Circular cell-permeable peptide

Abstract

The present disclosure is directed to cell-penetrating peptides, such as cyclic cell-penetrating peptides, with high cytoplasmic delivery efficiency and reduced toxicity that can effectively deliver cargo into cells to treat various conditions and diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 168,888 filed on 31 March 2021, U.S. Provisional Patent Application No. 63 / 171,860 filed on 7 April 2021, U.S. Provisional Patent Application No. 63 / 239,671 filed on 1 September 2021, U.S. Provisional Patent Application No. 63 / 290,960 filed on 17 December 2021, U.S. Provisional Patent Application No. 63 / 298,565 filed on 11 January 2022, and U.S. Provisional Patent Application No. 63 / 268,577 filed on 25 February 2022, the disclosures of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Nucleic acids and their synthetic analogues have enormous potential as therapeutic agents, particularly for targets that are difficult for conventional drug modalities to treat (e.g., deficiency / defective proteins caused by gene mutations).

[0003] However, a major problem in translating the potential of such therapies into clinical practice is the limited ability to access intracellular compartments when administered systemically. To facilitate intracellular delivery, transport systems such as polymers, cationic liposomes, or chemical modifications such as covalent bonding of cholesterol molecules are used. Nevertheless, intracellular delivery efficiency with these approaches is often low, and improved delivery systems to enhance the effectiveness of intracellular delivery remain undiscovered.

[0004] In the late 1980s, it was discovered that highly positively charged HIV Tat peptides could traverse mammalian cell membranes. Subsequently, other "cell-penetrating peptides" (CPPs) were discovered that could permeate cell membranes at low micromolar concentrations without causing significant membrane damage. (Qian et al. (2016) "Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides." Biochem. 55:2601-2612.) However, effective cytoplasmic delivery by many of these CPPs is limited by insufficient endosomal escape efficiency.

[0005] Therefore, compositions containing novel cell-permeable peptides and peptides with appropriate toxicity profiles are needed.

[0006] The compositions and methods disclosed herein address these and other needs. [Overview of the project]

[0007] One possible method for disrupting membrane barriers and delivering drugs to cells is to attach them to "cell-permeable peptides" (also referred to herein as CPPs or EEVs (endosomal escape vehicles)). Certain residues, such as arginine, which facilitate cytoplasmic delivery, are involved as important contributors to systemic organ toxicity.

[0008] A composition is provided comprising a novel cell-permeable peptide and a peptide having an appropriate toxicity profile.

[0009] The compositions and methods disclosed herein address these and other needs. [Brief explanation of the drawing]

