiments, the oligonucleotides that hybridize to at least a portion of exon 50 of the DMD gene are 15–35 nucleic acid lengths, 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 long.

[0380] In several embodiments, the oligonucleotide can hybridize to a nucleic acid sequence containing at least a portion of the 5' adjacent intron of exon 50 of the DMD gene. In several embodiments, the oligonucleotide can hybridize to a nucleic acid sequence containing at least a portion of the 3' adjacent intron of exon 50 of the DMD gene. In several embodiments, the oligonucleotide can hybridize to a nucleic acid sequence within exon 50 of the DMD gene. In several embodiments, the antisense oligonucleotide can hybridize to a nucleic acid sequence spanning an intron-exon junction or an exon-intron junction of exon 50 of the DMD gene.

[0381] In the sequences shown herein, the last nucleotide of the acceptor intron sequence preceding exon 50 is represented as nucleotide "-1", the first nucleotide of exon 50 is represented as nucleotide "0", and the first nucleotide of the donor intron sequence following exon 50 is represented as "-1".

[0382] In several embodiments, the nucleic acid sequence of exon 50 of the DMD gene is shown as sequence number 1 (5' to 3') below. [ka]

[0383] In several embodiments, 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 nucleotide of SEQ ID NO: 1 (for example, the oligonucleotide may be 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 2 (6mer, 27mer, 28mer, 29mer, or 30mer), where the first nucleotide of the oligonucleotide is -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, +2 2, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, ​​+49, +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, + Hybridizes to the nucleotides located at positions 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.In several embodiments, the oligonucleotide comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides complementary to the consecutive nucleotide of SEQ ID NO: 1 (for example, the oligonucleotide may be 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27 (It is mer, 28mer, 29mer or 30mer), where the first nucleotide of the oligonucleotide is -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, +2 6, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, ​​+49, +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, + Hybridizes to the nucleotides located at positions 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. In several embodiments, the oligonucleotide comprises 20, 21, 22, 23, 24, or 25 consecutive nucleotides complementary to the consecutive nucleotide of SEQ ID NO: 1 (for example, the oligonucleotide is 20mer, 21mer, 22mer, 23mer, 24mer, or 25mer), where the first nucleotide of the oligonucleotide hybridizes to the nucleotides at positions -5, -4, -3, -2, and -1 in exon 50 of the DMD gene.In several embodiments, the oligonucleotide comprises 20, 21, 22, 23, 24, or 25 consecutive nucleotides complementary to the consecutive nucleotide of SEQ ID NO: 1 (for example, the oligonucleotide is 20mer, 21mer, 22mer, 23mer, 24mer, or 25mer), where the first nucleotide of the oligonucleotide hybridizes to a nucleotide located at position 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 DMD gene. In several embodiments, the oligonucleotide comprises 20, 21, 22, 23, 24, or 25 consecutive nucleotides complementary to the consecutive nucleotide of SEQ ID NO: 1 (for example, the oligonucleotide is 20mer, 21mer, 22mer, 23mer, 24mer, or 25mer), where the first nucleotide of the oligonucleotide is +50 of exon 50 of the DMD gene of SEQ ID NO: 1. Hybridizes to nucleotides at the positions +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. As used herein, “first nucleotide” refers to the first 5' nucleotide of an oligonucleotide cargo.

[0384] The nucleic acid sequence of exon 50 of the DMD gene (in bold and underlined capital letters), along with the 50nt (ununderlined) adjacent upstream intron at the 5' end and the 50nt (ununderlined) adjacent downstream intron at the 3' end, are shown in Sequence ID No. 2 below.

[0385] Exon-50 (italic lowercase = intron; Uppercase / Bold and Underline (=exon). Note: The first nucleotide in the exon sequence is numbered "0". [ka]

[0386] The upstream (5') intron sequence (residues -50 to -1) and the downstream (3') intron sequence (residues -1 to -50) are shown in italicized lowercase letters, and the exon sequence (residues +0 to +108) is shown in bold and underlined uppercase letters. In several embodiments, the nucleic acid sequence of dystrophin exon 50 of the human Duchenne muscular dystrophy (DMD) gene consists of 109 nucleotides shown in bold and underlined uppercase letters.

[0387] In several embodiments, the oligonucleotide comprises one or more nucleotides complementary to one or more nucleotides in the 5' intron sequence adjacent to exon 50 of the DMD gene. In several embodiments, the oligonucleotide comprises one or more nucleotides in the 5' intron sequence adjacent to exon 50 and one or more nucleotides complementary to one or more nucleotides in the exon sequence of exon 50. In several embodiments, the oligonucleotide comprises one or more nucleotides complementary to one or more nucleotides in the 3' intron sequence following exon 50. In several embodiments, the oligonucleotide comprises one or more nucleotides in the 3' intron sequence adjacent to exon 50 and one or more nucleotides complementary to one or more nucleotides in the exon sequence of exon 50.

[0388] In several embodiments, the oligonucleotide hybridizes to a nucleic acid sequence spanning an intron-exon junction or exon-intron junction in exon 50 of the DMD gene. In multiple embodiments, the oligonucleotide is complementary to the target nucleic acid sequence, comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least nineteen, at least twenty or more consecutive nucleotides (i.e., nucleotides starting at position -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1) of the upstream (5') intron preceding exon 50.

[0389] In several embodiments, the oligonucleotide is complementary to the target nucleic acid sequence, which includes at least the first nucleotide at the 5' end of exon 50 (i.e., position 0). In multiple embodiments, the oligonucleotide is complementary to the target nucleic acid sequence, comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty, at least thirty-one, at least thirty-two, at least thirty-three, at least thirty-five

[0390] In several embodiments, oligonucleotides that hybridize to exon 50 of the DMD gene are shown as 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 respect to them.

[0391] In several embodiments, the oligonucleotide hybridizes to a nucleic acid sequence spanning a portion of exon 50 of the DMD gene. In several embodiments, the exon sequence of SEQ ID NO: 2 is shown in bold and underlined (residues 0 to +108). In several embodiments, the oligonucleotide has a nucleic acid sequence 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 respect to them.

[0392] In several embodiments, the oligonucleotide comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides complementary to the consecutive nucleotide of SEQ ID NO: 1 (for example, the oligonucleotide is 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27mer, 28mer, 29mer, or 30mer), where the first nucleotide of the oligonucleotide is -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7, +8, +9 of exon 50 of SEQ ID NO: 1. The nucleotides hybridize to the nucleotides at positions +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, and +35, as shown 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 to them. In multiple embodiments, the first nucleotide of the oligonucleotide 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, +100, The nucleotides hybridize to the nucleotides at positions +101, +102, +103, +104, +105, +106, +107, +108, and +109, as shown in Tables 11A-11D, 12A-12D, or 13, their reverse complementary sequences, or sequences having 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 to them.Exon 50 has the following sequence: [ka]

[0393] In some embodiments, the oligonucleotide hybridizes to the sequence of exon 50 of the DMD gene, selected from one of the nucleic acid sequences shown in Tables 11A-11D, 12A-12D, or 13. In some embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene is selected from one of the nucleic acid sequences shown 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 respect to them. In some embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes one or more modified nucleic acids, one or more modified nucleotide linkages, or a combination thereof. In several embodiments, the oligonucleotide hybridizing to exon 50 comprises one or more morpholine rings, one or more phosphorodiamidate links, or a combination thereof. In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) comprising a sequence selected from one of the following: nucleic acid sequences in Tables 11A-11D, 12A-12D, or Table 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 respect to them. [Table 21-1] [Table 21-2] Table 22-1 Table 22-2 Table 23-1 Table 23-2 Table 24-1 Table 24-2 Table 25 Table 26 Table 27 Table 28 Table 29

[0394] In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-AGTGGTCAGTCCAGGAGCTAGGTC-3' (24-mer-76), 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 to them. In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-GTGGTCAGTCCAGGAGCTAGG-3' (21-mer-78), 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 respect to them.In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-GGTCAGTCCAGGAGCTAGGTCA-3' (22-mer-75), 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 to them. In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-TAGTGGTCAGTCCAGGAGCTAGGT-3' (24-mer-77), 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 respect to them.In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-GCTCCAATAGTGGTCAGTCCAG-3' (22-mer-86), 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 to them. In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-ACCGCCTTCCACTCAGAGCTCAGA-3' (24-mer-14), 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 respect to them.In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-TTACCGCCTTCCACTCAGAGCTCA-3' (24-mer-16), 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 respect to them. In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-GGAGCTAGGTCAGGCTGCTTTG-3' (22-mer-65), 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 respect to them.In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-GTGGTCAGTCCAGGAGCTAGGTC-3' (23-mer-76), 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 respect to them. In several embodiments, the oligonucleotide that hybridizes to exon 50 of the DMD gene is an antisense phosphorodiamidate morpholino oligonucleotide (PMO) containing the sequence 5'-AGGAGCTAGGTCAGGCTGCTTT-3' (22-mer-66), 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 to them.