[0010] [Figure 1-1] Figure 1 shows a non-limiting example of cCPP containing an arginine substitute for cargo delivery. [Figure 1-2]Figure 1 shows a non-limiting example of cCPP containing an arginine substitute for cargo delivery. [Figure 1-3] Figure 1 shows a non-limiting example of cCPP containing an arginine substitute for cargo delivery. [Figure 1-4] Figure 1 shows a non-limiting example of cCPP containing an arginine substitute for cargo delivery. [Figure 1-5] Figure 1 shows a non-limiting example of cCPP containing an arginine substitute for cargo delivery. [Figure 1-6] Figure 1 shows a non-limiting example of cCPP containing an arginine substitute for cargo delivery. [Figure 2-1] Figure 2 shows the structure of compound 1b-PMO(EEV-PMO-MDX-1), a conjugation product formed between compound 1b and PMO. [Figure 2-2] Figure 2 shows the structure of compound 1b-PMO(EEV-PMO-MDX-1), a conjugation product formed between compound 1b and PMO. [Figure 2-3] Figure 2 shows the structure of compound 1b-PMO(EEV-PMO-MDX-1), a conjugation product formed between compound 1b and PMO. [Figure 3] Figure 3 shows the changes in cell viability of the human fibroblast cell line WI38 after treatment with various concentrations of EEV12 and compound 1b. [Figure 4] Figure 4 shows the quantification of lactate dehydrogenase (LDH) released from human fibroblast cell line WI38 after treatment with various concentrations of EEV12 and compound 1b. [Figure 5] Figure 5 shows the changes in cell viability of primary human renal proximal tubular epithelial cells (RPTECs) after treatment with various concentrations of EEV12 and compound 1b. [Figure 6] Figure 6 shows the quantification of lactate dehydrogenase (LDH) release from primary human renal proximal tubular epithelial cells (RPTECs) treated with various concentrations of EEV12 and compound 1b. [Figure 7]Figure 7 shows the changes in cell viability of human umbilical vein endothelial cells (HUVECs) after treatment with various concentrations of EEV12 and compound 1b. [Figure 8] Figure 8 shows the changes in cell viability of human peripheral blood mononuclear cells (hPBMCs) after treatment with various concentrations of EEV12 and compound 1b. [Figure 9] Figure 9 shows the quantification of serum histamine levels by LC / MS in male C57BL / 6 mice after intravenous injection of various amounts of EEV12 and compound 1b. [Figure 10A] Figures 10A to 10E show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 7 days after intravenous injection of various amounts of EEV-MDX-PMO-1 or EEV-MDX-PMO-2. Figure 10A shows the quantification of exon 23 skipping in the flank. Figure 10B shows the quantification of exon 23 skipping in the heart. Figure 10C shows the quantification of exon 23 skipping in the diaphragm. Figure 10D shows the quantification of exon 23 skipping in the tibialis anterior muscle. Figure 10E shows the quantification of exon 23 skipping in the quadriceps muscle. [Figure 10B] Figures 10A to 10E show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 7 days after intravenous injection of various amounts of EEV-MDX-PMO-1 or EEV-MDX-PMO-2. Figure 10A shows the quantification of exon 23 skipping in the flank. Figure 10B shows the quantification of exon 23 skipping in the heart. Figure 10C shows the quantification of exon 23 skipping in the diaphragm. Figure 10D shows the quantification of exon 23 skipping in the tibialis anterior muscle. Figure 10E shows the quantification of exon 23 skipping in the quadriceps muscle. [Figure 10C]Figures 10A to 10E show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 7 days after intravenous injection of various amounts of EEV-MDX-PMO-1 or EEV-MDX-PMO-2. Figure 10A shows the quantification of exon 23 skipping in the flank. Figure 10B shows the quantification of exon 23 skipping in the heart. Figure 10C shows the quantification of exon 23 skipping in the diaphragm. Figure 10D shows the quantification of exon 23 skipping in the tibialis anterior muscle. Figure 10E shows the quantification of exon 23 skipping in the quadriceps muscle. [Figure 10D] Figures 10A to 10E show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 7 days after intravenous injection of various amounts of EEV-MDX-PMO-1 or EEV-MDX-PMO-2. Figure 10A shows the quantification of exon 23 skipping in the flank. Figure 10B shows the quantification of exon 23 skipping in the heart. Figure 10C shows the quantification of exon 23 skipping in the diaphragm. Figure 10D shows the quantification of exon 23 skipping in the tibialis anterior muscle. Figure 10E shows the quantification of exon 23 skipping in the quadriceps muscle. [Figure 10E] Figures 10A to 10E show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 7 days after intravenous injection of various amounts of EEV-MDX-PMO-1 or EEV-MDX-PMO-2. Figure 10A shows the quantification of exon 23 skipping in the flank. Figure 10B shows the quantification of exon 23 skipping in the heart. Figure 10C shows the quantification of exon 23 skipping in the diaphragm. Figure 10D shows the quantification of exon 23 skipping in the tibialis anterior muscle. Figure 10E shows the quantification of exon 23 skipping in the quadriceps muscle. [Figure 11]Figure 11 shows Western blot quantification of dystrophin production in mdx mice 7 days after intravenous injection of various amounts of EEV-MDX-PMO-1 or EEV-MDX-PMO-2 as described in Example 4. The given values ​​are related to dystrophin levels in specified tissues in wild-type C57BL / 10 mice. [Figure 12-1] Figure 12 shows the structure of the conjugation product EEV-MDX-PMO-3 formed between compound 4b and PMO. [Figure 12-2] Figure 12 shows the structure of the conjugation product EEV-MDX-PMO-3 formed between compound 4b and PMO. [Figure 13] Figure 13 shows PCR agarose gel images of exon 23 skipping in mdx mice 3 days after intravenous injection of 40 mpk EEV-MDX-PMO-2 and 40 mpk EEV-MDX-PMO-3. [Figure 14A] Figures 14A to 14C show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 3 or 7 days after intravenous injection of various amounts of EEV-MDX-PMO-2, EEV-MDX-PMO-3, or R6-PMO, respectively. Figure 14A shows the quantification of exon 23 skipping in the quadriceps muscle. Figure 14B shows the quantification of exon 23 skipping in the diaphragm. Figure 14C shows the quantification of exon 23 skipping in the heart. [Figure 14B] Figures 14A to 14C show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 3 or 7 days after intravenous injection of various amounts of EEV-MDX-PMO-2, EEV-MDX-PMO-3, or R6-PMO, respectively. Figure 14A shows the quantification of exon 23 skipping in the quadriceps muscle. Figure 14B shows the quantification of exon 23 skipping in the diaphragm. Figure 14C shows the quantification of exon 23 skipping in the heart. [Figure 14C]Figures 14A to 14C show the quantification of exon 23 skipping measured by RT-PCR in mdx mice 3 or 7 days after intravenous injection of various amounts of EEV-MDX-PMO-2, EEV-MDX-PMO-3, or R6-PMO, respectively. Figure 14A shows the quantification of exon 23 skipping in the quadriceps muscle. Figure 14B shows the quantification of exon 23 skipping in the diaphragm. Figure 14C shows the quantification of exon 23 skipping in the heart. [Figure 15A] Figures 15A to 15D show the Western blot quantification of dystrophin production in mdx mice 3 days after intravenous injection of 40 mg / kg of EEV-MDX-PMO-2 or EEV-MDX-PMO-3. All values ​​are given as dystrophin levels compared to dystrophin levels in wild-type C57BL / 10 mice. Figure 15A shows the relative quantification of dystrophin levels in the heart. Figure 15B shows the relative quantification of dystrophin levels in the diaphragm. Figure 15C shows the relative quantification of dystrophin levels in the tibialis anterior muscle. Figure 15D shows the relative quantification of dystrophin levels in the quadriceps muscle. [Figure 15B] Figures 15A to 15D show the Western blot quantification of dystrophin production in mdx mice 3 days after intravenous injection of 40 mg / kg of EEV-MDX-PMO-2 or EEV-MDX-PMO-3. All values ​​are given as dystrophin levels compared to dystrophin levels in wild-type C57BL / 10 mice. Figure 15A shows the relative quantification of dystrophin levels in the heart. Figure 15B shows the relative quantification of dystrophin levels in the diaphragm. Figure 15C shows the relative quantification of dystrophin levels in the tibialis anterior muscle. Figure 15D shows the relative quantification of dystrophin levels in the quadriceps muscle. [Figure 15C]Figures 15A to 15D show the Western blot quantification of dystrophin production in mdx mice 3 days after intravenous injection of 40 mg / kg of EEV-MDX-PMO-2 or EEV-MDX-PMO-3. All values ​​are given as dystrophin levels compared to dystrophin levels in wild-type C57BL / 10 mice. Figure 15A shows the relative quantification of dystrophin levels in the heart. Figure 15B shows the relative quantification of dystrophin levels in the diaphragm. Figure 15C shows the relative quantification of dystrophin levels in the tibialis anterior muscle. Figure 15D shows the relative quantification of dystrophin levels in the quadriceps muscle. [Figure 15D] Figures 15A to 15D show the Western blot quantification of dystrophin production in mdx mice 3 days after intravenous injection of 40 mg / kg of EEV-MDX-PMO-2 or EEV-MDX-PMO-3. All values ​​are given as dystrophin levels compared to dystrophin levels in wild-type C57BL / 10 mice. Figure 15A shows the relative quantification of dystrophin levels in the heart. Figure 15B shows the relative quantification of dystrophin levels in the diaphragm. Figure 15C shows the relative quantification of dystrophin levels in the tibialis anterior muscle. Figure 15D shows the relative quantification of dystrophin levels in the quadriceps muscle. [Figure 16A] Figures 16A and 16B illustrate the conjugation chemistry for linking a therapeutic moiety (TM), such as an antisense compound (AC), to a cell-permeable peptide (CPP). Other chemistry methods, such as thiol-maleimide or copper-catalyzed click chemistry, may also be used. [Figure 16B] Figures 16A and 16B illustrate the conjugation chemistry for linking a therapeutic moiety (TM), such as an antisense compound (AC), to a cell-permeable peptide (CPP). Other chemistry methods, such as thiol-maleimide or copper-catalyzed click chemistry, may also be used. [Figure 17] Figure 17 shows the conjugation chemistry for linking the sample oligonucleotide and cCPP with an additional linker modality containing a polyethylene glycol (PEG) moiety. [Figure 18A] Figures 18A to 18C show the synthesis schemes for EEV-PMO-DMD44-1 (Figure 18A), EEV-PMO-DMD44-2 (Figure 18B), and EEV-PMO-DMD44-3 (Figure 18C). [Figure 18B] Figures 18A to 18C show the synthesis schemes for EEV-PMO-DMD44-1 (Figure 18A), EEV-PMO-DMD44-2 (Figure 18B), and EEV-PMO-DMD44-3 (Figure 18C). [Figure 18C] Figures 18A to 18C show the synthesis schemes for EEV-PMO-DMD44-1 (Figure 18A), EEV-PMO-DMD44-2 (Figure 18B), and EEV-PMO-DMD44-3 (Figure 18C). [Figure 19] Figure 19 shows the recovery of dystrophin protein expression in DMDΔ45 muscle cells treated with EEV-PMO-DMD44-1, EEV-PMO-DMD44-2, or EEV-PMO-DMD44-3, as quantified by Western blotting. [Figure 20A] Figures 20A and 20B show exon skipping and drug concentrations in the tissues of hDMD mice treated with EEV-PMO-DMD44-1 (Figure 20A) and EEV-PMO-DMD44-2 (Figure 20B) via IV injection. [Figure 20B] Figures 20A and 20B show exon skipping and drug concentrations in the tissues of hDMD mice treated with EEV-PMO-DMD44-1 (Figure 20A) and EEV-PMO-DMD44-2 (Figure 20B) via IV injection. [Figure 21A] Figures 21A and 21B show exon skipping (Figure 21A) and drug exposure (Figure 21B) for EEV-PMO-DMD44-1 in the NHP model. Figures 21C and 21D show exon skipping (Figure 21C) and drug exposure (Figure 21D) for EEV-PMO-DMD44-2 in the NHP model. [Figure 21B]Figures 21A and 21B show exon skipping (Figure 21A) and drug exposure (Figure 21B) for EEV-PMO-DMD44-1 in the NHP model. Figures 21C and 21D show exon skipping (Figure 21C) and drug exposure (Figure 21D) for EEV-PMO-DMD44-2 in the NHP model. [Figure 21C] Figures 21A and 21B show exon skipping (Figure 21A) and drug exposure (Figure 21B) for EEV-PMO-DMD44-1 in the NHP model. Figures 21C and 21D show exon skipping (Figure 21C) and drug exposure (Figure 21D) for EEV-PMO-DMD44-2 in the NHP model. [Figure 21D] Figures 21A and 21B show exon skipping (Figure 21A) and drug exposure (Figure 21B) for EEV-PMO-DMD44-1 in the NHP model. Figures 21C and 21D show exon skipping (Figure 21C) and drug exposure (Figure 21D) for EEV-PMO-DMD44-2 in the NHP model. [Figure 22A] Figures 22A to 22F show RNA splicing measurements for Mbnl1 (including exon 5; Figure 23A), Bin1 (including exon 11; Figure 22B), IR (including exon 11; Figure 22C), DMD (including exon 78; Figure 22D), LDB3 (including exon 11; Figure 22E), and Sos1 (including exon 25; Figure 22F). T-test results for treated vs. untreated DM1 myotubes were *p<0.05;**p<0.01;***p<0.001. [Figure 22B] Figures 22A to 22F show RNA splicing measurements for Mbnl1 (including exon 5; Figure 23A), Bin1 (including exon 11; Figure 22B), IR (including exon 11; Figure 22C), DMD (including exon 78; Figure 22D), LDB3 (including exon 11; Figure 22E), and Sos1 (including exon 25; Figure 22F). T-test results for treated vs. untreated DM1 myotubes were *p<0.05;**p<0.01;***p<0.001. [Figure 22C]Figures 22A to 22F show RNA splicing measurements for Mbnl1 (including exon 5; Figure 23A), Bin1 (including exon 11; Figure 22B), IR (including exon 11; Figure 22C), DMD (including exon 78; Figure 22D), LDB3 (including exon 11; Figure 22E), and Sos1 (including exon 25; Figure 22F). T-test results for treated vs. untreated DM1 myotubes were *p<0.05;**p<0.01;***p<0.001. [Figure 22D] Figures 22A to 22F show RNA splicing measurements for Mbnl1 (including exon 5; Figure 23A), Bin1 (including exon 11; Figure 22B), IR (including exon 11; Figure 22C), DMD (including exon 78; Figure 22D), LDB3 (including exon 11; Figure 22E), and Sos1 (including exon 25; Figure 22F). T-test results for treated vs. untreated DM1 myotubes were *p<0.05;**p<0.01;***p<0.001. [Figure 22E] Figures 22A to 22F show RNA splicing measurements for Mbnl1 (including exon 5; Figure 23A), Bin1 (including exon 11; Figure 22B), IR (including exon 11; Figure 22C), DMD (including exon 78; Figure 22D), LDB3 (including exon 11; Figure 22E), and Sos1 (including exon 25; Figure 22F). T-test results for treated vs. untreated DM1 myotubes were *p<0.05;**p<0.01;***p<0.001. [Figure 22F] Figures 22A to 22F show RNA splicing measurements for Mbnl1 (including exon 5; Figure 23A), Bin1 (including exon 11; Figure 22B), IR (including exon 11; Figure 22C), DMD (including exon 78; Figure 22D), LDB3 (including exon 11; Figure 22E), and Sos1 (including exon 25; Figure 22F). T-test results for treated vs. untreated DM1 myotubes were *p<0.05;**p<0.01;***p<0.001. [Figure 23A]Figures 23A and 23B show exon skipping and recovery of dystrophin in patient-derived muscle cells using compounds for exon 44 skipping (Figure 23A), and exon skipping in cardiac and skeletal muscle in transgenic mice carrying the full-length human DMD gene (Figure 23B). [Figure 23B] Figures 23A and 23B show exon skipping and recovery of dystrophin in patient-derived muscle cells using compounds for exon 44 skipping (Figure 23A), and exon skipping in cardiac and skeletal muscle in transgenic mice carrying the full-length human DMD gene (Figure 23B). [Figure 24A] Figures 24A to 24C show the tissue concentrations and percentages of exon skipping for compounds used for exon 44 skipping in the heart (Figure 24A), tibialis anterior muscle (Figure 24B), and diaphragm (Figure 25C) in transgenic mice carrying the full-length human DMD gene. [Figure 24B] Figures 24A to 24C show the tissue concentrations and percentages of exon skipping for compounds used for exon 44 skipping in the heart (Figure 24A), tibialis anterior muscle (Figure 24B), and diaphragm (Figure 25C) in transgenic mice carrying the full-length human DMD gene. [Figure 24C] Figures 24A to 24C show the tissue concentrations and percentages of exon skipping for compounds used for exon 44 skipping in the heart (Figure 24A), tibialis anterior muscle (Figure 24B), and diaphragm (Figure 25C) in transgenic mice carrying the full-length human DMD gene. [Figure 25] Figure 25 shows the time course of plasma levels after administration of compounds for exon 44 skipping to NHP. [Figure 26] Figure 26 shows significant levels of exon skipping in both skeletal muscle and heart of NHP patients administered compounds for exon skipping. [Figure 27]Figure 27 shows the modified nucleotides used in the antisense oligonucleotides described herein. [Figure 28A] Figures 28A to 28D show the structures of morpholino subunit monomers used in the synthesis of phosphorodiamidate-linked morpholino oligomers. Figure 28A shows the structure of adenine morpholino monomer. Figure 28B shows the structure of cytosine morpholino monomer. Figure 28C shows the structure of guanine morpholino monomer. Figure 28D shows the structure of thymine morpholino monomer. [Figure 28B] Figures 28A to 28D show the structures of morpholino subunit monomers used in the synthesis of phosphorodiamidate-linked morpholino oligomers. Figure 28A shows the structure of adenine morpholino monomer. Figure 28B shows the structure of cytosine morpholino monomer. Figure 28C shows the structure of guanine morpholino monomer. Figure 28D shows the structure of thymine morpholino monomer. [Figure 28C] Figures 28A to 28D show the structures of morpholino subunit monomers used in the synthesis of phosphorodiamidate-linked morpholino oligomers. Figure 28A shows the structure of adenine morpholino monomer. Figure 28B shows the structure of cytosine morpholino monomer. Figure 28C shows the structure of guanine morpholino monomer. Figure 28D shows the structure of thymine morpholino monomer. [Figure 28D] Figures 28A to 28D show the structures of morpholino subunit monomers used in the synthesis of phosphorodiamidate-linked morpholino oligomers. Figure 28A shows the structure of adenine morpholino monomer. Figure 28B shows the structure of cytosine morpholino monomer. Figure 28C shows the structure of guanine morpholino monomer. Figure 28D shows the structure of thymine morpholino monomer. [Figure 29A]Figures 29A to 29D illustrate the conjugation chemistry for linking AC to a cyclic cell-permeable peptide (cCPP). Figure 29A shows amide bond formation between a peptide having a carboxylic acid group or a TFP-activated ester and the primary amine residue at the 5' end of AC. Figure 29B shows the conjugation of a 3' secondary amine or primary amine-modified AC with a peptide-TFP ester via amide bond formation. Figure 29C shows the conjugation of a peptide-azide to a 5' cyclooctin-modified AC by copper-free azide-alkyne cycloaddition. Figure 29D shows another conjugation (click reaction) between a 3'-modified cyclooctin AC or 3'-modified azide AC and a cCPP containing a linker-azide or linker-alkyne / cyclooctin moiety, respectively, by copper-free azide-alkyne cycloaddition or copper-catalyzed azide-alkyne cycloaddition. [Figure 29B] Figures 29A to 29D illustrate the conjugation chemistry for linking AC to a cyclic cell-permeable peptide (cCPP). Figure 29A shows amide bond formation between a peptide having a carboxylic acid group or a TFP-activated ester and the primary amine residue at the 5' end of AC. Figure 29B shows the conjugation of a 3' secondary amine or primary amine-modified AC with a peptide-TFP ester via amide bond formation. Figure 29C shows the conjugation of a peptide-azide to a 5' cyclooctin-modified AC by copper-free azide-alkyne cycloaddition. Figure 29D shows another conjugation (click reaction) between a 3'-modified cyclooctin AC or 3'-modified azide AC and a cCPP containing a linker-azide or linker-alkyne / cyclooctin moiety, respectively, by copper-free azide-alkyne cycloaddition or copper-catalyzed azide-alkyne cycloaddition. [Figure 29C]Figures 29A to 29D illustrate the conjugation chemistry for linking AC to a cyclic cell-permeable peptide (cCPP). Figure 29A shows amide bond formation between a peptide having a carboxylic acid group or a TFP-activated ester and the primary amine residue at the 5' end of AC. Figure 29B shows the conjugation of a 3' secondary amine or primary amine-modified AC with a peptide-TFP ester via amide bond formation. Figure 29C shows the conjugation of a peptide-azide to a 5' cyclooctin-modified AC by copper-free azide-alkyne cycloaddition. Figure 29D shows another conjugation (click reaction) between a 3'-modified cyclooctin AC or 3'-modified azide AC and a cCPP containing a linker-azide or linker-alkyne / cyclooctin moiety, respectively, by copper-free azide-alkyne cycloaddition or copper-catalyzed azide-alkyne cycloaddition. [Figure 29D] Figures 29A to 29D illustrate the conjugation chemistry for linking AC to a cyclic cell-permeable peptide (cCPP). Figure 29A shows amide bond formation between a peptide having a carboxylic acid group or a TFP-activated ester and the primary amine residue at the 5' end of AC. Figure 29B shows the conjugation of a 3' secondary amine or primary amine-modified AC with a peptide-TFP ester via amide bond formation. Figure 29C shows the conjugation of a peptide-azide to a 5' cyclooctin-modified AC by copper-free azide-alkyne cycloaddition. Figure 29D shows another conjugation (click reaction) between a 3'-modified cyclooctin AC or 3'-modified azide AC and a cCPP containing a linker-azide or linker-alkyne / cyclooctin moiety, respectively, by copper-free azide-alkyne cycloaddition or copper-catalyzed azide-alkyne cycloaddition. [Figure 30] Figure 30 shows the conjugation chemistry for linking AC and cCPP with an additional linker modality containing a polyethylene glycol (PEG) moiety. Other bonding chemistry may be used. [Figure 31A]Figure 31A shows a schematic diagram of GYS1 knockdown by exon skipping, and Figure 31B shows a schematic diagram of IRF-5 knockdown by exon skipping. [Figure 31B] Figure 31A shows a schematic diagram of GYS1 knockdown by exon skipping, and Figure 31B shows a schematic diagram of IRF-5 knockdown by exon skipping. [Figure 32A] Figures 32A to 32D show the levels of GYS1 mRNA expression in untreated mice, PMO-treated mice, and mice treated with various concentrations of EEV-PMO in a GAA knockout mouse model. Figure 32A is a gel showing the level of GYS1 expression in the mouse diaphragm, and Figure 32B is a plot of the data from Figure 32A. Figure 32C is an SDS-PAGE gel showing the level of GYS1 expression in the mouse cardiomyocyte, and Figure 32D is a plot of the data from Figure 32C. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 32B] Figures 32A to 32D show the levels of GYS1 mRNA expression in untreated mice, PMO-treated mice, and mice treated with various concentrations of EEV-PMO in a GAA knockout mouse model. Figure 32A is a gel showing the level of GYS1 expression in the mouse diaphragm, and Figure 32B is a plot of the data from Figure 32A. Figure 32C is an SDS-PAGE gel showing the level of GYS1 expression in the mouse cardiomyocyte, and Figure 32D is a plot of the data from Figure 32C. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 32C]Figures 32A to 32D show the levels of GYS1 mRNA expression in untreated mice, PMO-treated mice, and mice treated with various concentrations of EEV-PMO in a GAA knockout mouse model. Figure 32A is a gel showing the level of GYS1 expression in the mouse diaphragm, and Figure 32B is a plot of the data from Figure 32A. Figure 32C is an SDS-PAGE gel showing the level of GYS1 expression in the mouse cardiomyocyte, and Figure 32D is a plot of the data from Figure 32C. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 32D] Figures 32A to 32D show the levels of GYS1 mRNA expression in untreated mice, PMO-treated mice, and mice treated with various concentrations of EEV-PMO in a GAA knockout mouse model. Figure 32A is a gel showing the level of GYS1 expression in the mouse diaphragm, and Figure 32B is a plot of the data from Figure 32A. Figure 32C is an SDS-PAGE gel showing the level of GYS1 expression in the mouse cardiomyocyte, and Figure 32D is a plot of the data from Figure 32C. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 33A] Figures 33A–33D plot the levels of GYS1 protein expression in the heart (Figure 33A), diaphragm (Figure 33B), quadriceps (Figure 33C), and triceps (Figure 33D) of untreated mice, PMO-treated mice, and EEV-PMO-treated mice at various time points after treatment. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 33B] Figures 33A–33D plot the levels of GYS1 protein expression in the heart (Figure 33A), diaphragm (Figure 33B), quadriceps (Figure 33C), and triceps (Figure 33D) of untreated mice, PMO-treated mice, and EEV-PMO-treated mice at various time points after treatment. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 33C]Figures 33A–33D plot the levels of GYS1 protein expression in the heart (Figure 33A), diaphragm (Figure 33B), quadriceps (Figure 33C), and triceps (Figure 33D) of untreated mice, PMO-treated mice, and EEV-PMO-treated mice at various time points after treatment. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 33D] Figures 33A–33D plot the levels of GYS1 protein expression in the heart (Figure 33A), diaphragm (Figure 33B), quadriceps (Figure 33C), and triceps (Figure 33D) of untreated mice, PMO-treated mice, and EEV-PMO-treated mice at various time points after treatment. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 34A] Figures 34A-34C are plots showing the levels of IRF5 expression in the liver (Figure 34A), small intestine (Figure 34B), and anterior tibialis muscle (Figure 34C) of mice treated with various concentrations of EEV-PMO. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 34B] Figures 34A-34C are plots showing the levels of IRF5 expression in the liver (Figure 34A), small intestine (Figure 34B), and anterior tibialis muscle (Figure 34C) of mice treated with various concentrations of EEV-PMO. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 34C] Figures 34A-34C are plots showing the levels of IRF5 expression in the liver (Figure 34A), small intestine (Figure 34B), and anterior tibialis muscle (Figure 34C) of mice treated with various concentrations of EEV-PMO. (P>0.05=NS; P≦0.05=*; P≦0.01=**; P≦0.001=***) [Figure 35] Figure 35 is a plot showing the levels of IRF5 expression in in vitro experiments in which mouse macrophage cells were treated with various concentrations of EEV-PMO-IRF5-1. (P>0.05=NS;P≦0.05=*;P≦0.01=**;P≦0.001=***) [Figure 36]Figure 36 is a plot showing the levels of IRF5 expression in in vitro experiments in which mouse macrophage cells were treated with various EEV-PMO constructs and then stimulated with R848. (P>0.05=NS;P≦0.05=*;P≦0.01=**;P≦0.001=***). [Figure 37A] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37B-1] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37B-2] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37B-3] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37C-1]Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37C-2] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37C-3] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37D-1] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37D-2] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37D-3]Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37E-1] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37E-2] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 37E-3] Figures 37A to 37E show the sequences (Figure 37A) and structures (Figures 37B to 37E) of various EEV-PMO compounds. Figure 37B shows the structure of EEV-PMO-IRF5-1. Figure 37C shows the structure of EEV-PMO-IRF5-3. Figure 37D shows the structure of EEV-PMO-IRF5-4. Figure 37E shows the structure of EEV-PMO-IRF5-2. [Figure 38] Figure 38 is a bar graph showing the levels of IRF5 expression in RAW 264.7 monocyte / macrophage cells after treatment with various concentrations of compounds followed by R848 stimulation. [Figure 39] Figure 39 is a bar graph showing the level of exon 4 skipping in RAW 264.7 monocyte / macrophage cells after treatment with various concentrations of compounds. NT = Untreated. [Figure 40]Figure 40 is a bar graph showing transcription levels in RAW 264.7 monocyte / macrophage cells after treatment with various concentrations of compounds. [Figure 41] Figures 41A to 41D show dose-dependent modifications of MBNL1 downstream genes in the quadriceps femoris muscle one week after injection of EEV-PMO-DM1-3 into DHSA-LR mice, with Figure 41A showing Atp2a1, Figure 41B showing Nfix, Figure 41C showing Clcn1, and Figure 41D showing Mbnl1. [Figure 42] Figures 42A to 42D show dose-dependent modifications of MBNL1 downstream genes in the gastrocnemius muscle of DHSA-LR mice one week after injection of EEV-PMO-DM1-3, with Figure 42A showing Atp2a1, Figure 42B showing Nfix, Figure 42C showing Clcn1, and Figure 42D showing Mbnl1. [Figure 43] Figures 43A to 43D show dose-dependent modifications of MBNL1 downstream genes in the tibialis anterior muscle one week after injection of EEV-PMO-DM1-3 into HSA-LR mice (Figure 43A shows Atp2a1, Figure 43B shows Nfix, Figure 43C shows Clcn1, and Figure 43D shows Mbnl1). [Figure 44] Figures 44A to 44D show dose-dependent modifications of MBNL1 downstream genes in the anterior triceps muscle one week after injection of EEV-PMO-DM1-3 into HSA-LR mice (Figure 44A shows Atp2a1, Figure 44B shows Nfix, Figure 44C shows Clcn1, and Figure 44D shows Mbnl1). [Figure 45] Figures 45A to 45D show an overlay of the data shown in Figures 41A to 41D, 42A to 42D, 43A to 43D, and 44A to 44D. [Figure 46] Figures 46A–46D show that administration of EEV-PMO-DM1-3 resulted in approximately 50–70% HSA mRNA knockdown in the skeletal muscle of HSA-LR mice, with Figure 46A showing the quadriceps, Figure 46B the gastrocnemius, Figure 46C the triceps, and Figure 46D the tibialis anterior. Statistical significance is calculated by one-way ANOVA against the HSA-LR vehicle treatment group (n=3). Dosage is based on PMO. [Figure 47]Figures 47A to 47F are graphs showing the dose-dependent responses of drug levels in various muscle tissues in mice administered with EEV-PMO-DM1-3. Figure 47A is the quadriceps femoris muscle, Figure 47B is the triceps muscle, Figure 47C is the heart, Figure 47D is the gastrocnemius muscle, Figure 47E is the tibialis anterior muscle, and Figure 47F is the diaphragm muscle. [Figure 48] Figure 48 shows PMO-DM1, the major metabolite detected in vivo. [Figure 49] Figures 49A to 49C show the exposure of EEV-PMO-DM1-3 in the brain (Figure 49A), liver (Figure 49B), and kidney (Figure 49C) after administration at 15, 30, 60, and 90 mpk. [Figure 50] Figure 50 shows that EEV-PMO-DM1-3 treatment reduces CUG foci in the TA muscle of HSA-LR mice after 1 week. [Figure 51] Figure 51 is a graph showing that EEV-PMO-DM1-3 treatment reduces CUG foci in the TA muscle of HSA-LR mice after 1 week. [Figure 52] Figure 52 shows the dose-dependent reduction of myotonia in HSA-LR mice 7 days after treatment with EEV-PMO-DM1-3 at 15, 30, 60, and 90 mpk. [Figure 53A] Figures 53A to 53C show the duration of the effect of 80 mpk EEV-PMO-DM1-3 (60 mpk oligo) on the inclusion of Atp2a1 exon 22 in HSA-LR mice. Tibialis anterior muscle (Figure 53A), triceps muscle (Figure 53B), and quadriceps femoris muscle (Figure 53C) [Figure 53B] Figures 53A to 53C show the duration of the effect of 80 mpk EEV-PMO-DM1-3 (60 mpk oligo) on the inclusion of Atp2a1 exon 22 in HSA-LR mice. Tibialis anterior muscle (Figure 53A), triceps muscle (Figure 53B), and quadriceps femoris muscle (Figure 53C) [Figure 53C]Figures 53A - 53C show the duration of the effect of 80 mpk EEV - PMO - DM1 - 3 (60 mpk oligo) on the inclusion of Atp2a1 exon 22 in HSA - LR mice. Tibialis anterior muscle (Figure 53A), triceps muscle (Figure 53B), and quadriceps femoris muscle (Figure 53C) [Figure 54A] Figures 54A - 54C show the duration of the effect of 80 mpk EEV - PMO - DM1 - 3 (60 mpk oligo) on the inclusion of Nfix exon 7 in HSA - LR mice. Tibialis anterior muscle (Figure 54A), triceps muscle (Figure 54B), and quadriceps femoris muscle (Figure 54C). [Figure 54B] Figures 54A - 54C show the duration of the effect of 80 mpk EEV - PMO - DM1 - 3 (60 mpk oligo) on the inclusion of Nfix exon 7 in HSA - LR mice. Tibialis anterior muscle (Figure 54A), triceps muscle (Figure 54B), and quadriceps femoris muscle (Figure 54C). [Figure 54C] Figures 54A - 54C show the duration of the effect of 80 mpk EEV - PMO - DM1 - 3 (60 mpk oligo) on the inclusion of Nfix exon 7 in HSA - LR mice. Tibialis anterior muscle (Figure 54A), triceps muscle (Figure 54B), and quadriceps femoris muscle (Figure 54C). [Figure 55A] Figures 55A - 55C show the duration of the effect of 80 mpk EEV - PMO - DM1 - 3 (60 mpk oligo) on the inclusion of Mbnl1 exon 5 in HSA - LR mice. Tibialis anterior muscle (Figure 55A), triceps muscle (Figure 55B), and quadriceps femoris muscle (Figure 55C). [Figure 55B] Figures 55A - 55C show the duration of the effect of 80 mpk EEV - PMO - DM1 - 3 (60 mpk oligo) on the inclusion of Mbnl1 exon 5 in HSA - LR mice. Tibialis anterior muscle (Figure 55A), triceps muscle (Figure 55B), and quadriceps femoris muscle (Figure 55C). [Figure 55C] Figures 55A - 55C show the duration of the effect of 80 mpk EEV - PMO - DM1 - 3 (60 mpk oligo) on the inclusion of Mbnl1 exon 5 in HSA - LR mice. Tibialis anterior muscle (Figure 55A), triceps muscle (Figure 55B), and quadriceps femoris muscle (Figure 55C). [Figure 56A]Figures 56A–56C show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on exon 22 inclusion in the gastrocnemius muscle of HSA-LR mice. Atp2a1 (Figure 56A), Nfix (Figure 56B), and Mbnl1 (Figure 56C). [Figure 56B] Figures 56A–56C show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on exon 22 inclusion in the gastrocnemius muscle of HSA-LR mice. Atp2a1 (Figure 56A), Nfix (Figure 56B), and Mbnl1 (Figure 56C). [Figure 56C] Figures 56A–56C show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on exon 22 inclusion in the gastrocnemius muscle of HSA-LR mice. Atp2a1 (Figure 56A), Nfix (Figure 56B), and Mbnl1 (Figure 56C). [Figure 57] Figure 57 shows the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on the gastrocnemius, triceps, tibialis anterior, and quadriceps muscles of HSA-LR mice. [Figure 58] Figures 58A to 58D show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) in the muscle tissue of HSA-LR mice. Figure 58A (quadriceps), Figure 58B (gastrocnemius), Figure 58C (triceps), and Figure 58 (tibialis anterior). [Figure 59A] Figures 59A to 59D show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on Clcn1 exon 7a inclusion in HSA-LR mice. Figure 59A (tibialis anterior), Figure 59B (triceps), Figure 59C (quadriceps femoris), and Figure 59D (gastrocnemius). [Figure 59B] Figures 59A to 59D show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on Clcn1 exon 7a inclusion in HSA-LR mice. Figure 59A (tibialis anterior), Figure 59B (triceps), Figure 59C (quadriceps femoris), and Figure 59D (gastrocnemius). [Figure 59C]Figures 59A to 59D show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on Clcn1 exon 7a inclusion in HSA-LR mice. Figure 59A (tibialis anterior), Figure 59B (triceps), Figure 59C (quadriceps femoris), and Figure 59D (gastrocnemius). [Figure 59D] Figures 59A to 59D show the duration of the effect of 80mpk EEV-PMO-DM1-3 (60mpk oligo) on Clcn1 exon 7a inclusion in HSA-LR mice. Figure 59A (tibialis anterior), Figure 59B (triceps), Figure 59C (quadriceps femoris), and Figure 59D (gastrocnemius). [Figure 60A] Figures 60A to 60D show that EEV-PMO-DM1-3 showed a tendency towards HSA mRNA knockdown at 1 week and 4 weeks after injection. Figure 60A (tibialis anterior muscle), Figure 60B (triceps muscle), Figure 60C (quadriceps femoris muscle), and Figure 60D (gastrocnemius muscle). [Figure 60B] Figures 60A to 60D show that EEV-PMO-DM1-3 showed a tendency towards HSA mRNA knockdown at 1 week and 4 weeks after injection. Figure 60A (tibialis anterior muscle), Figure 60B (triceps muscle), Figure 60C (quadriceps femoris muscle), and Figure 60D (gastrocnemius muscle). [Figure 60C] Figures 60A to 60D show that EEV-PMO-DM1-3 showed a tendency towards HSA mRNA knockdown at 1 week and 4 weeks after injection. Figure 60A (tibialis anterior muscle), Figure 60B (triceps muscle), Figure 60C (quadriceps femoris muscle), and Figure 60D (gastrocnemius muscle). [Figure 60D] Figures 60A to 60D show that EEV-PMO-DM1-3 showed a tendency towards HSA mRNA knockdown at 1 week and 4 weeks after injection. Figure 60A (tibialis anterior muscle), Figure 60B (triceps muscle), Figure 60C (quadriceps femoris muscle), and Figure 60D (gastrocnemius muscle). [Figure 61]Figures 61A–61D show the reduction in drug levels in muscle tissue after 1 to 4 weeks with 80mpk EEV-PMO-DM1-3. Figure 61A (tibialis anterior), Figure 61B (gastrocnemius), Figure 61C (triceps), and Figure 61D (gastrocnemius). EEV-PMO-DM1-3 (60mpk oligonucleotide, 80mpk total drug) completely corrects missplicing in the gastrocnemius, triceps, tibialis anterior, and quadriceps muscles after 1 week of treatment. [Figure 62] Figures 62A and 62B show that a decrease in drug levels was observed in the liver and kidneys after 1 to 4 weeks with an 80 mpk dose of EEV-PMO-DM1-3. [Figure 63] Figures 63A to 63C show that EEV-PMO-DM1-3 promotes significant biomarker splicing modifications in muscle cells derived from DM1 patients. [Figure 64] Figures 64A to 64C show that EEV-PMO-DM1-3 reduces nuclear focus in muscle cells derived from DM1 patients. [Figure 65] Figures 65A and 65B show that no tolerability issues were observed with PMO-DM1 and EEV-PMO-DM1-3 up to approximately 800 micromoles. [Modes for carrying out the invention]

[0011] Endosome escape vehicle (EEV) This specification provides an endosome escape vehicle (EEV) that can be used to transport cargo across the cell membrane, for example, to deliver cargo to the cytosol or nucleus of a cell. The cargo may include a macromolecule, such as a peptide or oligonucleotide, or a small molecule. The EEV may include a cell-permeable peptide (CPP), such as a cyclic cell-permeable peptide (cCPP) conjugated to an extracyclic peptide (EP). The EP may be interchangeably referred to as a regulatory peptide (MP). The EP may include a nuclear localization signal (NLS) sequence. The EP can bind to the cargo. The EP can bind to the cCPP. The EP can bind to both the cargo and the cCPP. The coupling between the EP, cargo, cCPP, or combinations thereof may be non-covalent or covalent. The EP can bind to the N-terminus of the cCPP via a peptide bond. The EP can bind to the C-terminus of the cCPP via a peptide bond. The EP can bind to the cCPP via the side chains of amino acids in the cCPP. EP can be conjugated to cCPP via a lysine side chain that can be conjugated to the glutamine side chain in cCPP. EP can be conjugated to the 5' or 3' end of an oligonucleotide cargo. EP can be conjugated to a linker. An extracyclic peptide can be conjugated to the amino group of the linker. EP can be coupled to the linker via cCPP and / or a side chain on EP, and via the C-terminus of EP and cCPP. For example, EP may contain terminal lysine, which can then be coupled to a glutamine-containing cCPP via an amide bond. If EP contains terminal lysine and can conjugate cCPP using the lysine side chain, the C-terminus or N-terminus can be conjugated to a linker on the cargo.

[0012] 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 (including all ranges and values ​​between these). An EP may contain 6 to 9 amino acid residues. An EP may contain 4 to 8 amino acid residues.

[0013] Each amino acid in the exocyclic 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 so as to mimic the structure and reactivity of the natural amino acid. A non-natural amino acid can be a modified amino acid and / or an amino acid analog that is not one of the 20 common natural amino acids or the rare natural amino acids selenocysteine or pyrrolysine. A non-natural amino acid may be a D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives, or combinations thereof. These amino acids and other amino acids are listed in Table 1 along with their abbreviations used herein. For example, the amino acid can be A, G, P, K, R, V, F, H, Nal, or citrulline.

[0014] EP may contain at least one positively charged amino acid residue, for example, a side chain containing a guanidine group or its protonated form, and may contain at least one lysine residue and / or at least one amine acid residue. EP may contain one or two amino acid residues having a side chain containing a guanidine group or its protonated form. The amino acid residue having a side chain containing a guanidine group can be an arginine residue. The protonated form may mean its salt throughout the present disclosure.

[0015] EP may contain at least two, at least three, or at least four or more lysine residues. EP may contain two, three, or four lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group such as 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 may be substituted with a trifluoroacetyl (-COCF3). Protecting groups may be included to enable amide conjugation. Protecting groups may be removed after EP has been conjugated to cCPP.

[0016] EP may contain at least two amino acid residues having hydrophobic side chains. The amino acid residues having hydrophobic side chains can be selected from valine, proline, alanine, leucine, isoleucine, and methionine. The amino acid residues having hydrophobic side chains may be valine or proline.

[0017] EP may contain at least one positively charged amino acid residue, for example, at least one lysine residue and / or at least one arginine residue. EP may contain at least two, at least three, at least four, or more lysine residues and / or arginine residues.

[0018] EP is KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH, KHKK, KKHK , KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, The formula may include KKKKR, KBKBK, RKKKKG, KRKKKG, KKKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

[0019] EP may contain KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP may contain PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

[0020] EP may consist of KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP may consist of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

[0021] EP may include amino acid sequences identified in the art as nuclear localization sequences (NLS). EP may consist of amino acid sequences identified in the art as nuclear localization sequences (NLS). EP may include an NLS containing the amino acid sequence PKKKRKV. EP may consist of an NLS containing the amino acid sequence PKKKRKV. EP may include an NLS containing amino acids selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK. EP may consist of NLS containing amino acids selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK.

[0022] All extracyclic sequences may also contain an N-terminal acetyl group. Therefore, for example, EP may have the structure:Ac-PKKKRKV.

[0023] Cell-permeable peptides (CPPs) Cell-permeable peptides (CPPs) may contain 6 to 20 amino acid residues. A cell-permeable peptide may be a cyclic cell-permeable peptide (cCPP). cCPPs can permeate the cell membrane. An extracyclic peptide (EP) can be conjugated to a cCPP, and the resulting construct can be called an endosomal escape vehicle (EEV). cCPPs can permeate the cell membrane by transporting a cargo (e.g., a therapeutic moiety (TM), such as an oligonucleotide, peptide, or small molecule). cCPPs can deliver the cargo to the cytosol of the cell. cCPPs can deliver the cargo to the cellular location where the target (e.g., pre-mRNA) is located. To conjugate a cCPP with a cargo (e.g., a peptide, oligonucleotide, or small molecule), at least one bond or lone pair of electrons on the cCPP can be substituted.

[0024] The total number of amino acid residues in a cCPP is in the range of 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, including all and partial ranges between these. A cCPP may contain 6 to 13 amino acid residues. A cCPP disclosed herein may contain 6 to 10 amino acids. For example, a cCPP containing 6 to 10 amino acid residues is given by formula IA to IE:

[0025] [ka] It can have a structure consisting of any of the following, where AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9 and AA 10 This is an amino acid residue.

[0026] cCPP can contain 6 to 8 amino acids. cCPP can contain 8 amino acids.

[0027] Each amino acid in cCPP may be 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. Suitable examples of amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives, or combinations thereof. These amino acids, along with other amino acids, are listed in Table 1, along with their abbreviations as used herein.

[0028] [Table 1] * Single-letter abbreviations: In this specification, when capitalized, they represent the L-amino acid form; when lowercase, they represent the D-amino acid form.

[0029] cCPP may contain 4 to 20 amino acids, where (i) at least one amino acid has a side chain containing a guanidine group or a protonated form thereof, and (ii) at least one amino acid does not have a side chain, or

[0030] [ka] (iii) having a side chain containing or a protonated form thereof, and at least two amino acids independently having a side chain containing an aromatic group or a heteroaromatic group.

[0031] At least two amino acids may not have side chains, or

[0032] [ka] Or it may have a side chain including its protonated form. As used herein, if a side chain is absent, the amino acid has two hydrogen atoms (e.g., -CH2-) linking the amine and carboxylic acid on a carbon atom.

[0033] The amino acid without a side chain may be glycine or β-alanine.

[0034] cCPP may contain 6 to 20 amino acid residues forming the cCPP, where (i) at least one amino acid may be glycine, β-alanine, or 4-aminobutyric acid residue, (ii) at least one amino acid may have a side chain containing an aryl or heteroaryl group, and (iii) at least one amino acid may contain a guanidine group

[0035] [ka] Or it has a side chain containing the protonated form thereof.

[0036] cCPP may comprise 6 to 20 amino acid residues forming the cCPP, where (i) at least two amino acid residues may independently be glycine, β-alanine, or 4-aminobutyric acid residues, (ii) at least one amino acid may have a side chain containing an aryl or heteroaryl group, and (iii) at least one amino acid may have a guanidine group.

[0037] [ka] Or it has a side chain containing the protonated form thereof.

[0038] cCPP may comprise 6 to 20 amino acid residues forming the cCPP, where (i) at least 3 amino acids may independently be glycine, β-alanine, or 4-aminobutyric acid residues, (ii) at least 1 amino acid may have a side chain containing an aromatic group or a heteroaromatic group, and (iii) at least 1 amino acid may have a guanidine group.

[0039] [ka] Alternatively, it may have a side chain containing the protonated form thereof.

[0040] Glycine and related amino acid residues cCPP may contain (i) 1, 2, 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 2 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 3, 4, or 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof.

[0041] cCPP may contain (i) 1, 2, 3, 4, 5, or 6 glycine residues. cCPP may contain (i) 2 glycine residues. cCPP may contain (i) 3 glycine residues. cCPP may contain (i) 4 glycine residues. cCPP may contain (i) 5 glycine residues. cCPP may contain (i) 6 glycine residues. cCPP may contain (i) 3, 4, or 5 glycine residues. cCPP may contain (i) 3 or 4 glycine residues. cCPP may contain (i) 2 or 3 glycine residues. cCPP may contain (i) 1 or 2 glycine residues.

[0042] cCPP may contain (i) 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof.

[0043] cCPP may contain at least 3 glycine residues. cCPP may contain (i) 3, 4, 5, or 6 glycine residues. cCPP may contain (i) 3 glycine residues. cCPP may contain (i) 4 glycine residues. cCPP may contain (i) 5 glycine residues. cCPP may contain (i) 6 glycine residues. cCPP may contain (i) 3, 4, or 5 glycine residues. cCPP may contain (i) 3 or 4 glycine residues.

[0044] In this embodiment, none of the glycine, β-alanine, or 4-aminobutyric acid residues in the cCPP are adjacent. Two or three glycine, β-alanine, 4-, or aminobutyric acid residues may be adjacent. Two glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent.

[0045] In this embodiment, none of the glycine residues in the cCPP are adjacent. Each glycine residue in the cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be adjacent. Two glycine residues may be adjacent.

[0046] Amino acid side chains having aromatic or heteroaromatic groups cCPP may contain (ii) 2, 3, 4, 5, or 6 amino acid residues, each independently having a side chain containing an aromatic group or a heteroaromatic group. cCPP may contain (ii) 2 amino acid residues, each independently having a side chain containing an aromatic group or a heteroaromatic group. cCPP may contain (ii) 3 amino acid residues, each independently having a side chain containing an aromatic group or a heteroaromatic group. cCPP may contain (ii) 4 amino acid residues, each independently having a side chain containing an aromatic group or a heteroaromatic group. cCPP may contain (ii) 5 amino acid residues, each independently having a side chain containing an aromatic group or a heteroaromatic group. cCPP may contain (ii) 6 amino acid residues, each independently having a side chain containing an aromatic group or a heteroaromatic group. cCPP may contain (ii) 2, 3, or 4 amino acid residues, each independently having a side chain containing an aromatic group or a heteroaromatic group. cCPP may contain (ii) two or three amino acid residues each independently having a side chain containing an aromatic group or a heteroaromatic group.