[0395] In some embodiments, the oligonucleotides hybridizing to exon 50 of the DMD gene include a sequence containing 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T), its reverse complementary sequence, or sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with them. In some embodiments, the oligonucleotides hybridizing to exon 50 of the DMD gene include a sequence containing at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleic acids of a nucleotide sequence containing 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 15 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 16 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 17 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In some embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene comprises a sequence containing 18 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In some embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene comprises a sequence containing 19 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T).In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 20 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 21 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 22 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 23 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 24 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene includes a sequence containing 25 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X=U or T). In several embodiments, the oligonucleotide hybridizing to exon 50 of the DMD gene comprises a sequence containing 26 consecutive nucleic acids of the nucleotide sequence 5'-XX ACC GCC XXC CAC XCA GAG CXC AGA-3' (where X = U or T). In several embodiments, the nucleotide sequence may vary at up to 1, 2, 3, 4, or 5 positions. In several embodiments, the oligonucleotide comprises one or more modified oligonucleotides.In some embodiments, the oligonucleotide comprises at least one phosphorodiamidate morpholino oligonucleotide (PMO). In some embodiments, each nucleotide in the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0396] In several embodiments, any oligonucleotide described herein (including oligonucleotides in Tables 11A-11D, Tables 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 thereto) is a phosphorothioate (PS) nucleotide. The oligonucleotide comprises at least one modified nucleotide or nucleic acid selected from otidol, phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), peptide nucleic acid (PNA), nucleotides containing a 2'-O-methyl (2'-OMe) modified skeleton, 2'O-methoxy-ethyl (2'-MOE) nucleotide, 2',4'-restricted ethyl (cEt) nucleotide, and 2'-deoxy-fluoro-beta-D-arabino nucleic acid (2'F-ANA). In several embodiments, hybridization of the oligonucleotide with the target sequence promotes or induces splicing of exon 50. In several embodiments, the oligonucleotide comprises at least one phosphorodiamidate morpholino oligonucleotide (PMO). In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1,4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides are modified. In some embodiments, each nucleotide in the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0397] Cytosol delivery efficiency Modifying the endosomal escape vehicle (EEV) or its components (such as a cyclic cell-permeable peptide (cCPP), extracyclic peptide (EP), or linker) may improve cytosolic delivery efficiency. This improvement in cytosolic uptake efficiency can be measured by comparing the cytosolic delivery efficiency of the modified EEV with that of a control sequence, which is otherwise identical but does not contain specific modifications to, for example, cCPP, EP, or linker.

[0398] In several embodiments, the EEV-cargo conjugate exhibits improved cytosolic uptake efficiency compared to the cargo alone. Cytosolic uptake efficiency can be measured by comparing the cytosolic delivery efficiency of the EEV-cargo conjugate with that of the cargo alone. In several embodiments, the cargo is an oligonucleotide. In several embodiments, the oligonucleotide is a therapeutic oligonucleotide. In several embodiments, the oligonucleotide is an antisense oligonucleotide. In several embodiments, the oligonucleotide is a PMO.

[0399] As used herein, cytosolic delivery efficiency refers to the ability of an EEV, EEV-cargo conjugate, or cargo to cross the cell membrane and enter the cell's cytosol. The cytosolic delivery efficiency of an EEV, EEV-cargo conjugate, or cargo is not necessarily dependent on the receptor or cell type. Cytosolic delivery efficiency may refer to absolute or relative cytosolic delivery efficiency.

[0400] Absolute cytosolic delivery efficiency is the ratio of the cytosolic concentration of EEV, EEV-cargo conjugate, or cargo to the concentration of EEV, EEV-cargo conjugate, or cargo in the growth medium. Relative cytosolic delivery efficiency refers to the cytosolic concentration of EEV, EEV-cargo conjugate, or cargo compared to the cytosolic concentration of a control of EEV, EEV-cargo conjugate, or cargo. Quantification can be achieved by fluorescently labeling EEV, EEV-cargo conjugate, or cargo (e.g., using FITC dyes) and measuring the fluorescence intensity using techniques well known in the art.

[0401] Relative cytosolic delivery efficiency is determined by comparing (i) the amount of EEV, EEV-cargo conjugate, or cargo internalized by a certain cell type (e.g., HeLa cells) with (ii) the amount of control EEV, EEV-cargo conjugate, or cargo internalized by the same cell type. To measure relative cytosolic delivery efficiency, the cell type can be incubated in the presence of EEV, EEV-cargo conjugate, or cargo for a specified period (e.g., 30 minutes, 1 hour, 2 hours, etc.), and then the amount of cCPP internalized by the cells can be quantified using methods known in the art (e.g., fluorescence microscopy). Separately, the same concentration of control EEV, EEV-cargo conjugate, or cargo can be incubated in the presence of the cell type for the same period, and the amount of control EEV, EEV-cargo conjugate, or cargo internalized by the cells can be quantified.

[0402] Modified EEVs, EEV-cargo conjugates, or cargo ICs targeting intracellular targets 50 Measure the modified EEV, EEV-cargo conjugate, or cargo IC compared to the control sequence. 50 By comparing these factors, the relative cytosol delivery efficiency can be determined.

[0403] Resplied target protein As used herein, “target protein” refers to the amino acid sequence resulting from the transcription and translation of a target gene. As used herein, “resplified target protein” refers to the protein encoded as a result of oligonucleotides being attached to the target pre-mRNA transcribed from the target gene. “Wild-type target protein” refers to the naturally occurring, correctly translated protein isomer resulting from the appropriate splicing of the target pre-mRNA encoded by the wild-type target gene. A respliced ​​target protein may contain one or more amino acid substitutions, deletions, and / or insertions compared to the wild-type target protein. In some embodiments, the respliced ​​target protein retains some degree of wild-type target protein activity. In some embodiments, the respliced ​​target protein is homologous to the wild-type target protein. In several embodiments, the respliced ​​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 several embodiments, the respliced ​​target protein is substantially homologous to the wild-type target protein. In several embodiments, the amino acid sequence of the respliced ​​target protein is at least 50% identical to the amino acid sequence of the wild-type target protein. In several embodiments, the amino acid sequence of the respliced ​​target protein is at least 75% identical to the amino acid sequence of the wild-type target protein. In several embodiments, the amino acid sequence of the respliced ​​target protein is at least 90% identical to the amino acid sequence of the wild-type target protein. In several embodiments, the respliced ​​target protein is a truncated form of the wild-type target protein.

[0404] In multiple embodiments, the respliced ​​target protein can restore one or more phenotypes lost due to a disease associated with the transcription and translation of the target gene, or improve one or more symptoms of a disease associated with the transcription and translation of the target gene. In multiple embodiments, the respliced ​​target protein can restore one or more phenotypes of a disease associated with the expression of the target protein, or improve one or more symptoms of the disease. In multiple embodiments, the respliced ​​target protein is an active fragment of the wild-type target protein. In some embodiments, the respliced ​​target protein functions in substantially the same manner as the wild-type target protein. In some embodiments, the respliced ​​target protein allows a cell to function substantially the same as a similar cell expressing the wild-type target protein. In some embodiments, the respliced ​​target protein does not cure a disease associated with the target gene or target protein, but improves one or more symptoms of the disease. In several embodiments, the respliced ​​target protein improves the target protein function 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%.

[0405] In several embodiments, the respliced ​​target protein may have an amino acid sequence in which one or more amino acids are reduced from the size 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.