[0047] cCPP may contain (ii) 2, 3, 4, 5, or 6 amino acid residues, each independently having a side chain containing an aromatic group. cCPP may contain (ii) 2 amino acid residues, each independently having a side chain containing an aromatic group. cCPP may contain (ii) 3 amino acid residues, each independently having a side chain containing an aromatic group. cCPP may contain (ii) 4 amino acid residues, each independently having a side chain containing an aromatic group. cCPP may contain (ii) 5 amino acid residues, each independently having a side chain containing an aromatic group. cCPP may contain (ii) 6 amino acid residues, each independently having a side chain containing an aromatic group. cCPP may contain (ii) 2, 3, or 4 amino acid residues, each independently having a side chain containing an aromatic group. cCPP may contain (ii) 2 or 3 amino acid residues, each independently having a side chain containing an aromatic group.

[0048] The aromatic group may be a 6- to 14-membered aryl group. The aryl group may be phenyl, naphthyl, or anthracenyl, each of which is optionally substituted. The heteroaromatic group may be a 6- to 14-membered heteroaryl group having 1, 2, or 3 heteroatoms selected from N, O, and S. The heteroaryl group may be pyridyl, quinolyl, or isoquinolyl.

[0049] Each amino acid residue having a side chain containing an aromatic or heteroaromatic group can independently be bis(homonafthylalanine), homonaphthylalanine, naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, 3-(3-benzothienyl)-alanine, or tyrosine, each of which may be optionally substituted with one or more substituents. Each amino acid having a side chain containing an aromatic or heteroaromatic group can independently be,

[0050] [ka] The following can be selected from the formula, where the H at the N-terminus and / or the H at the C-terminus are replaced by peptide bonds.

[0051] Each amino acid residue having a side chain containing an aromatic group or a heteroaromatic group can independently be a residue of phenylalanine, naphthylalanine, phenylglycine, 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 aromatic group can each 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-antryl)-alanine. The amino acid residues having a side chain containing an aromatic group can each independently be phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, or homophenylalanine, each of which can be optionally substituted with one or more substituents. Each amino acid residue having an aromatic side chain can independently be a phenylalanine, naphthylalanine, homophenylalanine, homophenylalanine, bis(homonaphthylalanine), or bis(homonaphthylalanine) residue, each of which may be optionally substituted with one or more substituents. Each amino acid residue having an aromatic side chain can independently be a phenylalanine or naphthylalanine residue, each of which may be optionally substituted with one or more substituents. At least one amino acid residue having an aromatic side chain can be a phenylalanine residue. At least two amino acid residues having aromatic side chains can be phenylalanine residues. Each amino acid residue having an aromatic side chain can be a phenylalanine residue.

[0052] In this embodiment, amino acids having side chains containing aromatic or heteroaromatic groups are not adjacent to each other. Two amino acids having side chains containing aromatic or heteroaromatic groups may be adjacent to each other. Two adjacent amino acids may have opposite stereochemistry. Two adjacent amino acids may have the same stereochemistry. Three amino acids having side chains containing aromatic or heteroaromatic groups may be adjacent to each other. Three adjacent amino acids may have the same stereochemistry. Three adjacent amino acids may have alternating stereochemistry.

[0053] Amino acid residues containing aromatic or heteroaromatic groups may be L-amino acids. Amino acid residues containing aromatic or heteroaromatic groups may be D-amino acids. Amino acid residues containing aromatic or heteroaromatic groups may be a mixture of D-amino acids and L-amino acids.

[0054] The optional substituents may be, for example, any atom or group that does not significantly (e.g., by more than 50%) reduce the cytoplasmic delivery efficiency of cCPP compared to a sequence that is otherwise identical except for the absence of substituents. The optional substituents may be hydrophobic or hydrophilic substituents. The optional substituents may be hydrophobic substituents. The substituents may increase the solvent contact surface area (as defined herein) of the hydrophobic amino acid. The substituents may be halogens, alkyls, alkenyls, alkylynylenes, cycloalkyls, cycloalkenyls, cycloalkynyls, heterocyclyls, aryls, heteroaryls, alkoxys, aryloxys, acyls, alkylcarbamoyls, alkylcarboxamidyls, alkoxycarbonyls, alkylthios, or arylthios. The substituents may be halogens.

[0055] While we do not wish to be constrained by theory, it is thought that amino acids with aromatic or heteroaromatic groups having higher hydrophobic values ​​(i.e., amino acids with side chains containing aromatic or heteroaromatic groups) can improve the cytoplasmic delivery efficiency of cCPP compared to amino acids with lower hydrophobic values. Each hydrophobic amino acid may independently have a hydrophobic value greater than that of glycine. Each hydrophobic amino acid may independently have a hydrophobic value greater than that of alanine. Each hydrophobic amino acid may independently have a hydrophobic value greater than or equal to that of phenylalanine. Hydrophobicity can be measured using hydrophobic scales known in the art. Table 2 lists the hydrophobic values ​​for various amino acids reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. USA 1984; 81(1): 140-144), Engleman et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986; 1986(15): 321-53), Kyte and Doolittle (J. Mol. Biol. 1982; 157(1): 105-132), Hoop and Woods (Proc. Natl. Acad. Sci. USA 1981; 78(6): 3824-3828), and Janin (Nature. 1979; 277(5696): 491-492) (each of these in its entirety is incorporated herein by reference). Hydrophobicity can be measured using the hydrophobic scale reported by Engleman et al.

[0056] [Table 2]

[0057] The size of the aromatic or heteroaromatic group can be selected to improve the cytoplasmic delivery efficiency of cCPP. Without wishing to be bound by theory, it is believed that a larger aromatic or heteroaromatic group on the side chain of an amino acid can improve the cytoplasmic delivery efficiency compared to the same sequence except having smaller hydrophobic amino acids. The size of the hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, the steric effect of the hydrophobic amino acid, the solvent accessible surface area (SASA) of the side chain, or a combination thereof. The size of the hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, and the larger hydrophobic amino acid has a side chain with a molecular weight of at least about 90 g / mol, or at least about 130 g / mol, or at least about 141 g / mol. The size of the amino acid can be measured in terms of the SASA of the hydrophobic side chain. The hydrophobic amino acid can have a side chain with a SASA greater than alanine or greater than glycine. The larger hydrophobic amino acid can have a side chain with a SASA greater than alanine or greater than glycine. The hydrophobic amino acid can have an aromatic or heteroaromatic group with a SASA greater than about piperidine-2-carboxylic acid, greater than about tryptophan, greater than about phenylalanine, or greater than about naphthylalanine. The first hydrophobic amino acid (AA H1 ) can have a side chain with a SASA of at least about 200 Å 2 , at least about 210 Å 2 , at least about 220 Å 2 , at least about 240 Å 2 , at least about 250 Å 2 , at least about 260 Å 2 , at least about 27, at least about 280 Å 2 , at least about 290 Å 2 , at least about 300 Å 2 , at least about 310 Å 2 , at least about 320 Å 2 , at least about 330 Å 2 , or at least about 340 Å 2 . The second hydrophobic amino acid (AA H2 ) can have a side chain with a SASA of at least about 200 Å 2 , at least about 210 Å 2, at least about 220 Å 2 , at least about 240 Å 2 , at least about 250 Å 2 , at least about 260 Å 2 , at least about 270 Å 2 , at least about 280 Å 2 , at least about 290 Å 2 , at least about 300 Å 2 , at least about 310 Å 2 , at least about 320 Å 2 , or at least about 330 Å 2 It may have a side chain containing SASA. AA H1 and AA H2 The side chain is at least about 350 Å. 2 , at least about 360 Å 2 It is at least approximately 370 Å 2 , at least about 380 Å 2 , at least about 390 Å 2 It is at least about 400 Å 2 , at least about 410 Å 2 It is at least approximately 420 Å 2 , at least about 430 Å 2 It is at least approximately 440 Å 2 It is at least about 450 Å 2 , at least about 460 Å 2 It is at least approximately 470 Å 2 , at least about 480 Å 2 It is at least approximately 490 Å 2 , about 500Å 2 Larger, at least about 510 Å 2 , at least about 520 Å 2 , at least about 530 Å 2 , at least about 540 Å 2 , at least about 550 Å 2 , at least about 560 Å 2 , at least about 570 Å 2 , at least about 580 Å 2 , at least about 590 Å 2 , at least about 600 Å 2 , at least about 610 Å 2 , at least about 620 Å2 , at least about 630 Å 2 , at least about 640 Å 2 Approximately 650 Å 2 Larger, at least about 660 Å 2 , at least about 670 Å 2 , at least about 680 Å 2 , at least about 690 Å 2 , or at least about 700 Å 2 The total SASA may be AA H2 , AA H1 The hydrophobic side chain may have a hydrophobic amino acid residue having a side chain with SASA that is less than or equal to SASA. For example, but not limited to, cCPPs having a Nal-Arg motif may show improved cytoplasmic delivery efficiency compared to cCPPs that are identical except for having a Phe-Arg motif. cCPPs having a Phe-Nal-Arg motif may show improved cytoplasmic delivery efficiency compared to cCPPs that are identical except for having a Nal-Phe-Arg motif, and cCPPs having a phe-Nal-Arg motif may show improved cytoplasmic delivery efficiency compared to cCPPs that are identical except for having a nal-Phe-Arg motif.

[0058] As used herein, “hydrophobic surface area” or “SASA” refers to the surface area (reported as square angstroms) of a solvent-contactable amino acid side chain, for example, SASA can be calculated using the “rolling ball” algorithm developed by Shrake & Rupley (J Mol Biol. 79(2):351-71), which is incorporated herein by reference in its entirety for all purposes. This algorithm uses a “sphere” of solvent with a specific radius to examine the surface of the molecule. A typical value for the sphere is 1.4 Å, which approximates the radius of a water molecule.

[0059] The SASA values ​​for specific side chains are shown in Table 3 below. The SASA values ​​described herein are based on the theoretical values ​​listed in Table 3 below, as reported by Tien et al. (PLOS ONE 8(11):e80635.https: / / doi.org / 10.1371 / journal.pone.0080635), which are incorporated herein by reference in their entirety for all purposes.

[0060] [Table 3]

[0061] 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:

[0062] [ka] It refers to.

[0063] When used herein, the protonated form of guanidine is structural:

[0064] [ka] It refers to.

[0065] 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 reproduce the hydrogen bond donating and accepting activity of a guanidinium group.

[0066] Guanidine substituents enhance the cell permeability and delivery of therapeutic agents while reducing toxicity associated with the guanidine group or its protonated form. cCPP may contain at least one amino acid having a side chain containing a guanidine or guanidinium substituent. cCPP may contain at least two amino acids having side chains containing a guanidine or guanidinium substituent. cCPP may contain at least three amino acids having side chains containing a guanidine or guanidinium substituent.

[0067] The guanidine or guanidinium group may be an isoconjunction of guanidine or guanidinium. The guanidine or guanidinium substituent may be less basic than guanidine.

[0068] As used herein, guanidine substituents are

[0069] [ka] Or it refers to its protonated form.

[0070] This disclosure relates to cCPP comprising 4 to 20 amino acid residues, wherein (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof, and (ii) at least one amino acid residue has no side chain, or

[0071] [ka] or having a side chain containing the protonated form thereof, (iii) having at least two amino acid residues independently having a side chain containing an aromatic group or a heteroaromatic group.

[0072] At least two amino acid residues do not need to have a side chain, or

[0073] [ka] Or it may have a side chain including its protonated form. As used herein, if a side chain is absent, the amino acid residue has two hydrogen atoms on the carbon atom linking the amine and the carboxylic acid (e.g., -CH2-).

[0074] The cCPP is the following part:

[0075] [ka] Alternatively, it may contain at least one amino acid having a side chain that includes one of its protonated forms.

[0076] cCPP may contain at least two amino acids, each amino acid independently comprising the following parts

[0077] [ka] or including one of its protonated forms. At least two amino acids are

[0078] [ka] Alternatively, it may have a side chain containing the same portion selected from its protonated form. At least one amino acid is

[0079] [ka] Or it may have a side chain containing the protonated form thereof. At least two amino acids are

[0080] [ka] Or it may have a side chain containing its protonated form. One, two, three, or four amino acids,

[0081] [ka] Or it may have a side chain containing the protonated form thereof. One amino acid is

[0082] [ka] Or it may have a side chain containing the protonated form thereof. Two amino acids,

[0083] [ka] Alternatively, it may have a side chain containing the protonated form thereof.

[0084] [ka] Alternatively, its protonated form can be attached to the end of an amino acid side chain.

[0085] [ka] It can be bound to the end of an amino acid side chain.

[0086] cCPP may contain 2, 3, 4, 5, or 6 amino acid residues, each independently having a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 2 amino acid residues, each independently having a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 3 amino acid residues, each independently having a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 4 amino acid residues, each independently having a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 5 amino acid residues, each independently having a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 6 amino acid residues, each independently having a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 2, 3, 4, or 5 amino acid residues that independently have a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 2, 3, or 4 amino acid residues that independently have a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain 2 or 3 amino acid residues that independently have a side chain containing (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may contain at least 1 amino acid residue that has a side chain containing (iii) a guanidine group or a protonated form thereof. cCPP may contain 2 amino acid residues that have a side chain containing (iii) a guanidine group or a protonated form thereof. cCPP may contain 3 amino acid residues that have a side chain containing (iii) a guanidine group or a protonated form thereof.

[0087] An amino acid residue may independently have a side chain containing a non-adjacent guanidine group, a guanidine substituent, or a protonated form thereof. Two amino acid residues may independently have a side chain containing a guanidine group, a guanidine substituent, or their protonated forms may be adjacent. Three amino acid residues may independently have a side chain containing a guanidine group, a guanidine substituent, or their protonated forms may be adjacent. Four amino acid residues may independently have a side chain containing a guanidine group, a guanidine substituent, or their protonated forms may be adjacent. Adjacent amino acid residues may have the same stereochemistry. Adjacent amino acids may have alternating stereochemistry.

[0088] Amino acid residues independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be L-amino acids. Amino acid residues independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be D-amino acids. Amino acid residues independently having a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be a mixture of L or D-amino acids.

[0089] 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 residue of the same protonated form. Each amino acid residue having a side chain containing a guanidine group or its protonated form may independently be arginine or a residue of the same protonated form.

[0090] Each amino acid having a side chain containing a guanidine substituent or its protonated form is independently

[0091] [ka] Alternatively, it may be its protonated form.

[0092] While not bound by theory, it is hypothesized that guanidine substituents, compared to arginine, possess reduced basicity and, in some cases, are uncharged at physiological pH (e.g., -N(H)C(O)), thus maintaining bidentate hydrogen bonding interactions with phospholipids on the plasma membrane, which are thought to promote effective membrane binding and subsequent internalization. The removal of positive charge is also thought to reduce the toxicity of cCPP.

[0093] Those skilled in the art will understand that the N-terminus and / or C-terminus of the above-mentioned non-natural fragrance hydrophobic amino acids form an amide bond when incorporated into the peptides disclosed herein.

[0094] cCPP may comprise a first amino acid having a side chain containing an aromatic or heteroaromatic group, and a second amino acid having a side chain containing an aromatic or heteroaromatic group, wherein the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain containing an aromatic or heteroaromatic group, and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain containing an aromatic or heteroaromatic group. By convention, the term “first amino acid” often refers to the N-terminal amino acid of a peptide sequence; however, as used herein, “first amino acid” is used to distinguish a reference amino acid from another amino acid in the cCPP (e.g., “second amino acid”), and therefore the term “first amino acid” may refer to the amino acid located at the N-terminus of a peptide sequence.

[0095] cCPP may include the N-terminus of a second glycine molecule that forms a peptide bond with an amino acid having a side chain containing an aromatic group or a heteroaromatic group, and the C-terminus of the second glycine molecule that forms a peptide bond with an amino acid having a side chain containing a guanidine group or a protonated form thereof.

[0096] cCPP may comprise a first amino acid having a side chain containing a guanidine group or its protonated form, and a second amino acid having a side chain containing a guanidine group or its protonated form, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain containing a guanidine group or its protonated form, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain containing a guanidine group or its protonated form.

[0097] cCPP may contain residues of asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine. cCPP may contain an asparagine residue. cCPP may contain a glutamine residue.

[0098] Each cCPP may independently contain the following residues: 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, and 3-(9-antryl)-alanine.

[0099] While we do not wish to be bound by theory, it is thought that the chirality of amino acids in cCPP may affect cytoplasmic uptake efficiency. cCPP may contain at least one D amino acid. cCPP may contain 1 to 15 D amino acids. cCPP may contain 1 to 10 D amino acids. cCPP may contain 1, 2, 3, or 4 D amino acids. cCPP may contain 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids having alternating D and L chiralities. cCPP may contain 3 adjacent amino acids having the same chirality. cCPP may contain 2 adjacent amino acids having the same chirality. At least 2 amino acids may have opposite chiralities. At least 2 amino acids with opposite chiralities may be adjacent to each other. At least 3 amino acids may have alternating stereochemistry with respect to each other. At least 3 amino acids with alternating chiralities with respect to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry with respect to each other. At least four amino acids with alternating chirality with respect to each other can be adjacent to each other. At least two amino acids can have the same chirality. At least two amino acids with the same chirality can be adjacent to each other. At least two amino acids have the same chirality, and at least two amino acids have opposite chirality. At least two amino acids with opposite chirality can be adjacent to at least two amino acids with the same chirality. Therefore, adjacent amino acids in a cCPP can have any of the following sequences: DL, LD, DLLD, LDDL, LDLLD, DLDDL, DLLDL, or LDDLD. All amino acid residues forming a cCPP can be L-amino acids. All amino acid residues forming a cCPP can be D-amino acids.

[0100] At least two amino acids may have different chiralities. At least two amino acids with different chiralities may be adjacent to each other. At least three amino acids may have different chiralities with respect to adjacent amino acids. At least four amino acids may have different chiralities with respect to adjacent amino acids. At least two amino acids have the same chirality, and at least two amino acids have different chiralities. One or more amino acid residues forming a cCPP may be achiral. A cCPP may contain a motif of 3, 4, or 5 amino acids, of which two amino acids with the same chirality may be separated by an achiral amino acid. A cCPP may contain the following sequences: DXD, DXDX, DXDXD, LXL, LXLX, or LXLXL (wherein X is an achiral amino acid). The achiral amino acid may be glycine.

[0101] [ka] Alternatively, an amino acid having a side chain containing a protonated form thereof may be adjacent to an amino acid having a side chain containing an aromatic group or a heteroaromatic group.

[0102] [ka] An amino acid having a side chain containing guanidine or its protonated form may be adjacent to at least one amino acid having a side chain containing guanidine or its protonated form. 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 aromatic group or a heteroaromatic group.

[0103] [ka] Or, two amino acids having a side chain containing a protonated form may be adjacent to each other. Two amino acids having a side chain containing guanidine or its protonated form are adjacent to each other. cCPP consists of at least two adjacent amino acids having a side chain that may contain an aromatic group or a heteroaromatic group,

[0104] [ka] Or it may have at least two non-adjacent amino acids having a side chain containing the protonated form. cCPP has at least two adjacent amino acids having a side chain containing an aromatic group or a heteroaromatic group,

[0105] [ka] Or it may have at least two non-adjacent amino acids having a side chain containing the protonated form. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, for example, any of the sequences described in the previous paragraph.

[0106] [ka] At least two amino acids having a side chain containing a guanidine group or its protonated form are alternating with at least two amino acids having a side chain containing a guanidine group or its protonated form.

[0107] cCPP is given by equation (A):

[0108] [ka] or may include the structure of its protonated form, During the ceremony, R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid. At least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid. R4, R5, R6, and R7 are independently H or amino acid side chains. At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidyl)alanine. AA SC These are amino acid side chains, q is 1, 2, 3, or 4. The cyclic peptide in formula (A) is not FfΦRrRrQ.

[0109] cCPP is given by equation (I):

[0110] [ka] or may include the structure of its protonated form, During the ceremony, R1, R2, and R3 may each be an amino acid residue having a side chain containing H or an aromatic group. At least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid. R4 and R7 are independently H or amino acid side chains. AA SC These are amino acid side chains, q is 1, 2, 3, or 4. Each m is independently an integer, either 0, 1, 2, or 3.

[0111] R1, R2, and R3 can each be independently H,-alkylene-aryl, or-alkylene-heteroaryl. R1, R2, and R3 can each be independently H,-C 1~3 Alkylene-aryl, or -C 1~3 It may be an alkylene heteroaryl. R1, R2, and R3 may each be independently H or an alkylene aryl. R1, R2, and R3 may each be independently H or a 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. R1, R2, and R3 are each independently H, -C 1~3 Alkylene-Ph or -C 1~3 It may be alkylene-naphthyl. R1, R2, and R3 may each be independently H, -CH2Ph, or -CH2naphthyl. R1, R2, and R3 may each be independently H or -CH2Ph.

[0112] R1, R2, and R3 can each independently be the side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butylphenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 3-(9-antryl)-alanine.

[0113] R1 may be a tyrosine side chain. R1 may be a phenylalanine side chain. R1 may be a 1-naphthylalanine side chain. R1 may be a 2-naphthylalanine side chain. R1 may be a tryptophan side chain. R1 may be a 3-benzothienylalanine side chain. R1 may be a 4-phenylphenylalanine side chain. R1 may be a 3,4-difluorophenylalanine side chain. R1 may be a 4-trifluoromethylphenylalanine side chain. R1 may be a 2,3,4,5,6-pentafluorophenylalanine side chain. R1 may be a homophenylalanine side chain. R1 may be a β-homophenylalanine side chain. R1 may be a 4-tert-butylphenylalanine side chain. R1 may be a 4-pyridinylalanine side chain. R1 may be a 3-pyridinylalanine side chain. R1 may be a side chain of 4-methylphenylalanine. R1 may be a side chain of 4-fluorophenylalanine. R1 may be a side chain of 4-chlorophenylalanine. R1 may be a side chain of 3-(9-antryl)-alanine.

[0114] R2 may be a tyrosine side chain. R2 may be a phenylalanine side chain. R2 may be a 1-naphthylalanine side chain. R1 may be a 2-naphthylalanine side chain. R2 may be a tryptophan side chain. R2 may be a 3-benzothienylalanine side chain. R2 may be a 4-phenylphenylalanine side chain. R2 may be a 3,4-difluorophenylalanine side chain. R2 may be a 4-trifluoromethylphenylalanine side chain. R2 may be a 2,3,4,5,6-pentafluorophenylalanine side chain. R2 may be a homophenylalanine side chain. R2 may be a β-homophenylalanine side chain. R2 may be a 4-tert-butylphenylalanine side chain. R2 may be a 4-pyridinylalanine side chain. R2 may be a 3-pyridinylalanine side chain. R2 may be a side chain of 4-methylphenylalanine. R2 may be a side chain of 4-fluorophenylalanine. R2 may be a side chain of 4-chlorophenylalanine. R2 may be a side chain of 3-(9-antryl)-alanine.

[0115] R3 can be a tyrosine side chain. R3 can be a phenylalanine side chain. R3 can be a 1-naphthylalanine side chain. R3 can be a 2-naphthylalanine side chain. R3 can be a tryptophan side chain. R3 can be a 3-benzothienylalanine side chain. R3 can be a 4-phenylphenylalanine side chain. R3 can be a 3,4-difluorophenylalanine side chain. R3 can be a 4-trifluoromethylphenylalanine side chain. R3 can be a 2,3,4,5,6-pentafluorophenylalanine side chain. R3 can be a homophenylalanine side chain. R3 can be a β-homophenylalanine side chain. R3 can be a 4-tert-butylphenylalanine side chain. R3 can be a 4-pyridinylalanine side chain. R3 can be a 3-pyridinylalanine side chain. R3 may be a side chain of 4-methylphenylalanine. R3 may be a side chain of 4-fluorophenylalanine. R3 may be a side chain of 4-chlorophenylalanine. R3 may be a side chain of 3-(9-antryl)-alanine.

[0116] R4 can be H,-alkylene-aryl, or-alkylene-heteroaryl. R4 can be H,-C 1~3 Alkylene-aryl, or -C 1~3 It may be an alkylene heteroaryl. R4 may be H or an alkylene aryl. R4 may be H or an alkylene aryl. 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. R4 is H, -C 1~3 Alkylene-Ph or -C 1~3It may be alkylene-naphthyl. R4 may be H or the side chain of an amino acid in Table 1 or Table 3. R4 may be H or an amino acid residue having a side chain containing an aromatic group. R4 may be H, -CH2Ph, or -CH2 naphthyl. R4 may be H or -CH2Ph.

[0117] R5 may be H, -alkylene-aryl, -alkylene-heteroaryl. R5 may be H, -C 1~3 alkylene-aryl, or -C 1~3 alkylene-heteroaryl. R5 may be H or -alkylene-aryl. R5 may be H or -C 1~3 alkylene-aryl. C 1~3 Alkylene may be methylene. Aryl may be 6- to 14-membered aryl. Heteroaryl may be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl may be selected from phenyl, naphthyl, or anthracenyl. Aryl may be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl may be pyridyl, quinolyl, and isoquinolyl. R5 may be H, -C 1~3 alkylene-Ph or -C 1~3 alkylene-naphthyl. R5 may be H or the side chain of an amino acid in Table 1 or Table 3. R4 may be H or an amino acid residue having a side chain containing an aromatic group. R5 may be H, -CH2Ph, or -CH2 naphthyl. R4 may be H or -CH2Ph.

[0118] R6 may be H, -alkylene-aryl, -alkylene-heteroaryl. R6 may be H, -C 1~3 alkylene-aryl, or -C 1~3 alkylene-heteroaryl. R6 may be H or -alkylene-aryl. R6 may be H or -C 1~3 alkylene-aryl. C 1~3The alkylene can be methylene. The aryl can be a 6- to 14-membered aryl. The heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R6 can be H, -C 1~3 alkylene-Ph or -C 1~3 alkylene-naphthyl. R6 can be H or the side chain of an amino acid in Table 1 or Table 3. R6 can be H or an amino acid residue having a side chain containing an aromatic group. R6 can be H, -CH2Ph, or -CH2naphthyl. R6 can be H or -CH2Ph.

[0119] R7 can be H, -alkylene-aryl, -alkylene-heteroaryl. R7 can be H, -C 1~3 alkylene-aryl, or -C 1~3 alkylene-heteroaryl. R7 can be H or -alkylene-aryl. R7 can be H or -C 1~3 alkylene-aryl. C 1~3 The alkylene can be methylene. The aryl can be a 6- to 14-membered aryl. The heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R7 can be H, -C 1~3 alkylene-Ph or -C 1~3 alkylene-naphthyl. R7 can be H or the side chain of an amino acid in Table 第1若しくは表3のアミノ酸の側鎖であり得る。R7は、H又は芳香族基を含む側鎖を有するアミノ酸残基であり得る。R7は、H、-CH2Ph、又は-CH2ナフチルであり得るアミノ酸の側鎖であり得る。R7は、H又は-CH2Phであり得る。

[0120] [[ID=)) One, two, or three of R1, R2, R3, R4, R5, R6, and R7 may be -CH2Ph. One of R1, R2, R3, R4, R5, R6, and R7 may be -CH2Ph. Two of R1, R2, R3, R4, R5, R6, and R7 may be -CH2Ph. Three of R1, R2, R3, R4, R5, R6, and R7 may be -CH2Ph. At least one of R1, R2, R3, R4, R5, R6, and R7 may be -CH2Ph. Four or fewer of R1, R2, R3, R4, R5, R6, and R7 may be -CH2Ph.

[0121] One, two, or three of R1, R2, R3, and R4 are -CH2Ph. One of R1, R2, R3, and R4 is -CH2Ph. Two of R1, R2, R3, and R4 are -CH2Ph. Three of R1, R2, R3, and R4 are -CH2Ph. At least one of R1, R2, R3, and R4 is -CH2Ph.

[0122] One, two, or three of R1, R2, R3, R4, R5, R6, and R7 may be H. One of R1, R2, R3, R4, R5, R6, and R7 may be H. Two of R1, R2, R3, R4, R5, R6, and R7 are H. Three of R1, R2, R3, R5, R6, and R7 may be H. At least one of R1, R2, R3, R4, R5, R6, and R7 may be H. Three or fewer of R1, R2, R3, R4, R5, R6, and R7 may be -CH2Ph.

[0123] One, two, or three of R1, R2, R3, and R4 are H. One of R1, R2, R3, and R4 is H. Two of R1, R2, R3, and R4 are H. Three of R1, R2, R3, and R4 are H. At least one of R1, R2, R3, and R4 is H.

[0124] At least one of R4, R5, R6, and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R4, R5, R6, and R7 may be a side chain of 4-guanidino-2-aminobutanoic acid. At least one of R4, R5, R6, and R7 may be a side chain of arginine. At least one of R4, R5, R6, and R7 may be a side chain of homoarginine. At least one of R4, R5, R6, and R7 may be a side chain of N-methylarginine. At least one of R4, R5, R6, and R7 may be a side chain of N,N-dimethylarginine. At least one of R4, R5, R6, and R7 may be a side chain of 2,3-diaminopropionic acid. At least one of R4, R5, R6, and R7 may be a side chain of 2,4-diaminobutanoic acid or lysine. At least one of R4, R5, R6, and R7 may be a side chain of N-methyllysine. At least one of R4, R5, R6, and R7 may be a side chain of N,N-dimethyllysine. At least one of R4, R5, R6, and R7 may be a side chain of N-ethyllysine. At least one of R4, R5, R6, and R7 may be a side chain of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least one of R4, R5, R6, and R7 may be a side chain of citrulline. At least one of R4, R5, R6, and R7 may be a side chain of N,N-dimethyllysine or β-homoarginine. At least one of R4, R5, R6, and R7 may be a side chain of 3-(1-piperidinyl)alanine.

[0125] At least two of R4, R5, R6, and R7 may be side chains of 3-guanidino-2-aminopropionic acid. At least two of R4, R5, R6, and R7 may be side chains of 4-guanidino-2-aminobutanoic acid. At least two of R4, R5, R6, and R7 may be side chains of arginine. At least two of R4, R5, R6, and R7 may be side chains of homoarginine. At least two of R4, R5, R6, and R7 may be side chains of N-methylarginine. At least two of R4, R5, R6, and R7 may be side chains of N,N-dimethylarginine. At least two of R4, R5, R6, and R7 may be side chains of 2,3-diaminopropionic acid. At least two of R4, R5, R6, and R7 may be 2,4-diaminobutanoic acid or lysine side chains. At least two of R4, R5, R6, and R7 may be N-methyllysine side chains. At least two of R4, R5, R6, and R7 may be N,N-dimethyllysine side chains. At least two of R4, R5, R6, and R7 may be N-ethyllysine side chains. At least two of R4, R5, R6, and R7 may be N,N,N-trimethyllysine or 4-guanidinophenylalanine side chains. At least two of R4, R5, R6, and R7 may be citrulline side chains. At least two of R4, R5, R6, and R7 may be N,N-dimethyllysine or β-homoarginine side chains. At least two of R4, R5, R6, and R7 may be side chains of 3-(1-piperidinyl)alanine.

[0126] At least three of R4, R5, R6, and R7 may be side chains of 3-guanidino-2-aminopropionic acid. At least three of R4, R5, R6, and R7 may be side chains of 4-guanidino-2-aminobutanoic acid. At least three of R4, R5, R6, and R7 may be side chains of arginine. At least three of R4, R5, R6, and R7 may be side chains of homoarginine. At least three of R4, R5, R6, and R7 may be side chains of N-methylarginine. At least three of R4, R5, R6, and R7 may be side chains of N,N-dimethylarginine. At least three of R4, R5, R6, and R7 may be side chains of 2,3-diaminopropionic acid. At least three of R4, R5, R6, and R7 may be 2,4-diaminobutanoic acid and lysine side chains. At least three of R4, R5, R6, and R7 may be N-methyllysine side chains. At least three of R4, R5, R6, and R7 may be N,N-dimethyllysine side chains. At least three of R4, R5, R6, and R7 may be N-ethyllysine side chains. At least three of R4, R5, R6, and R7 may be N,N,N-trimethyllysine and 4-guanidinophenylalanine side chains. At least three of R4, R5, R6, and R7 may be citrulline side chains. At least three of R4, R5, R6, and R7 may be N,N-dimethyllysine and β-homoarginine side chains. At least three of R4, R5, R6, and R7 may be side chains of 3-(1-piperidinyl)alanine.

[0127] AA SC This can be a side chain of asparagine, glutamine, or homoglutamine residues. AA SC cCPP is a side chain of a glutamine residue. SCFor example, cCPP may further include linkers conjugated to asparagine, glutamine, or homoglutamine residues. Therefore, cCPP may further include linkers conjugated to asparagine, glutamine, or homoglutamine residues. cCPP may further include linkers bound to glutamine residues.

[0128] q can be 1, 2, or 3. q can be 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.

[0129] 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.

[0130] The cCPP in equation (A) is given by equation (I)

[0131] [ka] The formula may include the structure or its protonated form, where AA SC R1, R2, R3, R 4- R7, m, and q are as defined herein.

[0132] The cCPP in formula (A) is either formula (Ia) or formula (Ib):

[0133] [ka] Structure or may include its protonated form, AA SC R1, R2, R3, R4, and m are as defined herein.

[0134] [ka]

[0135] The cCPP in formula (A) is given by formulas (I-1), (I-2), (I-3), or (I-4):

[0136] [ka] Structure or may include its protonated form, where AA SC and m are as defined herein.

[0137] The cCPP in formula (A) is given by formula (I-5) or (I-6):

[0138] [ka] The formula may include the structure or its protonated form, where AA SC This is defined herein.

[0139] The cCPP in equation (A) is given by equation (I-1):

[0140] [ka] This may include the structure or its protonated form. During the ceremony, A.A. SC and m are as defined herein.

[0141] The cCPP in equation (A) is given by equation (I-2):

[0142] [ka] This may include the structure or its protonated form. During the ceremony, A.A. SC and m are as defined herein.

[0143] The cCPP in equation (A) is given by equation (I-3):

[0144] [ka] This may include the structure of, or its protonated form. During the ceremony, A.A.SC and m are as defined herein.

[0145] The cCPP in equation (A) is given by equation (I-4):

[0146] [ka] This may include the structure of, or its protonated form. During the ceremony, A.A. SC and m are as defined herein.

[0147] The cCPP in equation (A) is given by equation (I-5):

[0148] [ka] This may include the structure of, or its protonated form. During the ceremony, A.A. SC and m are as defined herein.

[0149] The cCPP in equation (A) is given by equation (I-6):

[0150] [ka] The formula may include the structure of or its protonated form, where AA SC and m are as defined herein.

[0151] cCPP may contain one of the following sequences: FGFGRGR, GfFGrGr, FfΦGRGR, FfFGRGR, or FfΦGrGr. cCPP may have one of the following sequences: FGFΦ, GfFGrGrQ, FfΦGRGRQ, FfFGRGRQ, or FfΦGrGrQ.