[0406] In several embodiments, the respliced ​​target protein may have one or more improved properties compared to the target protein. In several embodiments, the respliced ​​target protein may have one or more improved properties compared to the wild-type target protein. In several embodiments, enzyme activity or stability may be improved by promoting different splicing of the target pre-mRNA. In several embodiments, the respliced ​​target protein may have the same or substantially similar sequence as a wild-type target protein isomer having improved properties compared to another wild-type target protein isomer.

[0407] In several embodiments, one or more properties of the target protein are absent (removed) or reduced in the respliced ​​target protein. In several embodiments, one or more properties of the wild-type target protein are absent (eliminated) or reduced in the respliced ​​target protein. Non-limiting examples of properties that may be reduced or eliminated include immunogenicity, angiogenesis, thrombosis, aggregation, and ligand-binding activity.

[0408] In several embodiments, the respliced ​​target protein contains one or more amino acid relaxations compared to the wild-type target protein. In several embodiments, the substitutions may be conservative or non-conservative. Examples of conservative amino acid substitutions include substituting one amino acid from any of the following groups: basic amino acids (arginine, lysine, histidine), acidic amino acids (glutamic acid, aspartic acid), polar amino acids (glutamine, asparagine), hydrophobic amino acids (leucine, isoleucine, valine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), and small amino acids (glycine, alanine, serine, threonine, methionine). In several embodiments, the charge of a residue is reversed by substituting structurally similar amino acids (e.g., glutamine to glutamic acid or vice versa, aspartic acid to aspartic acid or vice versa). In several embodiments, tyrosine is substituted for phenylalanine or vice versa. Other non-limiting examples of amino acid substitutions are described, for example, in H. Neurath and RLHill, 1979, *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.

[0409] In some embodiments, the respliced ​​target protein may contain substitutions, deletions, and / or insertions at one or more (e.g., several) positions compared to the wild-type target protein. In some embodiments, the number of amino acid substitutions, deletions, and / or insertions in the amino acid sequence of the respliced ​​target protein may be 200 or less, 150 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0410] Treatment method In several embodiments, the EEV-cargo conjugate is administered to patients diagnosed with Duchenne muscular dystrophy (DMD). In several embodiments, the cargo is an oligonucleotide. In several embodiments, the oligonucleotide is a therapeutic oligonucleotide. In several embodiments, the oligonucleotide is an antisense oligonucleotide. In several embodiments, the EEV-cargo conjugate is administered to patients in doses ranging from 0.1 mg / kg to 1000 mg / kg (including all values ​​and ranges within that range).

[0411] A method is provided for treating Duchenne muscular dystrophy (DMD) in a subject requiring such treatment, comprising administering an EEV-cargo conjugate disclosed herein. In some embodiments, the target gene is DMD. In some embodiments, the target sequence includes at least a portion of exon 50 of the DMD gene, at least a portion of the 3' intron adjacent to exon 50 of the DMD gene, at least a portion of the 5' intron adjacent to exon 50 of the DMD gene, or a combination thereof.

[0412] In some embodiments, treatment means partial or complete reduction, improvement, alleviation, inhibition, delay of onset, reduction of severity and / or frequency of one or more symptoms in a subject.

[0413] In several embodiments, a method is provided for modifying the expression of a target gene in a subject requiring such modification, the method comprising administering an EEV-cargo conjugate disclosed herein. In several embodiments, the treatment results in reduced expression of the target protein. In several embodiments, the treatment results in the expression of a respliced ​​target protein. In several embodiments, the treatment results in the preferential expression of the wild-type target protein isomer.

[0414] In several embodiments, the treatment reduces the expression of the target protein 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 mean level of the target protein in the subject before treatment or the mean level of the target protein in one or more untreated control individuals with similar diseases. In several embodiments, the treatment increases the expression of the respliced ​​target protein 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 mean level of the target protein in the subject before treatment or the mean level of the target protein in one or more untreated control individuals with similar diseases. In several embodiments, treatment increases or decreases the expression of the wild-type target protein isomer 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 mean level of the target protein in the subject before treatment or the mean level of the target protein in one or more untreated control individuals with similar diseases.

[0415] Terms such as “improve,” “increase,” “decrease,” and “decrease,” as used herein, indicate values ​​compared to a control. In embodiments, a preferred control is a baseline measurement, e.g., a measurement in the same individual before the commencement of the treatment described herein, or a measurement in a control individual (or more control individuals) in the absence of the treatment described herein. A “control individual” is an individual suffering from the same disease, being approximately the same age and / or sex as the individual being treated (to ensure that the disease stage in the treated individual and the control individual(s) is comparable).

[0416] The individual being treated (also referred to as the “patient” or “subject”) is an individual (fetus, infant, child, adolescent, or adult) that has or is at risk of developing the disease. The individual may have a disease mediated by abnormal gene expression or abnormal gene splicing. In some embodiments, an individual with the disease may have wild-type target protein expression or activity levels that are 1% to 99% of the normal protein expression or activity levels in an individual without the disease. In some embodiments, the 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 dystrophin expression or activity levels. In some embodiments, an individual may have target protein expression or activity levels that are 1% to 500% higher than the normal wild-type target protein expression or activity levels. In several embodiments, the range includes, but is not limited to, target protein expression or activity levels that are 1-10%, 10-50%, 50-100%, 100-200%, 200-300%, 300-400%, 400-500%, or 500-1000% higher.

[0417] In several embodiments, the individual is one who has recently been diagnosed with the disease. Typically, early treatment (treatment initiated as soon as possible after diagnosis) is important to minimize the impact of the disease and maximize the benefits of treatment.

[0418] In several embodiments, the efficacy of the EEV-cargo conjugate is evaluated in animal models of DMD. Animal models are valuable resources for studying disease pathogenesis and provide a means to test dystrophin-related activity. In several embodiments, both dystrophin-negative mdx mice and dogs with golden retriever muscular dystrophy (GRMD) (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003) are used to evaluate the EEV-cargo conjugate. In several embodiments, the C57BL / 10ScSn-Dmdmdx / (Bl10 / mdx) or D2.B10-Dmdmdx / J(D2 / mdx) mouse models are used to evaluate the EEV-cargo conjugate. In several embodiments, transgenic mice possessing the human DMD gene but lacking the mouse Dmd gene (hDMD / Dmd-null mice) are used to evaluate the EEV-cargo conjugate. These mice can be generated by crossing male hDMD mice (available from Jackson Laboratory, Bar Harbor, ME) with female DMD-null mice. The following references describe these models, and they are incorporated herein by reference in their entirety: J Neuromuscul Dis. 2018;5(4):407-417.; Proc Natl Acad Sci US A. 1984;81(4):1189-92.; Am J Pathol. 2010;176(5):2414-24.; J Clin Invest. 2009;119(12):3703-12; International Publication No. WO2019014772. These and other animal models can also be used to measure the functional activity of various dystrophin proteins.

[0419] In several embodiments, an in vitro model is used to evaluate the efficacy of the compositions of this disclosure. In several embodiments, the in vitro model is an immortalized muscle cell model. This model is described in the following paper, which is incorporated herein by reference in its entirety: Nguyen et al. J Pers Med. 2017 Dec;7(4):13.

[0420] Manufacturing method EEV-cargo conjugates can be prepared by various methods known to those skilled in the art of organic synthesis, or by variations thereof as understood by those skilled in the art. EEV-cargo conjugates can be prepared from readily available starting materials. Reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0421] Variations of EEV-cargo conjugates include the addition, deletion, or movement of various components of the EEV-cargo conjugate. Similarly, the chirality of the molecule can change if 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, as well as 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 Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0422] The starting materials and reagents used in the preparation of EEV-Cargo Conjugate and its compositions are from 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), and Schering. It is available from private suppliers such as Plough (Kenilworth, NJ) or Boehringer Ingelheim (Ingelheim, Germany), or from publications 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 Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc.).It is either prepared by methods known to those skilled in the art, following the procedures described in references such as (1989). Other materials, such as pharmaceutical carriers, can be obtained from private sources.

[0423] The reaction to produce EEV-cargo conjugates can be carried out in solvents that can be selected by those skilled in the field of organic synthesis. The solvent may be substantially inactive with the starting materials (reactants), intermediates, or products under the conditions under which the reaction takes place, i.e., temperature and pressure. The reaction can be carried out in one solvent or a mixture of two or more solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) It can be monitored by spectroscopic means such as infrared spectroscopy, spectrophotometric methods (e.g., UV-Vis), or mass spectrometry, or by chromatography such as high-performance liquid chromatography (HPLC) or thin-layer chromatography.

[0424] EEV and its components (including EP and cCPP), as well as EEV-cargo conjugates, can be prepared by solid-phase peptide synthesis, in which case the amino acid α-N terminus is protected by an acidic or basic protecting group. Such protecting groups should be stable to peptide linkage conditions and easily removable without disrupting the elongated peptide chain or racemizing any of the chiral centers contained in the chain. Suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, and 2-cyano-t-butyloxycarbonyl. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group can be used. Other side-chain protecting groups include 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc, and adamantyloxycarbonyl for side-chain amino groups such as lysine and arginine; benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl, and acetyl (Ac) for tyrosine; t-butyl, benzyl, and tetrahydropyranyl for serine; trityl, benzyl, Cbz, p-toluenesulfonyl, and 2,4-dinitrophenyl for histidine; formyl for tryptophan; benzyl and t-butyl for aspartic acid and glutamic acid; and triphenylmethyl (trityl) for cysteine. In solid-phase peptide synthesis, the α-C-terminal amino acid is bonded to a suitable solid support or resin. A suitable solid support useful for the above synthesis is a material that is inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and is insoluble in the medium used.The solid support for the synthesis of α-C-terminal carboxypeptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin, available from Applied Biosystems (Foster City, Calif.). The α-C terminal amino acid is coupled to the resin by mediation coupling at a temperature of 10°C to 50°C for about 1 to 24 hours in a solvent such as dichloromethane or DMF, with or without the addition of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl). This coupling is performed using N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU). When the solid support is a 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, such as piperidine, before coupling with the α-C terminal amino acid as described above. One method for coupling to a deprotected 4(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin is with O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. Coupling of successive protected amino acids can be performed in an automated polypeptide synthesizer. In one example, the α-N terminus of the amino acid of the peptide chain being extended is protected with Fmoc. Removal of the Fmoc protecting group from the α-N terminal side of the peptide being extended is achieved by treatment with a secondary amine, such as piperidine. Next, each protected amino acid is introduced in a 3x molar excess, and coupling is performed in DMF.The coupling agents may be O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). After solid-phase synthesis is complete, the polypeptide is cleaved from the resin and deprotected, either sequentially or in a single operation. Cleavage and deprotection of the polypeptide can be achieved in a single operation by treating the polypeptide bound to the resin with a cleavage reagent containing thioanisole, water, ethanedithiol, and trifluoroacetic acid. If the α-C terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be cleaved by transesterification with methanol, for example, followed by aminolysis or direct amide transfer. The protected peptide may be purified at this point, or the process may proceed directly to the next step. Removal of the side-chain protecting group can be achieved using the above cleavage cocktail. Completely deprotected peptides can be purified by a series of chromatographic steps using one or all of the following types: ion exchange with a weakly basic resin (acetic acid type); hydrophobic adsorption chromatography with underivativeated polystyrene-divinylbenzene (e.g., Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography with carboxymethylcellulose; partition chromatography, e.g., Sephadex G-25, LH-20, or countercurrent partitioning; high-performance liquid chromatography (HPLC), particularly reversed-phase HPLC with octyl- or octadecylsilyl-silica bonded phase column packing.

[0425] The above polymers, such as PEG groups, can be conjugated to oligonucleotides under any preferred conditions used to react the activated polymer molecule with a protein. Any means known in the art can be used, including acylation, reductive alkylation, Michael addition, thiol alkylation, or other chemoselective conjugation / ligation methods to a reactive group on an oligonucleotide (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimide, or hydrazino group) via a reactive group on the PEG moiety (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimide, or hydrazino group). Activated groups that can be used to link a water-soluble polymer to one or more proteins include, but are not limited to, sulfones, maleimides, sulfhydryls, thiols, triflates, toresylates, azidiline, oxiranes, 5-pyridyls, and alpha-halogenated acyl groups (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). When conjugating to oligonucleotides by reductive alkylation, the selected polymer should have a single reactive aldehyde so that the degree of polymerization is controlled. 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).

[0426] To directly and covalently link oligonucleotides to cyclic peptides, appropriate amino acid residues of the CPP can be reacted with an organic derivatizing agent capable of reacting with the side chain, N-terminus, or C-terminus of a selected amino acid. Examples of reactive groups on the peptide or conjugate moiety include aldehydes, amino acids, esters, thiols, α-haloacetyls, maleimide, or hydrazino groups. Examples of derivatizing agents include maleimidobenzoylsulfosuccinimide (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, or other agents known in the art.

[0427] Methods for synthesizing oligonucleotides are known in the art. This disclosure is not limited to methods for synthesizing oligonucleotides. In some embodiments, reactive phosphate groups useful for forming nucleoside linkages include, for example, phosphodiesters and phosphorothioate nucleoside linkages. Methods for preparing and / or purifying precursors or oligonucleotides are not limitations to the compositions or methods provided herein. Methods for the synthesis and preparation of DNA, RNA, and oligonucleotides are well known to those skilled in the art.

[0428] Oligomerization of modified and unmodified nucleosides can be carried out by conventional methods in accordance with the literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and / or the literature procedures for RNA (Scaringe, Methods (2001), 23, 206-217; Gait et al., Applications of Chemically Synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36; Gallo et al., Tetrahedron (2001), 57, 5707-5713).

[0429] The oligonucleotides provided herein can be prepared by simple and conventional methods via well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several distributors, including, for example, Applied Biosystems (Foster City, CA). Any other synthetic means known in the art may be employed additionally or alternatively. Similar techniques are well known for preparing oligonucleotides such as phosphorothioates and alkylated derivatives. The present invention is not limited to methods for synthesizing oligonucleotides.

[0430] Methods for purifying and analyzing oligonucleotides are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. Such synthesis and analysis methods can be carried out in multiwell plates. The methods of the present invention are not limited to the methods for purifying oligomers.

[0431] Method of administration The in vivo administration of EEV-cargo conjugates and compositions containing them disclosed herein can be achieved by any suitable methods and techniques currently or hereafter known to those skilled in the art. For example, EEV-cargo conjugates may be formulated in a physiologically or pharmaceutically acceptable form and administered via any suitable route known in the art, including, for example, oral and parenteral administration routes. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal, and intrathecal administration, such as by injection. Administration of the EEV-cargo conjugates or compositions disclosed herein may be a single dose or may be continuous or at different intervals, as can be readily determined by those skilled in the art.

[0432] The EEV-cargo conjugates and compositions containing them disclosed herein can also be administered using liposome technology, sustained-release capsules, embedded pumps, and biodegradable containers. These delivery methods can advantageously provide a uniform dosage over extended periods. EEV-cargo conjugates can also be administered in their salt derivative forms or crystalline forms.

[0433] EEV-cargo conjugates can be formulated according to known methods for preparing pharmaceutically acceptable compositions. These formulations are well known to those skilled in the art and are described in detail in several readily available sources. For example, Remington's Pharmaceutical Science (1995) by EW Martin describes formulations that may be used in connection with the methods of this disclosure. Generally, EEV-cargo conjugates can be formulated by combining an effective amount of EEV-cargo conjugate with a suitable carrier to facilitate the effective administration of the EEV-cargo conjugate. The compositions used can also be in various forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and injectable solutions, and sprays. The form depends on the intended mode of administration and therapeutic use. The compositions also include conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, physiological saline, and equivalent carriers and diluents. To provide such a dose for a desired therapeutic treatment, the composition may contain one or more EEV-cargo conjugates in a total weight of 0.1% to 100% by weight, based on the total weight of the composition including the carrier or diluent.

[0434] Suitable formulations for administration include, for example, aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the recipient's blood; as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. The formulations can be supplied in unit dose or multi-dose containers, such as sealed ampoules and vials, and can be stored before use in a sterile liquid carrier, such as a freeze-dried state requiring only the conditions of water for injection. Immediate injection solutions and suspensions can be prepared from sterile powders, granules, tablets, etc. In addition to the components specifically mentioned above, it should be understood that the composition may contain other standard agents in the art, taking into account the type of formulation.