[0152] This disclosure also includes formula (II):

[0153] [ka] Regarding cCPP having the structure, During the ceremony, AA SC These are amino acid side chains, R 1a , R 1b , and R 1c Each of these is independently a 6- to 14-membered aryl or a 6- to 14-membered heteroaryl. R 2a , R 2b , R 2c and R 2d These are, independently, amino acid side chains. R 2a , R 2b , R 2c and R 2d At least one of them is

[0154] [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d At least one of these is guanidine or its protonated form, Each n'' is an integer, independently of the integers 0, 1, 2, 3, 4, or 5. Each n' is an integer from 0, 1, 2, or 3, independently. If n' is 0, R 2a , R 2b , R 2b or R 2d It does not exist.

[0155] R 2a , R 2b , R 2c and R 2d At least two of them are

[0156] [ka] Or it may be its protonated form. 2a , R 2b , R2c and R 2d Two or three of them are

[0157] [ka] Or it may be its protonated form. 2a , R 2b , R 2c and R 2d One of them is,

[0158] [ka] Or it may be its protonated form. 2a , R 2b , R 2c and R 2d At least one of them is

[0159] [ka] Or it may be its protonated form, R 2a , R 2b , R 2c and R 2d The remainder may be guanidine or its protonated form. 2a , R 2b , R 2c and R 2d At least two of them are

[0160] [ka] Or it may be its protonated form. 2a , R 2b , R 2c and R 2d The remainder may be guanidine or its protonated form.

[0161] R 2a , R 2b , R 2c and R 2d All of the above

[0162] [ka] Or it may be its protonated form. 2a , R 2b , R 2c and R 2d At least one of them

[0163] [ka] Or it may be its protonated form, R 2a , R 2b , R 2c and R 2d The remainder may be guaninide or its protonated form. At least two R 2a , R 2b , R 2c and R 2d The basis is,

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

[0165] R 2a , R 2b , R 2c and R 2d Each of these may independently be 2,3-diaminopropionic acid, 2,4-diaminobutyric acid side chain, ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homo-lysine, serine, homo-serine, threonine, allo-threonine, histidine, 1-methylhistidine, 2-aminobutanediic acid, aspartic acid, glutamic acid, or homo-glutamic acid.

[0166] AA SC teeth

[0167] [ka] This is possible, and in the formula, t can be an integer from 0 to 5. AA SC teeth

[0168] [ka] In the expression, t can be an integer from 0 to 5. t can be from 1 to 5. t is 2 or 3. t can be 2. t can be 3.

[0169] R 1a , R 1b , and R 1c Each of these can be an independent aryl with 6 to 14 members. 1a , R 1b , and R 1c Each of these can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected independently from N, O, or S. 1a , R 1b , and R 1c Each of these can be independently selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl, or isoquinolyl. 1a , R 1b , and R 1c Each of these can be independently selected from phenyl, naphthyl, or anthracenyl. 1a , R 1b , and R 1c Each of these can independently be phenyl or naphthyl. 1a , R 1b , and R 1c Each of these can be independently selected from pyridyl, quinolyl, or isoquinolyl.

[0170] Each n' can independently be 1 or 2. Each n' can be 1. Each n' can be 2. At least one n' can be 0. At least one n' can be 1. At least one n' can be 2. At least one n' can be 3. At least one n' can be 4. At least one n' can be 5.

[0171] Each n'' can be an integer from 1 to 3 independently. Each n'' can be 2 or 3 independently. Each n'' can be 2. Each n'' can be 3. At least one n'' can be 0. At least one n'' can be 1. At least one n'' can be 2. At least one n'' can be 3.

[0172] Each n'' can independently be 1 or 2, and each n' can independently be 2 or 3. Each n'' can be 1, and each n' can independently be 2 or 3. Each n'' can be 1, and each n' can be 2. Each n'' is 1, and each n' is 3.

[0173] The cCPP in equation (II) is given by equation (II-1):

[0174] [ka] It may have a structure, In the formula, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d AA SC n' and n'' are as defined herein.

[0175] The cCPP in equation (II) is given by equation (IIa):

[0176] [ka] It may have a structure, In the formula, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d AA SC And n' are as defined herein.

[0177] The cCPP in equation (II) is given by equation (IIb):

[0178] [ka] It may have a structure, In the formula, R 2a , R 2b AA SC And n' are as defined herein.

[0179] cCPP is given by equation (IIb):

[0180] [ka] It may have the structure or a protonated form thereof. During the ceremony, AA SC And n' are as defined herein.

[0181] The cCPP in equation (IIa) has the following structure:

[0182] [ka] It has one of the following, in the formula, AA SC And n are as defined herein.

[0183] The cCPP in equation (IIa) has the following structure:

[0184] [ka] It has one of the following, in the formula, AA SC And n are as defined herein.

[0185] The cCPP in equation (IIa) has the following structure:

[0186] [ka] It has one of the following, in the formula, AA SCAnd n are as defined herein.

[0187] The cCPP in equation (II) has the following structure:

[0188] [ka] It may have.

[0189] The cCPP in equation (II) has the following structure:

[0190] [ka] It may have.

[0191] cCPP has the structure of equation (III):

[0192] [ka] It may have, During the ceremony, AA SC These are amino acid side chains, R 1a , R 1b , and R 1c Each of these is independently a 6- to 14-membered aryl or a 6- to 14-membered heteroaryl. R 2a and R 2c Each of them is independent of H,

[0193] [ka] or its protonated form, R 2b and R 2d Each of these is independently guanidine or its protonated form. Each n'' is an independent integer between 1 and 3. Each n' is an independent integer between 1 and 5. Each p' is an independent integer between 0 and 5.

[0194] The cCPP in equation (III) is given by equation (III-1):

[0195] [ka] It may have a structure, During the ceremony, AA SC , R 1a , R 1b , R 1c , R 2a , R 2c , R 2b , R 2d n', n'' and p' are as defined herein.

[0196] The cCPP in equation (III) is given by equation (IIIa):

[0197] [ka] It may have a structure, During the ceremony, AA SC , R 2a , R 2c , R 2b , R 2d n', n'', and p' are as defined herein.

[0198] In equations (III), (III-1), and (IIIa), R a and R c It can be H. a and R c can be H, and R b and R d Each of these can independently be guanidine or its protonated form. a It can be H. b p' can be H. p' can be 0. R a and R c H can be H, and each p' can be 0.

[0199] In equations (III), (III-1), and (IIIa), R a and Rc can be H, and R b and R d Each of these can independently be guanidine or its protonated form, n'' can be 2 or 3, and each p' can be 0.

[0200] p' can be 0. p' can be 1. p' can be 2. p' can be 3. p' can be 4. p' can be 5.

[0201] cCPP is structured as follows:

[0202] [ka] It may have.

[0203] The cCPP in equation (A) can be selected from the following options.

[0204] [Table 4]

[0205] The cCPP in equation (A) can be selected from the following options.

[0206] [Table 5]

[0207] AA SC It can be conjugated into a linker.

[0208] Linker The cCPP of this disclosure may be conjugated to a linker. The linker may link cargo to the cCPP. The linker may be bound to the side chains of amino acids of the cCPP, and cargo may be bound to appropriate positions on the linker.

[0209] The linker may be any suitable part that can conjugate the cCPP to one or more further parts, such as an extracyclic peptide (EP) and / or cargo. Prior to conjugation to the cCPP and one or more further parts, the linker may have two or more functional groups, each of which can independently form a covalent bond to the cCPP and one or more further parts. If the cargo is an oligonucleotide, the linker may be covalently bonded to the 5' or 3' end of the cargo. The linker may be covalently bonded to the 5' end of the cargo. The linker may be covalently bonded to the 3' end of the cargo. If the cargo is a peptide, the linker may be covalently bonded to the N-terminus or C-terminus of the cargo. The linker may be covalently bonded to the backbone of the oligonucleotide or peptide cargo. The linker may be any suitable part that conjugates the cCPP described herein to a cargo such as an oligonucleotide, peptide or small molecule.

[0210] The linker may contain hydrocarbon linkers.

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

[0212] The linker is (i) one or more D or L amino acids, each of which is optionally substituted, (ii) an optionally substituted alkylene, (iii) an optionally substituted alkenylene, (iv) an optionally substituted alkynylene, (v) an optionally substituted carbocyclyl, (vi) an optionally substituted heterocyclyl, or (vii) one or more -(R 1- JR 2 )z”-subunit, where R 1 and R 2 Each of these is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl in each case, and each J is independently C, NR 3 , -NR 3C(O)-, S, and O, where R 3 However, these are independently selected from H, alkyl, alkenyl, alkynyl, carbocykryl, and heterocyclyl, each of which is optionally substituted, and z'' is an integer from 1 to 50, subunit, (viii)-(R 1 (J)z- or -(JR 1 )z-, where each R 1 However, in each case, independently, it is alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J is independently C, NR 3 , -NR 3 C(O)-, S, or O, where R 3 The elements may include H, alkyl, alkenyl, alkynyl, carbocykrill, or heterocyclyl, each of which is optionally substituted, and z'' is an integer from 1 to 50, or (ix) the linker may include one or more of (i) to (x).

[0213] The linker is one or more D or L amino acids and / or -(R 1- JR 2 )z”- and in the formula, R 1 and R 2 Each of these is independently alkylene in each case, and each J is independently C, NR 3 , -NR 3 C(O)-, S, and O, where R 4 However, independently, it may include those selected from H and alkyl, where z'' is an integer from 1 to 50, or a combination thereof.

[0214] The linker is -(OCH2CH2) z’ - (for example, as a spacer) may be included, and z' is an integer from 1 to 23, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. "-(OCH2CH2)z" can also be called polyethylene glycol (PEG).

[0215] The linker may contain one or more amino acids. The linker may contain peptides. The linker is -(OCH2CH2) z’ -(wherein z' is an integer from 1 to 23), and may include a peptide. The peptide may contain 2 to 10 amino acids. The linker may further include a functional group (FG) that can react via click chemistry. The FG may be an azide or an alkyne, and a triazole is formed when the cargo is conjugated to the linker.

[0216] The linker consists of (i) β-alanine residues and lysine residues, (ii)-(JR 1 )z-, or (iii) combinations thereof. Each R 1 J can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can independently be C, NR 3 , -NR 3 C(O)-, S, or O, where R 3 z'' is H, alkyl, alkenyl, alkynyl, carbocykryl, or heterocyclyl, each of which is optionally substituted, and z'' can be an integer from 1 to 50. Each R 1 may be alkylene, and each J may be O.

[0217] The linker consists of (i) residues of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof, and (ii)-(R 1- J)z”-or-(JR 1 ) may include z''-. Each R 1 J can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can independently be C, NR 3 , -NR 3 C(O)-, S, or O, where R 3 z'' is H, alkyl, alkenyl, alkynyl, carbocykryl, or heterocyclyl, each of which is optionally substituted, and z'' can be an integer from 1 to 50. Each R 1is 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.

[0218] The linker can be a trivalent linker. The linker has the following structure:

[0219] [ka] The formula may have a hydrocarbon linker (e.g., NRH-(CH2) n -COOH), PEG linker (e.g., NRH-(CH2O) n -COOH, where R is H, methyl or ethyl, or one or more amino acid residues, and Z is independently a protecting group. The linker is also a disulfide [NH2-(CH2O) n -SS-(CH2O) n -COOH] or cleavage sites such as caspase cleavage sites (Val-Cit-PABC) can be incorporated.

[0220] The hydrocarbon may be a residue of glycine or beta-alanine.

[0221] The linker is divalent and can link cCPP to the cargo. The linker is divalent and can link cCPP to the extracyclic peptide (EP).

[0222] The linker can be trivalent, and cCPP can be linked to the cargo and EP.

[0223] Linkers are divalent or trivalent C1-C 50The molecule may be alkylene, where 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl, or cycloalkenyl. The linker is divalent or trivalent C1-C 50 The material may be an alkylene, where 1 to 25 methylene groups are optionally and independently substituted with -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.

[0224] The linker is structured as follows:

[0225] [ka] It may have such that each AA is an amino acid residue independently. * is AA SC It is a connection point to AA SC is the side chain of the amino acid residue of cCPP, where x is an integer from 1 to 10, y is an integer from 1 to 5, and z is an integer from 1 to 10. x can be an integer from 1 to 5. x can be an integer from 1 to 3. x can be 1. y can be an integer from 2 to 4. y can be 4. z can be an integer from 1 to 5. z can be an integer from 1 to 3. z can be 1. Each AA can be independently selected from glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminohexanoic acid.

[0226] cCPP can be bound to the cargo via a linker ("L"). The linker can be conjugated to the cargo via a binding group ("M").

[0227] The linker is structured as follows:

[0228] [ka] The formula may have such a configuration, where x is an integer from 1 to 10, y is an integer from 1 to 5, z is an integer from 1 to 10, and each AA is an independent amino acid residue. * is AA SC It is a connection point to AA SC is the side chain of the amino acid residue of cCPP, and M is the binding group as defined herein.

[0229] The linker is structured as follows:

[0230] [ka] It may have, x' is an integer between 1 and 23, y is an integer between 1 and 5, and z' is an integer between 1 and 23. * is AA SC It is a connection point to AA SC is the side chain of the amino acid residue of cCPP, and M is the binding group as defined herein.

[0231] The linker is structured as follows:

[0232] [ka] It may have, x' is an integer between 1 and 23, y is an integer between 1 and 5, and z' is an integer between 1 and 23. * is AA SC It is a connection point to AA SC is the side chain of the amino acid residue of cCPP, and M is the binding group as defined herein.

[0233] The linker may have the following structure:

[0234] [ka] In the equation, x' is an integer between 1 and 23, y is an integer between 1 and 5, and z' is an integer between 1 and 23. * is AA SC It is a connection point to AA SC is the side chain of the amino acid residues in cCPP.

[0235] x can be an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (including all ranges and subranges between these).

[0236] x' can be an integer between 1 and 23, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 (including all ranges and subranges between these). x' can be an integer between 5 and 15. x' can be an integer between 9 and 13. x' can be an integer between 1 and 5. x' can be 1.

[0237] 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 3, y can be 4, y can be 5.

[0238] z can be an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (including all ranges and subranges between these).

[0239] z' can be an integer between 1 and 23, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 (including all ranges and subranges between these). z' can be an integer between 5 and 15. z' can be an integer between 9 and 13. z' can be 11.

[0240] As described above, a linker or M (where M is part of the linker) can be covalently bonded to the cargo at any suitable position on the cargo. A linker or M (where M is part of the linker) can be covalently bonded to the 3' end or 5' end of an oligonucleotide cargo. A linker or M (where M is part of the linker) can be covalently bonded to the N-terminus or C-terminus of a peptide cargo. A linker or M (where M is part of the linker) can be covalently bonded to the backbone of an oligonucleotide or peptide cargo.

[0241] The linker can bind to the side chains of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or to the modified side chains of glutamine or asparagine (e.g., reduced side chains having an amino group). The linker can also bind to the side chain of lysine on the cCPP.

[0242] The linker can bind to the side chains of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the peptide cargo, or to the modified side chains of glutamine or asparagine (e.g., reduced side chains having an amino group). The linker can also bind to the side chain of lysine on the peptide cargo.

[0243] The linker is structured as follows:

[0244] [ka] It may have, During the ceremony, M is a group that conjugates L to a cargo, such as an oligonucleotide. AA s These are the side chains or terminals of amino acids on cCPP, Each AA x These are independently amino acid residues, o is an integer from 0 to 10, p is an integer between 0 and 5.

[0245] The linker is structured as follows:

[0246] [ka] It may have, During the ceremony, M is a group that conjugates L to a cargo, such as an oligonucleotide. AA s These are the side chains or terminals of amino acids on cCPP, Each AA x These are independent amino acid residues, o is an integer from 0 to 10, p is an integer between 0 and 5.

[0247] M may include alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted.

[0248] [ka] It can be selected from the following, where R is alkyl, alkenyl, alkynyl, carbocykyl, or heterocyclyl.

[0249] M is

[0250] [ka] You can choose from the following: R 10 is alkylene, cycloalkyl, or

[0251] [ka] In the equation, a ranges from 0 to 10.

[0252] M is

[0253] [ka] It could be, R 10 teeth

[0254] [ka] It can be, and a is between 0 and 10. M is,

[0255] [ka] It is possible.

[0256] M is a heterobifunctional crosslinking agent, for example,

[0257] [ka] This is possible, and is disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-4.41.20, which is incorporated herein by reference in its entirety.

[0258] M can be -C(O)-.

[0259] AA s AA may be a side chain or terminal of an amino acid on cCPP. s Non-limiting examples 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). s In this specification, AA is defined as AA SC It is possible.

[0260] Each AA x These are independently natural or non-natural amino acids. One or more AAs x These can be natural amino acids. One or more AAs x These may be non-natural amino acids. One or more AAs xThis could be a β-amino acid. This β-amino acid could be β-alanine.

[0261] o can be an integer from 0 to 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. o can be 0, 1, 2, or 3. o can be 0. o can be 1. o can be 2. o can be 3.

[0262] p can be between 0 and 5, for example, 0, 1, 2, 3, 4, or 5. p can be 0. p can be 1. p can be 2. p can be 3. p can be 4. p can be 5.

[0263] The linker is structured as follows:

[0264] [ka] It may have, In the formula, M, AA s , each -(R 1- JR 2 )z”-, o and z” are defined herein. r may be 0 or 1.

[0265] r can be 0. r can be 1.

[0266] The linker is structured as follows:

[0267] [ka] It may have, in the formula, M, AA s Each of o, p, q, r, and z may be as defined herein.

[0268] z can be an integer from 1 to 50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 (including all ranges and values ​​in between). z'' can be an integer from 5 to 20. z'' can be an integer from 10 to 15.