[0435] EEV-cargo conjugates and compositions containing them may be delivered to cells either by direct contact with the cells or via carrier means. Carrier means are known in the art and include, for example, encapsulating the composition in a liposome. Another delivery means includes conjugating the EEV-cargo conjugate to a targeted protein or nucleic acid so as to be delivered to a target cell. U.S. Patent No. 6,960,648 and U.S. Patent Publications 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition, enabling the composition to travel across biological membranes. U.S. Patent Publication 20020035243 also describes a composition for transporting biological portions across cell membranes and delivering them into cells. EEV-cargo conjugates can also be incorporated into polymers, including poly(DL-lactide-co-glycolide) polymers for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a molar ratio of 20:80 (used in GLIADEL); chondroitin; chitin; and chitosan.

[0436] EEV-Cargo Conjugate and its compositions, including their pharmaceutically acceptable salts or prodrugs, can be administered intravenously, intramuscularly, or intraperitoneally by injection or infusion. Solutions of the activator or its salts can be prepared in water and optionally mixed with non-toxic surfactants. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as in oil. These preparations may contain preservatives to prevent microbial growth under normal storage and use conditions.

[0437] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, prepared for immediate preparation of sterile solutions or dispersions for injection or infusion, and optionally encapsulated in liposomes. The final dosage form must be sterile, fluid, and stable under manufacturing and storage conditions. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, for example, containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by liposome formation, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Optionally, inhibition of microbial activity may be provided by various other antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Often, isotonic agents, such as sugars, buffers, or sodium chloride, may be included. Sustained absorption of an injectable composition can be achieved by including an absorption-delaying agent, such as aluminum monostearate and gelatin.

[0438] Sterile solutions for injection are prepared by incorporating the EEV-cargo conjugate in the required amount, along with various other components listed above, into a suitable solvent, and then, if necessary, by sterilizing by filtration. In the case of sterile powders for the preparation of sterile solutions for injection, the preparation method includes vacuum drying and freeze-drying techniques, which yield powders of the active ingredients and any additional desired ingredients present in the previously sterile filtered solution.

[0439] The effective dosage of EEV-cargo conjugate and its pharmaceutical composition can be determined by comparing its in vitro activity with its in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known to those skilled in the art.

[0440] The dosage range for administering a composition should be large enough to produce the desired effect that affects the symptoms or disorder. The dosage should not be so large that it causes adverse side effects such as undesirable cross-reactions or anaphylactic reactions. Generally, the dosage may vary depending on the patient's age, condition, sex, and the severity of the disease, and may be determined by those skilled in the art. If there are any contraindications, the dosage may be adjusted by the individual physician. The dosage may vary and may be administered once or more times a day over a period of one or several days.

[0441] Pharmaceutical compositions comprising EEV-cargo conjugate in combination with a pharmaceutically acceptable carrier are also disclosed. Pharmaceutical compositions containing a certain amount of EEV-cargo conjugate, suitable for oral, topical, or parenteral administration are provided. The dose administered to a patient, particularly a human, should be sufficient to produce a therapeutic response in the patient within a reasonable timeframe without lethal toxicity and causing only tolerable side effects or pathological conditions. Those skilled in the art will understand that the dose depends on various factors, including the subject's condition (health), the subject's weight, the type of concomitant therapy (if any), the frequency of treatment, the treatability ratio, and the severity and stage of the pathological condition.

[0442] Kits containing EEV-cargo conjugate in one or more containers are also disclosed. The kits of this disclosure may optionally include pharmaceutically acceptable carriers and / or diluents. In some embodiments, the kit includes one or more other components, auxiliaries, or adjuvants described herein. In some embodiments, the kit includes one or more anticancer agents, such as drugs described herein. In some embodiments, the kit includes instructions or packaging materials describing a method of administering the EEV-cargo conjugate or a composition thereof. The containers of the kit may be made of any preferred material, e.g., glass, plastic, metal, etc., and may be of any preferred size, shape, or configuration. In some embodiments, the EEV-cargo conjugate is provided in the kit as a solid, such as in tablet, pill, or powder form. In some embodiments, the EEV-cargo conjugate is provided in the kit as a liquid or solution. In some embodiments, the kit includes an ampoule or syringe containing the EEV-cargo conjugate in liquid or solution form.

[0443] Several embodiments of the present invention have been described. However, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are also included in the following claims.

[0444] Specific definition When used in the specification and the attached claims, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise explicitly indicated by the context. For example, a reference to "a composition" includes mixtures of two or more such compositions, a reference to "an agent" includes mixtures of two or more such agents, and a reference to "the component" includes mixtures of two or more such components.

[0445] As used herein, the terms “cyclic cell-permeable peptide” or “cCPP” refer to peptides that facilitate the delivery of cargo to the cytosol of cells.

[0446] As used herein, the term “endosomal escape vehicle” (EEV) refers to a cCPP conjugated to a linker and / or extracyclic peptide (EP) by chemical linkage (i.e., covalent or noncovalent interaction).

[0447] As used herein, the term “EEV-cargo conjugate” refers to an endosomal escape vehicle (EEV) as defined herein, conjugated to an oligonucleotide by chemical linkage (i.e., covalent or noncovalent interaction). The oligonucleotide can be delivered to a cell by the EEV.

[0448] As used herein, the term “extracyclic peptide” (EP) refers to two or more amino acid residues linked by peptide bonds that can be conjugated to a cyclic cell-permeable peptide (cCPP). When conjugated to a cCPP, an EP can alter the tissue distribution and / or retention of a compound. Typically, an EP comprises at least one positively charged amino acid residue, e.g., at least one lysine residue and / or at least one arginine residue. Non-limiting examples of EPs are described herein. An EP may be a peptide identified in the art as a nuclear localization sequence (NLS). Non-limiting examples of nuclear localization include the nuclear localization sequence of the SV40 virus large T antigen (its smallest functional unit is the seven-amino acid sequence PKKKRKV), the nucleoplasmin binocular NLS having the sequence NLSKRPAAIKKAGQAKKKK, the c-myc nuclear localization sequence having the amino acid sequence PAAKRVKLD or RQRRNELKRSF, the IBB domain sequence of importin-alpha RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, the myoma T protein sequences VSRKRPRP and PPKKARED, the human p53 sequence PQPKKKPL, and mouse c-abl Examples include the sequence SALIKKKKKMAP of influenza IV, the sequences DRLRR and PKQKKRK of influenza virus NS1, the sequence RKLKKKIKKL of hepatitis virus delta antigen, the sequence REKKKFLKRR of mouse Mxl protein, the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase, and the sequence RKCLQAGMNLEARKTKK of steroid hormone receptor (human) glucocorticoid. International Publication No. 2001 / 038547 describes additional examples of NLS, which are incorporated herein by reference in their entirety.

[0449] As used herein, “linker” or “L” refers to one or more moieties (e.g., moieties that covalently link an extracyclic peptide (EP) and an oligonucleotide to a cyclic cell-permeable peptide (cCPP). The linker may include natural or non-natural amino acids or polypeptides. The linker may be a synthetic compound containing two or more suitable functional groups. The linker may include a polyethylene glycol (PEG) component. The linker may include one or more amino acid (AA) components. The linker may include one or more hydrophobic groups (X).

[0450] As used herein, the term “oligonucleotide” refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides in an oligonucleotide may be modified. Oligonucleotides may comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides may consist of native and / or modified nucleic acid bases, sugars, and covalent nucleoside linkages, and may further comprise non-nucleic acid conjugates. In several embodiments, oligonucleotides are therapeutic oligonucleotides.

[0451] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to natural or synthetic molecules containing two or more amino acids, where the carboxyl group of one amino acid is linked to the alpha-amino group of the other. Two or more amino acid residues may be linked by the carboxyl group and alpha-amino group of one amino acid. Two or more amino acids in a polypeptide may be linked by peptide bonds. Polypeptides may include peptide backbone modifications in which two or more amino acids are covalently linked by bonds other than peptide bonds. Polypeptides may contain one or more unnatural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide. The term polypeptide includes naturally occurring amino acids and artificially produced amino acids. The term polypeptide includes not only peptides, for example, containing 2 to 100 amino acid residues, but also proteins, including, but not limited to, therapeutic proteins such as antibodies, enzymes, receptors, and soluble proteins, containing more than 100 or 1000 amino acid residues.

[0452] As used herein, "polyethylene glycol" and "PEG" are used interchangeably. PEG refers to the -CH2CH2O- group of the chemical formula.

[0453] The term "PEG2" refers to 2-[2-[2-aminoethoxy]ethoxy]acetic acid.

[0454] The term "therapeutic polypeptide" refers to polypeptides that possess therapeutic, prophylactic, or other biological activity. Therapeutic polypeptides can be produced in any preferred manner. For example, therapeutic polypeptides may be isolated or purified from naturally occurring environments, chemically synthesized, recombinantly produced, or a combination thereof.

[0455] The term "small molecule" refers to organic compounds that possess pharmacological activity and have a molecular weight of less than 2000 daltons, less than 1000 daltons, or less than 500 daltons. Small molecule therapeutics are typically manufactured by chemical synthesis.

[0456] As used herein, the term “consecutive” refers to two amino acids connected by a covalent bond. For example, [ka] In relation to representative cyclic cell-permeable peptides (cCPPs), AA1 / AA2, AA2 / AA3, AA3 / AA4, and AA5 / AA1 are examples of consecutive amino acid pairs.

[0457] When used herein, a chemical species residue refers to a derivative of that chemical species present in a particular product. To form a product, at least one atom of a chemical species is replaced by bonding to another part, thereby causing the product to contain a derivative or residue of that chemical species. For example, the cyclic cell-permeable peptides (cCPPs) described herein incorporate amino acids (e.g., arginine) through the formation of one or more peptide bonds. The amino acids incorporated into the cCPP may be referred to as residues, or simply as amino acids. Therefore, arginine or arginine residues are, [ka] It refers to.

[0458] The term "protonated form" refers to the protonated form of an amino acid. For example, the guanidine group on the side chain of arginine can be protonated to form a guanidinium group. The structure of the protonated form of arginine is: [ka] That is the case.

[0459] As used herein, the term "chirality" refers to a molecule having multiple stereoisomers with different three-dimensional spatial arrangements of atoms, where one stereoisomer is a mirror image of the other that cannot be superimposed. Amino acids, with the exception of glycine, have a chiral carbon atom adjacent to the carboxyl group. The term "enantiomer" refers to a stereoisomer that is chiral. A chiral molecule may be an amino acid residue having "D" and "L" enantiomers. Molecules that do not have a chiral center, such as glycine, may be called "achiral."

[0460] As used herein, the term "hydrophobic" refers to a portion that is insoluble in water or has minimal solubility in water. Generally, a neutral and / or nonpolar portion, or a portion that is primarily neutral and / or nonpolar, is hydrophobic.

[0461] 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 under the definition of aromatic.

[0462] "Alkyl," "alkyl chain," or "alkyl group" refers to a fully saturated, linear or branched hydrocarbon chain radical having 1 to 40 carbon atoms, bonded to the rest of the molecule by single bonds. Alkyl groups containing any number of carbon atoms from 1 to 40 are included. Alkyl groups containing up to 40 carbon atoms are C1-C 40 Alkyl atoms, which are alkyl groups containing up to 10 carbon atoms, are C1-C 10 Alkyl groups containing up to six carbon atoms are C1-C6 alkyl groups, and alkyl groups containing up to five carbon atoms are C1-C5 alkyl groups. C1-C5 alkyl groups include C5 alkyl groups, C4 alkyl groups, C3 alkyl groups, C2 alkyl groups, and C1 alkyl groups (i.e., methyl groups). C1-C6 alkyl groups include all the parts described above for C1-C5 alkyl groups, and also include C6 alkyl groups. C1-C 10Alkyl includes all the parts described above for C1-C5 alkyl and C1-C6 alkyl, and further includes C7, C8, C9 and C 10 It also includes alkyl groups. Similarly, C1-C 12 Alkyl includes all the aforementioned parts, and further C 11 and C 12 Includes alkyl groups. C1-C 12 Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless otherwise specifically provided herein, alkyl groups may be substituted by choice.

[0463] "Alkylene," "alkylene chain," or "alkylene group" refers to a fully saturated, linear or branched divalent hydrocarbon chain radical having 1 to 40 carbon atoms. C2-C 40 Non-limiting examples of alkylenes include ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, and n-butynylene. Unless otherwise specified herein, alkylene chains may be optionally substituted.

[0464] An "alkenyl," "alkenyl chain," or "alkenyl group" refers to a linear or branched hydrocarbon chain radical having 2 to 40 carbon atoms and one or more carbon-carbon double bonds. Each alkenyl group is bonded to the rest of the molecule by a single bond. Alkenyl groups can contain any number of carbon atoms from 2 to 40. Alkenyl groups containing up to 40 carbon atoms are C2-C 40 Alkenyls are alkenyls that contain up to 10 carbon atoms, C2-C 10Alkenyls are C2-C6 alkenyls, and alkenyl groups containing up to six carbon atoms are C2-C5 alkenyls. C2-C5 alkenyls include C5 alkenyls, C4 alkenyls, C3 alkenyls, and C2 alkenyls. C2-C6 alkenyls include all the parts described above for C2-C5 alkenyls, and also include C6 alkenyls. 10 Alkenyls include all the parts described above for C2-C5 alkenyls and C2-C6 alkenyls, and further include C7, C8, C9 and C 10 This also includes alkenyls. Similarly, C2-C 12 Alkenil includes all the parts mentioned above, and further C 11 and C 12 Includes alkenyls. C2-C 12 Non-limiting examples of alkenyls include ethenyl(vinyl), 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, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl Examples include 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 specified herein, alkyl groups may be substituted by choice.

[0465] "Alkenylene," "alkenylene chain," or "alkenylene group" refers to a straight or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and one or more carbon-carbon double bonds. C2-C 40 Non-exclusive examples of alkenylenes include ethene, propene, and butene. Unless otherwise specified herein, the alkenylene chain may be of any choice.

[0466] "Alkoxy" or "alkoxy group" refers to an -OR group, where R is an alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl as defined herein. Unless otherwise specifically provided herein, alkoxy groups may be optionally substituted.

[0467] "Acyl" or "acyl group" refers to a -C(O)R group, where R is hydrogen, alkyl, alkenyl, alkynyl, carbocykyl, or heterocyclyl as defined herein. Unless otherwise specifically provided herein, acyls may be optionally substituted.

[0468] "Alkylcarbamoyl" or "Alkylcarbamoyl group" is -OC(O)-NR a R b It refers to the base, and in the formula, R a and R b These are the same or different, independently of each other, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or R as defined herein. a R b These may together form a cycloalkyl group or heterocyclyl group as defined herein. Unless otherwise specifically provided herein, the alkylcarbamoyl group may be optionally substituted.

[0469] "Alkylcarboxamidyl" or "Alkylcarboxamidyl group" is -C(O)-NR a R bIt refers to the base, and in the formula, R a and R b These are the same or different, independently, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, or heterocyclyl groups as defined herein, or R a R b These may together form a cycloalkyl group as defined herein. Unless otherwise specifically provided herein, the alkylcarboxamidyl group may be optionally substituted.

[0470] "Aryl" refers to a hydrocarbon cyclic radical comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl radicals can be monocyclic, bicyclic, tricyclic, or tetracyclic systems, and may include fused or bridging cyclic systems. Examples of aryl radicals include, but are not limited to, aryl radicals derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically provided herein, the term "aryl" refers to optionally substituted aryl radicals.

[0471] "Heteroaryl" refers to a 5-20 membered cyclic radical comprising a hydrogen atom, 1-13 carbon atoms, 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur, and at least one aromatic ring. For the purposes of the present invention, heteroaryl radicals can be monocyclic, bicyclic, tricyclic, or tetracyclic systems, and may include fused or bridging cyclic systems. The nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized, and the nitrogen atom may be optionally quaternized. Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxolyl, benzofuranil, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanil, benzonaphthofuranil, benzoxazolyl, benzodioxolyl, benzodioxynil, benzopyranil, benzopyranonil, benzofuranil, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, sinnolinil, dibenzofuranil, dibenzothiophenyl, furanil, furanonil, isothiazolyl, imidazolyl, indazolyl, indazolyl, isoindolyl, in Examples include, but are not limited to, dolinyl, isoindolinyl, isoquinolyl, indolidinyl, isoxazolyl, naphthilidinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxyranil, 1-oxidepyridinyl, 1-oxidepyrimidinyl, 1-oxidepyradinyl, 1-oxidepyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxadinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridadinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless otherwise specified herein, heteroaryl groups may be optionally substituted.