[0269] The linker is structured as follows:

[0270] [ka] It may have, During the ceremony, M, AA s and o are as defined herein.

[0271] Other non-limiting examples of suitable linkers include:

[0272] [ka]

[0273] [ka] These include, in the formula, M and AA s This is as defined herein.

[0274] This specification provides for a compound comprising cCPP and AC complementary to a target in a pre-mRNA sequence, further comprising L, wherein a linker is conjugated to AC via a binding group (M), and M is

[0275] [ka] A compound is provided.

[0276] This specification describes a compound comprising cCPP and an antisense compound (AC), for example, a cargo containing an antisense oligonucleotide complementary to a target in a pre-mRNA sequence, further comprising L, wherein the linker is conjugated to the AC via a binding group (M), and M is

[0277] [ka] Selected from, R 1 is alkylene, cycloalkyl, or

[0278] [ka] In the formula, t' is from 0 to 10, and each R is independently an alkyl, alkenyl, alkynyl, carbocykyl, or heterocyclyl. 1 teeth

[0279] [ka] A compound is provided in which t' is 2.

[0280] The linker is structured as follows:

[0281] [ka] It may have, During the ceremony, A.A. s m' is as defined herein, and m' is between 0 and 10.

[0282] The linker is, formula:

[0283] [ka] It could be.

[0284] The linker is, formula:

[0285] [ka] This can be the case, where "base" corresponds to the nucleic acid base at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0286] The linker is, formula:

[0287] [ka] This can be the case, where "base" corresponds to the nucleic acid base at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0288] The linker is, formula:

[0289] [ka] This can be the case, where "base" corresponds to the nucleic acid base at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0290] The linker is, formula:

[0291] [ka] This can be the case, where "base" corresponds to the nucleic acid base at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0292] The linker is, formula:

[0293] [ka] It could be.

[0294] Linker c can be covalently bonded to the cargo at any suitable position on the cargo. The linker is covalently bonded to the 3' end of the cargo or the 5' end of an oligonucleotide cargo. The linker can also be covalently bonded to the cargo backbone.

[0295] The linker can bind to the side chains of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or to the modified side chains of glutamine or asparagine (e.g., reduced side chains having an amino group). The linker can also bind to the side chain of lysine on the cCPP.

[0296] cCPP-Linker Conjugate cCPP may be conjugated to a linker as defined herein. The linker is an AA of cCPP as defined herein. SC It can be conjugated to.

[0297] The linker is -(OCH2CH2) z’ Subunits may be included (for example, as spacers), where z' is an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. (-(OCH2CH2)) z’ This is also called PEG. The cCPP-linker conjugate may have a structure selected from Table 4.

[0298] [Table 6]

[0299] The linker is -(OCH2CH2) z’ - May include a subunit (wherein z' is an integer from 1 to 23) and a peptide subunit. The peptide subunit may contain 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table 5.

[0300] [Table 7]

[0301] The cCPP-linker conjugate may have the structure shown in Figure 1 (e.g., compound 1a, compound 1b, compound 2a, or compound 3a) or the sequences listed in Table 4.

[0302] The cCPP-linker conjugate may have the sequences listed in Table 5.

[0303] The cCPP-linker conjugate may be Ac-PKKKRKV-K(cyclo[FfΦGrGrQ])-PEG12-K(N3)-NH2. An EEV comprising a permeable peptide (cCPP), a linker, and an extracyclic peptide (EP) is provided. The EEV is given by formula (B):

[0304] [ka] This may include the structure of, or its protonated form. During the ceremony, R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid. R4 and R7 are independently H or amino acid side chains. EP is an extracyclic peptide as defined herein, Each m is an independent integer between 0 and 3. n is an integer between 0 and 2. x' is an integer between 1 and 20. y is an integer between 1 and 5. q is 1 to 4, z' is an integer between 1 and 23.

[0305] R1, R2, R3, R4, R7, EP, m, q, y, x', z' are as described herein.

[0306] n can be 0. n can be 1. n can be 2.

[0307] EEV is given by formula (Ba) or (Bb):

[0308] [ka] The formula may include the structure or its protonated form, where EP, R 1 , R 2 , R 3 , R 4 m and z' are as defined above in equation (B).

[0309] EEV is given by equation (Bc):

[0310] [ka] The formula may include the structure or its protonated form, where EP, R 1 , R 2 , R 3 , R 4 , and m are as defined above in formula (B), AA is an amino acid as defined herein, M is as defined herein, n is an integer from 0 to 2, x is an integer from 1 to 10, y is an integer from 1 to 5, and z is an integer from 1 to 10.

[0311] EEV is given by formulas (B-1), (B-2), (B-3), or (B-4):

[0312] [ka]

[0313] [ka] The structure or protonated form may be as defined above in formula (B).

[0314] EEV may include formula (B), structure: Ac-PKKKRKVAEEA-K(cyclo[FGFGRGRQ])-PEG 12 -OH or Ac-PK-KKR-KV-AEEA-K(cyclo[GfFGrGrQ])-PEG 12 It may contain -OH.

[0315] EEV may include cCPP of the formula:

[0316] [ka]

[0317] EEV may include the formula:Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3).

[0318] EEV could be...

[0319] [ka]

[0320] EEV is

[0321] [ka] It is possible.

[0322] EEV can be Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-miniPEG-K(cyclo-Ff-Nal-GrGrQ)-PEG12-OH.

[0323] EEV is

[0324] [ka] It is possible.

[0325] EEV can be Ac-PKKKRKV-miniPEG-K(cyclo(Ff-Nal-GrGrQ)-PEG12-OH).

[0326] EEV is

[0327] [ka]

[0328] EEV is

[0329] [ka] It is possible. EEV is possible.

[0330] [ka]

[0331] EEV is

[0332] [ka]

[0333] EEV is

[0334] [ka] It is possible.

[0335] EEV is

[0336] [ka]

[0337] EEV could be...

[0338] [ka]

[0339] EEV could be...

[0340] [ka]

[0341] EEV can be.

[0342]

Chemical formula

[0343] EEV can be.

[0344]

Chemical formula

[0345] EEV is

[0346]

Chemical formula

[0347] EEV is Ac-rr-miniPEG2-Dap[cyclo(FfΦ-Cit-r-Cit-rQ)]-PEG12-OH Ac-frr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rfr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbfbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rrr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbrbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hbhbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbhbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hbrbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-frr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rfr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbfbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rrr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbrbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hbhbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbhbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hbrbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-KKKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH Ac-KGKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH Ac-KKGK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KGK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBKBK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KR-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBR-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PGKKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKGKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKGRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKGKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRGV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRKG-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 and You can choose from Ac-KRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2.

[0348] EEV can be selected from the following.

[0349] [Table 8]

[0350] EEV can be selected from the following.

[0351] [Table 9]

[0352] EEV can be selected from the following.

[0353] [Table 10]

[0354] EEV can be selected from the following.

[0355] [Table 11] It can be selected from the following.

[0356] Cargo can be a protein, and EEV is, Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[FGFRRRRQ])-PEG12 -OH Ac-rrr-PEG2-K(シクロ[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(シクロ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rrr-PEG2-K(シクロ[FfF-GRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(シクロ[FGFGRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(シクロ[GfFGrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(シクロ[FGFGRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(シクロ[FGFRRRRQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[FfF-GRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[FGFGRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[GfFGrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[FGFGRRRQ])-PEG 12 -OH Ac-rhr-PEG2-K(シクロ[FGFRRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(シクロ[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(シ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbr-PEG2-K(シ[FfF-GRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(。[FGFGRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(。[GfFGrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(。[FGFGRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(。[FGFRRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(シ[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(シ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(シ[FfF-GRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(FGFGRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(シ[GfFGrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(FGFGRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(。[FGFRRRRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(。[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(シ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, and Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH The following can be selected from the formula, where b is beta-alanine and the extraring sequence can be D or L stereochemistry.

[0357] cargo Cell-permeable peptides (CPPs), such as cyclic cell-permeable peptides (e.g., cCPPs), can be conjugated to a cargo. The cargo may be a therapeutic moiety. The cargo can be conjugated to the terminal carbonyl group of a linker. At least one atom of the cyclic peptide may be substituted by the cargo, or at least one lone pair of electrons may form a bond to the cargo. The cargo can be conjugated to cCPP by the linker. The cargo can be conjugated to AA by the linker. SC It can be conjugated to AA. At least one atom of cCPP may be substituted with the therapeutic moiety, or at least one lone pair of electrons of cCPP may form a bond to the therapeutic moiety. The hydroxyl group on the amino acid side chain of cCPP may be replaced by a bond to the cargo. The hydroxyl group on the glutamine side chain of cCPP may be replaced by a bond to the cargo. The cargo may be conjugated to cCPP by a linker. The cargo may be conjugated by a linker SC It can be conjugated to.

[0358] The cargo may comprise one or more detectable parts, one or more therapeutic parts, one or more targeted parts, or any combination thereof. The cargo may be a peptide, oligonucleotide, or small molecule. The cargo may be a peptide sequence or a non-peptidyl therapeutic agent. The cargo may be an antibody or its antigen-binding fragment, for example, an scFv or a nanobody, but not limited to these.

[0359] The cargo may contain one or more additional amino acids (e.g., K, UK, TRV), a linker (e.g., a bifunctional linker LC-SMCC), coenzyme A, phosphocoumarin aminopropionic acid (pCAP), 8-amino-3,6-dioxaoctanoic acid (miniPEG), L-2,3-diaminopropionic acid (Dap or J), L-β-naphthylalanine, L-pipecolic acid (Pip), sarcosine, trimesic acid, 7-amino-4-methylcoumarin (Amc), fluorescein isothiocyanate (FITC), L-2-naphthylalanine, norleucine, 2-aminobutyric acid, rhodamine B (Rho), dexamethasone (DEX), or a combination thereof.

[0360] The cargo may include any of the items listed in Table 6, or derivatives or combinations thereof.

[0361] [Table 12] * pCAP is phosphocoumarin aminopropionic acid, Ω is norleucine, U is 2-aminobutyric acid, D-pThr is D-phosphothreonine, and Pip is L-piperidine-2-carboxylate.

[0362] Detectable portion This compound may contain a detectable moiety. The detectable moiety can be bound to the cell-permeable peptide (CPP) by the amino group, carboxylate group, or side chain of any amino acid in the CPP (e.g., by the amino group, carboxylate group, or side chain of any amino acid in cCPP). The detectable moiety can be bound to the cyclic cell-permeable peptide (cCPP) by the side chain of any amino acid in cCPP. The cargo may contain a detectable moiety. The cargo may contain a therapeutic agent and a detectable moiety. The detectable moiety may contain any detectable label. Suitable examples of detectable labels include, but are not limited to, UV-Vis labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable moieties, chelate centers, heavy atoms, radioisotopes, isotope-detectable spin resonance labels, paramagnetic moieties, chromophores, or any combination thereof. The label may be detectable without the addition of further reagents.

[0363] The detectable portion may be the biocompatible portion, so that the compound may be suitable for use in various biological applications. “Biocompatible” and “biologically suitable,” as used herein, generally refer to compounds (together with any of their metabolites or degradation products) that are generally nontoxic to cells and tissues and do not cause any significant adverse effects on cells and tissues when incubated (e.g., cultured) in their presence.

[0364] The detectable portion may contain a luminescent phore such as a fluorescent label or a near-infrared label. Suitable examples of luminescent phores include, but are not limited to, metal porphyrins, benzoporphyrins, azabenzoporphyrins, naptoporphyrins, phthalocyanines, polycyclic aromatic hydrocarbons (such as diimines and pyrenes), azo dyes, xanthene dyes, boron dipyrometenes, aza-boron dipyrometenes, cyanine dyes, metal ligand complexes (such as bipyridines, bipyridyls, phenanthrolines, and coumarins), as well as ruthenium and iridium acetylacetonates, acridines, oxazine derivatives (such as benzophenoxazines), aza-annulenes, squaline, 8-hydroxyquinolines, polymethines, luminescent nanoparticles (such as quantum dots and nanocrystals), carbostyryls, terbium complexes, inorganic phosphors, ionophores (such as crown ether series or derivatized dyes), or combinations thereof. Specific examples of suitable luminescent phospholipids include Pd(II) octaethylporphyrin, Pt(II)-octaethylporphyrin, Pd(II) tetraphenylporphyrin, Pt(II) tetraphenylporphyrin, Pd(II) meso-tetraphenylporphyrin tetrabenzoporfin, and Pt(II) meso-tetraphenylmetrilbenzoporphyrin. (metrylbenzoporphyrin), Pd(II) octaethylporphyrin ketone, Pt(II) octaethylporphyrin ketone, Pd(II) meso-tetra(pentafluorophenyl)porphyrin, Pt(II) meso-tetra(pentafluorophenyl)porphyrin, Ru(II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru(dpp)3), Ru(II) tris(1,10-phenanthroline) (Ru(phen)3), tris(2,2'-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3), erythrosine B, fluorescein, fluorescein isothiocyanate (FITC), eosin, iridium(III) ((N-methyl-benzimidazole-2-yl)-7-(diethylamino)-coumarin)), (Enzothiazole)((Benzothiazole-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate), Lumogen dye, Macroflex fluorescent red, Macrolex fluorescent yellow, Texas Red, Rhodamine B, Rhodamine 6G, Sulfur Rhodamine, m-Cresol, Thymol Blue, Xylenol Blue, Cresol Red, Chlorophenol Blue, Bromocresol Green, Bromocresol Red, Bromothymol Blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, 4-Nitirophenol, Alizarin, Phenolphthalein, o-Cresolphthalein, Chlorophenol Red, Calmagite, Bromo-Xylenol, Phenol Red, Neutral Red, Nitrazine, 3,4,5,6-Tetrabromophenolphthalein Examples include, but are not limited to, fluorescein, Congo Red, fluor'sc'in, eosin, 2',7'-dichlorofluorescein, 5(6)-carboxy-fluorescein, carboxynaphthofluorescein, 8-hydroxypyrene-1,3,6-trisulfonic acid, seminaphthophorfodaflore, seminaphthofluorescein, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride, (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)tetraphenylboron, platinum(II) octaethylporphyrin, dialkylcarbocyanine, dioctadecylcyclooxacarbocyanine, fluorenylmethyloxycarbonyl chloride, 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), and derivatives or combinations thereof.

[0365] The detection portion may include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), or derivatives or combinations thereof.

[0366] The detectable portion can be bound to the cell-permeable peptide (CPP) by the amino group, carboxylate group, or side chain of any amino acid in the CPP (for example, by the amino group, carboxylate group, or side chain of any amino acid in cCPP).

[0367] treatment part The compounds of this disclosure may include a therapeutic moiety. The cargo may include a therapeutic moiety. A detectable moiety may be ligated to a therapeutic moiety, or a detectable moiety may also function as a therapeutic moiety. A therapeutic moiety refers to a group that, when administered to a subject, alleviates one or more symptoms of a disease or disorder. A therapeutic moiety may include a peptide, a protein (e.g., an enzyme, an antibody or a fragment thereof), a small molecule, or an oligonucleotide.

[0368] The therapeutic portion may include, and may not include, a wide range of drugs, such as antagonists (e.g., enzyme inhibitors) and agonists (e.g., transcription factors that produce increased expression of a desired gene product (as understood by those skilled in the art, antagonistic transcription factors may also be used)). Furthermore, the therapeutic portion may include drugs that can induce toxicity and / or toxicity to healthy and / or unhealthy cells in the body. In addition, the therapeutic portion may be capable of inducing and / or priming the immune system against potential pathogens.

[0369] The therapeutic component may include, for example, anticancer drugs, antiviral drugs, antibacterial drugs, anti-inflammatory drugs, immunosuppressants, anesthetics, or any combination thereof.

[0370] The therapeutic portion may include anticancer drugs. Examples of anticancer drugs include 13-cis-retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adolsil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-trans retinoic acid, Alpha-interferon, Altretamine, Ametopterin, Amifostin, Aminoglutethimide, Anag Lerido, Anandron, Anastrozole, Arabinosilcytosine, Aranesp, Aredia, Aromasin, Arsenic Trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxan, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Leucovorin Calcium, Campas, Camptosar, Camptothecin-11, Capecitabine, Carlac, Carboplatin, Carmustine, Carmustine Wafer, Casodex, CCNU, C DDP, CeeNU, Serubidin, Cetuximab, Chlorambucil, Cisplatin, Citroborum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Sitadren, Cytarabine, Cytarabine Liposome, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposome, DaunoXome, Decadron, Delta-Cortef, Deltazone, Deniloquine Difutitex, Depotyte, De Xamethasone, Dexamethasone Acetate, Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin Liposome, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin Alpha, Elbitax, Elvinia L-Asparaginase, Estramustine, Ethyol, Etopophos, EtoposideEtoposide phosphate, Evulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floroxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folic acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, Mechloretamine, Mechloretamine hydrochloride, Medralon, Medrol, Megres, Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Menex, Methotrexate, Methotrexate sodium, Methylprednisolone, Mirocel, Letrozole, Neo Prolifegroprolife, which has carmustine implants, has the following products: Sarl, Neurasta, Numega, Nepugen, Nilandron, Niltamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide Acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orpred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon, Peg Aspargase, Pegfilgrastim, PEG-INTRON, PEG-L-Asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelon, Procarbazine, Procrit, Proleukin, and Carmustine Implants. 20, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (Interferon Alpha-2a), Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Salglamostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targetretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Salomide, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE,Toposar, Topotecan, Toremifene, Trastubumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, Vepsid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine Tartrate, VLB, VP-16, Vamon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic Acid, Zometa, Gliadelwafer, Gleevec GM-CSF, Goserelin, Granulocyte Colony-Stimulating Factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocortisone Acetate, Hydrocortisone, Sodium Hydrocortisone Phosphate, Sodium Hydrocortisone Succinate, Hydrocortisone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Diuxetane, Idamycin, Idarubicin, Ifex, IFN-Alpha, Ifosfamide, IL Examples include IL-11, imatinib mesylate, imidazole carboxamide, interferon alfa, interferon alfa-2b (PEG conjugate), interleukin-2, interleukin-11, intron A (interferon alfa-2b), leucovorin, Leukeran, Leukine, leuprolide, leulocristine, leustatin, liposomal Ara-C, Liquid Pred, lomustine, L-PAM, L-sarcolicin, methicorten, mitomycin, mitomycin-C, mitoxantrone, M-prednisol, MTC, MTX, mustagen, mustine, mutamycin, myrelan, irinotecan, isotretinoin, quidrolase, lanacort, L-asparaginase, and LCR. The therapeutic portion may also include, for example, biopharmaceuticals such as antibodies.