[0472] As used herein, the term “substituted” means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom is substituted with a non-hydrogen atom, such as, but not limited to, halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, 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 above groups in which one or more atoms are replaced by higher-order bonds (e.g., double or triple bonds) with heteroatoms such as oxygen in oxo, carbonyl, carboxyl, and ester groups, as well as nitrogen in imines, oximes, hydrazones, and nitriles. For example, "substituted" means that one or more atoms are replaced by -NR g R h , -NR g C(=O)R h , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g SO2R h -OC(=O)NR g R h , -OR g , -SR g -SOR g , -SO2R g , -OSO2R g , -SO2OR g ,=NSO2Rg , and -SO2NR g R h Contains any of the above groups that are replaced by -C(=O)R. "Substituted" also means that one or more hydrogen atoms are replaced by -C(=O)R g , -C(=O)OR g -C(=O)NR g R h ,-CH2SO2R g -CH2SO2NR g R h This refers to any of the above bases that are replaced by R. g and R h "A" is the same or different, independently of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further refers to any of the above groups in which one or more atoms are replaced by amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" can also mean an amino acid in which one or more atoms on the side chain are replaced by alkyl, alkenyl, alkynyl, acyl, alkylcarboxamidyl, alkoxycarbonyl, carbocykyl, heterocyclyl, aryl, or heteroaryl substituents. In addition, each of the aforementioned substituents may also be optionally substituted with one or more of the substituents listed above.

[0473] In this specification, "phenyl" refers to the cyclic atomic group represented by the formula C6H5, which can be covalently bonded to a molecule as a functional group. Phenyl may be abbreviated as "Ph" in this specification.

[0474] As used herein, “subject” refers to an individual. Therefore, “subject” may 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” may also include primates or mammals such as humans. Therefore, a subject may be a human or a veterinary patient. The term “patient” refers to a subject receiving treatment from a clinician, e.g., an internist.

[0475] The term “treatment” refers to the medical management of a patient with the intention of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at improving the disease, pathological condition, or disorder, and causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative care, i.e., treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; preventive care, i.e., treatment aimed at minimizing, or partially or completely inhibiting, the onset of the associated disease, pathological condition, or disorder; and supportive care, i.e., treatment used to complement other specific therapies aimed at improving the associated disease, pathological condition, or disorder.

[0476] The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of a disease or disorder. Such improvement requires only reduction or alteration and does not necessarily require elimination.

[0477] The term "pharmaceutically acceptable" means a compound, material, composition, and / or dosage form that, within reasonable medical judgment and in light of a reasonable benefit / risk ratio, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0478] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, assists or enhances, with respect to its intended use or purpose, the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristics of that compound or composition. For example, a carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the target.

[0479] The term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders that are reconstituted into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifiers, and dispersants. Microbial action can be prevented by including various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride. Injectable formulations can be sterilized, for example, by filtration through a bacterial capture filter, or by incorporating a sterilizing agent in the form of a sterile solid composition and dissolving or dispersing it in sterile water or another sterile injectable medium immediately before use. Suitable inert carriers may include sugars such as lactose.

[0480] The term "sequence identity" refers to the percentage of amino acids that are identical and in the same relative positions between two polypeptide sequences. Thus, one polypeptide sequence has a predetermined percentage of sequence identity compared to another polypeptide sequence. For sequence comparison, typically one sequence serves as a reference sequence and is compared to the test sequence. Those skilled in the art will understand that two sequences are generally considered "substantially identical" if they contain identical residues in corresponding positions. In several embodiments, sequence identity between two amino acid sequences may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277) in a version available as of the filing date. The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (emboss version of BLOSUM62) substitution matrix. The Needle-labeled "longest identity" output (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows: (identical residues × 100) / (length of alignment - total number of gaps in the alignment).

[0481] In several embodiments, sequence identity may be determined using a version of the Smith-Waterman algorithm available as of the filing date.

[0482] 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 predetermined percentage of sequence homology compared to another polypeptide sequence. As those skilled in the art will recognize, two sequences are generally considered "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues that have appropriately similar structural and / or functional properties. For example, as those skilled in the art will know, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids and / or having "polar" or "nonpolar" side chains, and substituting one amino acid for another of the same type can often be considered a "homologous" substitution.

[0483] As is well known in the art, amino acid sequences can be compared using any of the various algorithms available in commercially available computer programs such as BLASTP, gapped BLAST, and PSI-BLAST, which exist as of the filing date. Such programs are described in Altschul, et al., Basic local alignment search tool, J.Mol.Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol.132), Humana Press, 1999. In addition to identifying homologous sequences, the aforementioned programs typically provide an indicator of the degree of homology.

[0484] The term “antisense oligonucleotide” refers to a high molecular weight nucleic acid structure (also called oligonucleotide or polynucleotide) that is at least partially complementary to the target nucleic acid molecule to hybridize. Oligonucleotides can be short (in some embodiments, less than 50 ba...

Claims

1. EEV-Cargo Conjugate, (a) A cyclic cell-permeable peptide (cCPP), wherein the cCPP has the structure of formula (2): 【Chemistry 1】 or having a protonated form thereof, During the ceremony, R １ , R ２ , and R ３ Each of these can independently be H, or an amino acid residue having a side chain containing an aryl group or a heteroaryl group. R １ , R ２ , and R ３ At least two of these are independently aryl side chains or heteroaryl side chains of amino acids. R ４ and R ６ These are independently H or amino acid side chains. AA ＳＣ is an amino acid side chain, q is 1, 2, 3, or 4. m' and m'' are each independent integers from 0 to 3, and the cCPP is... (b) A linear extracyclic peptide (EP) containing 2 to 10 amino acid residues, (c) Linker of formula (A'): 【Chemistry 2】 (In the formula, ** is the binding site to the linear extracyclic peptide (EP), * indicates the binding site to the cyclic cell-permeable peptide (CPP), L １ and L ２ It is independently a linker arm, ^ indicates L-stereochemistry or D-stereochemistry. y' is an integer between 1 and 5. M is a reactive handle containing a functional group that reacts with the corresponding functional group on the cargo to form a bonding group (M'), (d) A cargo comprising an oligonucleotide that binds to exon 50 in the pre-mRNA transcript of the human dystrophin gene (DMD) and causes exon skipping, wherein the oligonucleotide binds to or comprises nucleic acid sequences shown in Tables 11A-11D, Tables 12A-12D, or Table 13, and comprises 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 respect to the cargo and The EEV-cargo conjugate, including the aforementioned EEV-cargo conjugate.

2. The aforementioned linker is given by formula (B'): 【Transformation 3】 Includes, During the ceremony, x' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. The EEV-cargo conjugate according to claim 1.

3. The aforementioned linker is given by formula (C'): 【Chemistry 4】 Includes, x' is an integer between 1 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. X ｏ ' is a hydrophobic component, The EEV-cargo conjugate according to claim 1.

4. The aforementioned linker is given by formula (D'): 【Transformation 5】 Includes, x' is an integer between 1 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. X ｏ ' is a hydrophobic component, K ＃ This is a D-lysine residue or an L-lysine residue. The EEV-cargo conjugate according to claim 1.

5. Formula (A-1): 【Transformation 6】 Includes, During the ceremony, EP is a linear extracyclic peptide, cCPP is a cyclic cell-permeable peptide, M' is a bonding group. The EEV-cargo conjugate according to claim 1.

6. Formula (B-1): 【Transformation 7】 Includes, During the ceremony, x' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. The EEV-cargo conjugate according to claim 5.

7. Formula (C-1): 【Transformation 8】 Includes, During the ceremony, x' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. X ｏ ' is a hydrophobic group, The EEV-cargo conjugate according to claim 5.

8. Formula (D-1): 【Chemistry 9】 Includes, During the ceremony, x' is an integer between 0 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. X ｏ ' is a hydrophobic group, K ＃ This is a D-lysine residue or an L-lysine residue. The EEV-cargo conjugate according to claim 5.

9. Formula (A-3): 【Chemistry 10】 Includes, During the ceremony, R １ , R ２ , and R ３ These are independently either H, or an aryl side chain or heteroaryl side chain of an amino acid residue. R １ , R ２ , and R ３ At least two of these are aryl or heteroaryl side chains of amino acid residues. R ４ , R ５ , R ６ , and R ７ These are independently H or amino acid residue side chains. The compound according to claim 1.

10. R １ , R ２ , and R ３ However, each is independently a side chain containing an aryl group or a heteroaryl group. R １ , R ２ , and R ３ At least two of these are phenylalanine side chains, 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 However, independently, H or amino acid side chains, q is 1, 2, 3, or 4. The EEV-cargo conjugate according to claim 9.

11. R １ , R ２ , and R ３ However, -CH ２ It is Ph, R ４ , R ５ , R ６ , and R ７ However, independently, H or amino acid side chains, The EEV-cargo conjugate according to claim 9.

12. R １ , R ２ , and R ３ One of them is H, R １ , R ２ , and R ３ Two of them are CH ２ It is Ph, R ４ , R ５ , R ６ , and R ７ However, independently, H or amino acid side chains, The EEV-cargo conjugate according to claim 9.

13. R １ , R ２ , and R ３ One of them is H, R １ , R ２ , and R ３ Two of them are CH ２ It is Ph, R ４ , R ５ , R ６ , and R ７ However, independently, H or the amino acid side chain of arginine, The EEV-cargo conjugate according to claim 9.

14. The EEV-cargo conjugate according to claim 9, wherein the cCPP has an array selected from fNalRrRrQ, Ff-Nal-Cit-r-Cit-r-Q, and Ff-Nal-GrGrQ.

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

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

17. AA ＳＣ However, the EEV-cargo conjugate according to claim 1, comprising glutamine (Q).

18. The aforementioned linker, 【Chemistry 11】 Selected from, in the formula, x' is an integer between 1 and 12. j' is 0, 1, or 2, where if x' is 0, then j' is 0. z' is an integer between 0 and 12. j'' is 0, 1, or 2, where if z' is 0, then j'' is 0. X ｏ ' is a hydrophobic component, K ＃ This is a D-lysine residue or an L-lysine residue. The EEV-cargo conjugate according to claim 9.

19. M' -NH-, -C(O)-, -O-, 【Chemistry 12】 An EEV-cargo conjugate according to any of the prior claims, comprising, in the formula, y'' being an integer from 1 to 4 and t' being an integer from 0 to 10.

20. The EEV-cargo conjugate according to claim 19, wherein M includes -C(O)-.

21. M, 【Chemistry 13】 The EEV-cargo conjugate according to claim 19, comprising, where t' is an integer from 0 to 10.

22. The EEV-cargo conjugate according to 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 according to claim 22, wherein z' is 2 or 12.

24. The EEV-cargo conjugate according to claim 22, wherein z' is 2.

25. The EEV-cargo conjugate according to claim 22, wherein z' is 12.

26. An EEV-cargo conjugate according to 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 according to claim 26, wherein x' is 0 or 2.

28. The EEV-cargo conjugate according to claim 26, wherein x' is 0.

29. The EEV-cargo conjugate according to claim 26, wherein x' is 2.

30. X ｏ The EEV-cargo conjugate according to any one of claims 3-4, 7-8, 19-21, and 22-25, wherein ' 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. X ｏ The EEV-cargo conjugate according to claim 30, wherein ' is L-2-naphthylalanine (L-Nal) or D-2-naphthylalanine (d-nal).

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

33. X ｏ The EEV-cargo conjugate according to claim 30, wherein ' is L-2-naphthylalanine (L-Nal).

34. X ｏ The EEV-cargo conjugate according to claim 30, wherein ' is D-2-naphthylalanine (d-nal).

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

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

37. The EEV-cargo conjugate according to any one of the prior claims, wherein the linker is covalently bonded to the 5' end, 3' end, or backbone of the oligonucleotide cargo.

38. The EEV-cargo conjugate according to any of the prior claims, wherein the EP comprises 2 to 10 amino acid residues, wherein at least one amino acid residue comprises a side chain comprising a guanidine group, a terminal amine, an imidazole, or a protonated form thereof.

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

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

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

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

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

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

45. The EEV-cargo conjugate according to any one of claims 1 to 40, wherein the EP includes one of the following order: PKKKRKV; KR; RR; KKK; KGK; KBK; KBR; KRK; KRR; RKK; RRR; KKKK; KKRK; KRKKK; KRRRK; RKKR; RRRR; KGKK; KKGK; KKKKK; KKKRK; KBKBK; KKKRKV; PGKKRKV; PKGKRKV; PKKGRRKV; PKKKGKV; PKKKRGV; or PKKKRKG.

46. The EEV-cargo conjugate according to any one of claims 1 to 40, wherein the EP has structure: Ac-PKKKRKV. 【Request Item 47】 【Table 1】 An EEV-cargo conjugate according to claim 1, selected from the above. 【Request Item 48】 【Table 2】 An EEV-cargo conjugate according to claim 1, selected from the above. 【Request Item 49】 【Table 3】 An EEV-cargo conjugate according to claim 1, selected from the above. 【Request Item 50】 【Table 4】 The EEV-cargo conjugate according to claim 1, comprising: 【Request Item 51】 【Table 5】 An EEV-cargo conjugate according to claim 1, selected from the above. 【Request Item 52】 【Table 6】 An EEV-cargo conjugate according to claim 1, selected from the above.

53. The following structure 【Chemistry 14】 The EEV-cargo conjugate according to claim 1, wherein the formula comprises, where # is a phosphorodiamidate morpholino oligonucleotide (PMO), and the 3' base is a nucleic acid base at the 3' end of the PMO.

54. The following structure 【Chemistry 15】 The EEV-cargo conjugate according to claim 1, wherein the formula comprises, where # is a phosphorodiamidate morpholino oligonucleotide (PMO), and the 3' base is a nucleic acid base at the 3' end of the PMO.

55. The following structure 【Chemistry 16】 The EEV-cargo conjugate according to claim 1, wherein the formula comprises, where # is a phosphorodiamidate morpholino oligonucleotide (PMO), and the 3' base is a nucleic acid base at the 3' end of the PMO.

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

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

58. The EEV-cargo conjugate according to any one of claims 1 to 57, wherein the oligonucleotide comprises at least one modified nucleotide or nucleic acid selected from phosphorothioate (PS) nucleotide, phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), peptide nucleic acid (PNA), nucleotide containing a 2'-O-methyl (2'-OMe) modified skeleton, 2'-O-methoxyethyl (2'-MOE) nucleotide, 2',4'-restricted ethyl (cEt) nucleotide, and 2'-deoxy-2'-fluoro-beta-D-arabino nucleic acid (2'F-ANA).

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

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

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

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

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

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

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

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

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

68. The EEV-cargo conjugate according to any one of claims 1 to 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 them.

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

70. The oligonucleotide is 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'-GTGGTCCAGTCCCAGGAGCTAGGTC-3', and 5'-AGGAGCTAGGTCAGGCTGCTTT-3' An EEV-cargo conjugate according to any one of claims 1 to 63, comprising an array selected from.

71. The oligonucleotide is 5'-AGTGGTCAGTCCAGGAGCTAGGTC-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

72. The oligonucleotide is 5'-GTGGTCAGTCCAGGAGCTAGG-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

73. The oligonucleotide is 5'-GGTCAGTCCAGGAGCTAGGTCA-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

74. The oligonucleotide is 5'-TAGTGGTCAGTCCAGGAGCTAGGT-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

75. The oligonucleotide is 5'-GCTCCAATAGTGGTCAGTCCAG-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

76. The oligonucleotide is 5'-ACCGCCTTCCACTCAGAGCTCAGA-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

77. The oligonucleotide is 5'-TTACCGCCTTCCACTCAGAGCTCA, -3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

78. The oligonucleotide is 5'-GGAGCTAGGTCAGGCTGCTTTG-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

79. The oligonucleotide is 5'-GTGGTCAGTCCAGGAGCTAGGTC-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

80. The oligonucleotide is 5'-AGGAGCTAGGTCAGGCTGCTTT-3' An EEV-cargo conjugate according to any one of claims 1 to 63, including the above.

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

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

83. The method according to claim 82, wherein administration includes parenteral administration.

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

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