[0371] The therapeutic portion may include antiviral agents such as ganciclovir, azidothymidine (AZT), and lamivudine (3TC).

[0372] The treatment areas include acetapzon, acetosulfone sodium, aramesin, alexidine, amdinocillin, amidinocillin pivoxil, amicycline, amifloxacin, amifloxacin mesylate, amikacin, amikacin sulfate, aminosalicylic acid, aminosalicylate sodium, amoxicillin, amphomycin, ampicillin, ampicillin sodium, aparcillin sodium, apramycin, aspartosin, astromycin sulfate, aviramycin, avoparcin, azithromycin, azurocillin, azurocillin sodium, and bacampi. Bacitracin hydrochloride, bacitracin methylenedisalicylate, bacitracin zinc, babelmycin, benzoylpas calcium, berithromycin, betamycin sulfate, biapenem, vinyramycin, bifenamin hydrochloride, bispirithione magnsulfex, buticacin, butyrosine sulfate, capreomycin sulfate, carbadox, carbenicillin disodium, carbenicillin indanil sodium, carbenicillin phenyl sodium, carbenicillin potassium, carmonam sodium, cefaclor, cefadroxil, cefam Ndol, Cephamandol Naphate, Cephamandol Sodium, Cephaparol, Cephatidine, Cefazalur Sodium, Cefazolin, Cefazolin Sodium, Cefbuperazone, Cefdinir, Cefepime, Cefepime Hydrochloride, Cefetecor, Cefixime, Cefmenoxime Hydrochloride, Cefmetazole, Cefmetazole Sodium, Cefonisid Monosodium, Cefonisid Sodium, Cefoperazone Sodium, Cefolanide, Cefotaxime Sodium, Cefotetan, Cefotetan Disodium, Cefotiam Hydrochloride, Cefoxy Citin, cefoxitin sodium, cefpimisole, cefpimisole sodium, cefpyramide, cefpyramide sodium, cefpirom sulfate, cefpodoxime proxetil, cefprodil, ceffloxazine, cefsulodine sodium, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroxime pivocetil, cefuroxime sodium, acecetril sodium, cephalexin, cephalexin hydrochloride, cephaloglysin, cephaloridine, cephalothin sodium,Cefapillin sodium, cefradin, cetocycline hydrochloride, cetophenicol, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate complex, chloramphenicol succinate sodium, chlorohexidine phosphanilate, chlorooxylenol, chlortetracycline bicarbonate, chlortetracycline hydrochloride, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride, ciloremycin, clarithromycin, clinafloxacin hydrochloride, clindamycin, clindamycin hydrochloride Clindamycin palmitate hydrochloride, clindamycin phosphate, clofazimine, cloxacillin benzathine, cloxacillin sodium, cloxiquin, colistin metasodium, colistin sulfate, coumamycin, coumamycin sodium, cyclacillin, cycloserine, dalphopristin, dapsone, daptomycin, demeclocycline, demeclocycline hydrochloride, demecycline, denofungin, diaperidine, dicloxacillin, dicloxacillin sodium, dihydrostreptomycin sulfate, dipyrithione, dilis Romycin, doxycycline, doxycycline calcium, doxycycline phosphatex, doxycycline helicate, doxycycline sodium, enoxacin, epicillin, epitetracycline hydrochloride, erythromycin, erythromycin assistrate, erythromycin estrate, erythromycin ethyl succinate, erythromycin single ceptate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, ethambutol hydrochloride, ethionamide, freloxacin , floxacillin, fludaranin flumequine, fosfomycin, fosfomycin tromethamine, fumoxicillin, furazolium chloride, furazolium tartrate, sodium fusidate, fusidic acid, gentamicin sulfate, gloximonam, gramicidin, haloprosin, hetacillin, hetacillin potassium, hexedine, ivafloxacin, imipenem, isoconazole, isepamycin, isoniazid, josamycin, kanamycin sulfate, kitasamycin, levoflartadone, levopropylcillin potassium, lexithromycin, lincomycin,Lincomycin hydrochloride, lomefloxacin, lomefloxacin hydrochloride, lomefloxacin mesylate, loracalbef, mafenide, meclocycline, meclocycline sulfosalicylate, megalomycin potassium phosphate, mekidox, meropenem, metacycline, metacycline hydrochloride, methenamine, methenamine hyprulate, methenamine mandelate, methicillin sodium, methioprim, metronidazole hydrochloride, metronidazole phosphate, mezlocillin, mezlocillin sodium, minocycline, minocycline hydrochloride, milcamycin hydrochloride, monet Ncin, monensin sodium, naphicillin sodium, nalidixate sodium, nalidixic acid, natamycin, nebramycin, palmitate neomycin, sulfate neomycin, undecylenate neomycin, netylmycin sulfate, neutomycin, nifladen, niflardezone, niflatel, niflatron, nifludazil, niflamide, niflupyrinol, nifluquinazole, nifurthiazol, nitrocycline, nitrofurantoin, nitromid, norfloxacin, novobiosin sodium, ofloxacin, onnetoprim, oxacin Oxacillin sodium, oxymonam, oxymonam sodium, oxolinic acid, oxytetracycline, oxytetracycline calcium, oxytetracycline hydrochloride, paldimycin, parachlorophenol, paulomycin, pefloxacin, pefloxacin mesylate, penamecillin, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V, penicillin V benzathine, penicillin V hydravamin, penicillin V potassium, pentizidone sodium, phenylaminosulfate Licylic acid, piperacillin sodium, pirbenicillin sodium, pyridicillin sodium, pirurimycin hydrochloride, pivampicillin hydrochloride, pivampicillin pamoate, pivampicillin probenate, polymyxin B sulfate, porphyromycin, propicacin, pyrazinamide, zinc pyrithione, quindecamine acetate, quinupristin, racefenicol, lamoplanin, ranimycin, reromycin, repromycin, rifabutin, rifametan, rifamexil, rifaamide, rifampin, rifapentin, rifaximin, lolitetracycline,Lolitetracycline nitrate, rosalamycin, rosalamycin butyrate, rosalamycin propionate, rosalamycin sodium phosphate, rosalamycin stearate, rosoxacin, roxalson, roxithromycin, sancycline, sanfetolinemushi sodium, salmonoxicillin, salpicillin, scopafungin, shisomecin, shisomecin sulfate, sparfloxacin, spectinomycin hydrochloride, spiramycin, starimycin hydrochloride, stefimycin, streptomycin sulfate, streptonicozide, sulfabenz, sulfabenzamide, sulfacetamide, sulfacetamide sodium, sulacitin, sulfadiazine, sulfadiazine sodium, sulfadoxine, sulfarene, sulfamerazine, sulfamethazine, sulfamethizol, sulfamethoxazole, sulfamonomethoxine, sulfamoxol, zinc sulfanilate, sulfanito It may contain antibacterial agents such as lan, sulfasalazine, sulfasomisole, sulfathiaazole, sulfazameth, sulfisoxazole, sulfisoxazole acetyl, sulfisoxazole diolamine, sulfomyxin, sulopenem, sultamicillin, sancillin sodium, talampicillin hydrochloride, teicoplanin, temafloxacin hydrochloride, temocillin, tetracycline, tetracycline hydrochloride, tetracycline phosphate complex, tetroxoprim, thianphenicol, tifencillin potassium, ticalcillin glesyl sodium, ticalcillin disodium, ticalcillin monosodium, ticlaton, thiodonium chloride, tobramycin, tobramycin sulfate, tosufloxacin, trimethoprim, trimethoprim sulfate, trisulfapyrimidine, troleandomycin, trospemycin sulfate, tyrosulisin, vancomycin, vancomycin hydrochloride, virginiamycin, or zolbamycin.

[0373] The treatment may include anti-inflammatory agents.

[0374] The therapeutic portion may include dexamethasone (Dex).

[0375] The therapeutic portion may include therapeutic proteins. For example, some people have deficiencies in certain enzymes (e.g., lysosomal storage disorders). Such enzymes / proteins can be delivered to human cells by ligating them to cyclic cell-permeable peptides (cCPPs) disclosed herein. The cCPPs of this disclosure have been tested with proteins (e.g., GFP, PTP1B, actin, calmodulin, troponin C) and have been shown to be functional.

[0376] The therapeutic component may be an anti-infective agent. The term "anti-infective agent" refers to a drug that can kill, inhibit, or otherwise slow the growth of an infectious pathogen. The term "infectious agent" refers to pathogenic microorganisms such as bacteria, viruses, fungi, and intracellular or extracellular parasites. Since infectious diseases are caused by infectious agents, they can be treated with anti-infective agents.

[0377] The infectious agent may be a Gram-negative bacterium. Gram-negative bacteria may belong to genera selected from Escherichia, Proteus, Salmonella, Klebsiella, Providencia, Enterobacter, Burkholderia, Pseudomonas, Acinetobacter, Aeromonas, Haemophilus, Yersinia, Neisseria, Erwinia, Rhodopseudomonas, and Burkholderia. The infectious agent may be a Gram-positive bacterium. Gram-positive bacteria may belong to genera selected from Lactobacillus, Azorhizobium, Streptococcus, Pediococcus, Photobacterium, Bacillus, Enterococcus, Staphylococcus, Clostridium, Butyrivibrio, Sphingomonas, Rhodococcus, and Streptomyces. The infectious agent may be mycobacteria of the genus Mycobacterium, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, and Mycobacterium leprae. The infectious agent may be of the genus Nocardia. The infectious agent can be selected from one of the following species: Nocardia asteroides, Nocardia brasiliensis, Nocardia brasiliensis, and Nocardia caviae.

[0378] The infectious agent may be a fungus. The fungus may be of the genus Mucor. The fungus may be of the genus Crytococcus. The fungus may be of the genus Candida. The fungus can be selected from one of the following: Mucor racemosus, Candida albicans, Crytococcus neoformans, or Aspergillus fumigatus.

[0379] The infectious agent may be a protozoan. The protozoan may belong to the genus Plasmodium (e.g., P. falciparum, P. vivax, P. ovale, or P. malariae). These protozoa cause malaria.

[0380] Examples of organisms include Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, and Helicobacter. Examples include Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.

[0381] The infectious agent may be a parasite. The parasite may be Cryptosporidium. The parasite may be an endoparasite. The endoparasite may be a heartworm, tapeworm, or flatworm. The parasite may be an epiparasite. The parasite causes diseases selected from acanthomolebiasis, babesiosis, balantidiosis, blastocystosis, coccidiosis, amoebic dysentery, giardiasis, isosporiasis, cystosporiasis, leishmaniasis, primary amoebic meningoencephalitis, malaria, rhinovirus infection, toxoplasmosis, trichomoniasis, trypanosoma, Chagas disease, or scabies.

[0382] The infectious agent can be a virus. Non-specific examples of viruses include idiopathic acute respiratory coronavirus 2 (SARS-CoV-2), idiopathic acute respiratory coronavirus (SARS-CoV), Middle East respiratory virus (MERS), influenza, hepatitis C virus, dengue virus, West Nile virus, Ebola virus, hepatitis B, human immunodeficiency virus (HIV), herpes simplex virus, herpes zoster, and Lassa virus.

[0383] Antiinfective agents may be antiviral agents. Non-limiting examples of antiviral agents include nucleosides or nucleotide reverse transcriptase inhibitors (e.g., zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stabudine (d4T), lamivudine (3TC), emtricitabine, abacavir succinate, erbucitabine, adefovir dipivoxil, lobucavir (BMS-180194), rodenosine (FddA), and tenofovir (Te (e.g., nofovir disoproxil and tenofovir disoproxil fumarate), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine, deravirazine, efavirenz, etravirine and rilpivirine), protease inhibitors (e.g., ritonavir, tipranavir, saquinavir, nelfinavir, indinavir, amprenavir, fosamprenavir, atazanavir, lopinavir, darunavir (TM) Examples include C-114), rasanavir and blekanavir (VX-385), cell entry inhibitors (e.g., CCR5 antagonists (e.g., maraviroc, bicriviroc, INCB9471 and TAK-652) and CXCR4 antagonists (AMD-11070)), fusion inhibitors (e.g., enfuvirtide), integrase inhibitors (e.g., raltegravir, BMS-707035 and elvitegravir), Tat inhibitors (e.g., didehydrocortistatin A (dCA)), maturation inhibitors (e.g., berivimat), immunomodulators (e.g., levamizole), and other antiviral agents (e.g., hydroxyurea, ribavirin, interleukin-2 (IL-2), interleukin-12 (IL-12), pensafuside, peramivir, zanamivir, oseltamivir phosphate, baloxavir marboxil).

[0384] Antiinfective agents can be antibiotics. Non-specific examples of antibiotics include aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin-resistant, netylmycin, streptomycin, and tobramycin), cavecephems (e.g., loracalbef), carbapenems (e.g., ertapenem, imipenem / cilastatin, and meropenem), and cephalosporins (e.g., cefadroxil, cefazolin, cephalexin, cefac). Lol, cephamandol, cephalexin, cefoxitin, cefprodil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone and cefepime), macrolides (e.g., azithromycin, clarithromycin, zithromycin, erythromycin and troleandmycin), monobacter Penicillins (e.g., amoxicillin, ampicillin, carbenicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin and ticalcillin), polypeptides (e.g., bacitracin, colistin and polymyxin B), quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin) Examples include (norfloxacin, ofloxacin, and trovafloxacin), sulfonamides (e.g., mafenide, sulfacetamide, sulfamethizol, sulfasalazine, sulfisoxazole, and trimethoprim-sulfamethoxazole), tetracyclines (e.g., demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline), and vancomycin. Antiinfective agents may be steroidal anti-inflammatory drugs.Non-specific examples of steroidal anti-inflammatory drugs include fluocinolone, triamcinolone, triamcinolone acetonide, betamethasone, betamethasone dipropionate, diflucortolone, fluticasone, cortisone, hydrocortisone, mometasone, methylprednisolone, beclomethasone dipropionate, clobetasol, prednisone, prednisolone, methylprednisolone, methylprednisolone, betamethasone, budesonide, and dexamethasone. Antiinfective agents may be nonsteroidal anti-inflammatory drugs. Non-steroidal anti-inflammatory drugs (NSAIDs) include, but are not limited to, cerocoxib, nimeslid, lofecoxib, meclofenamic acid, sodium meclofenamate, flunixin, fluprofen, flurbiprofen, sulindac, meloxicam, piroxicam, etodolac, fenoprofen, fenbuprofen, ketoprofen, suprofen, diclofenac, bromfenac sodium, phenylbutazone, thalidomide, and indomethacin.

[0385] Antiinfective drugs can be antifungal drugs. Non-specific examples of antifungal agents include amphotericin B, caspofungin, fluconazole, flucytosine, itraconazole, ketoconazole, amrolphine, butenafine, naftifine, terbinafine, erbiol, econazole, enoxol, itraconazole, isoconazole, imidazole, miconazole, sulconazole, clotrimazole, enilconazole, oxyconazole, thioconazole, terconazole, butoconazole, thiabendazole, voriconazole. Examples include saperconazole, sertaconazole, fenticonazole, posaconazole, bifonazole, flutrimazole, nystatin, pimaricin, natamycin, tolnaftate, mafenide, dapsone, actofunicone, griseofulvin, potassium iodide, crystal violet, cyclopirox, cyclopiroxolamine, haloprosin, undecylenate, silver sulfadiazine, undecylenic acid, undecylenic acid alkanolamide, and carbol-fuchsin.

[0386] The therapeutic portion may be an analgesic or pain reliever. Non-exclusive examples of analgesics or pain relievers include aspirin, acetaminophen, ibuprofen, naproxen, procaine, lidocaine, tetracaine, dibucaine, benzocaine, 2-(diethylamino)ethyl p-butylaminobenzoate HCl, mepivacaine, pipelocaine, and diclonin.

[0387] The therapeutic portion may be an antibody or an antigen-binding fragment. Antibodies and antigen-binding fragments may be derived from any suitable source, such as humans, mice, camelids (e.g., camels, alpacas, llamas), rats, ungulates, or non-human primates (e.g., monkeys, rhesus monkeys).

[0388] Furthermore, it should be understood that cargoes containing anti-infective agents and other therapeutic components described herein include possible salts thereof, and the pharmaceutically acceptable salts are particularly relevant to therapeutic use. Examples of salts include acid addition salts and basic salts. Examples of acid addition salts include hydrochloride, fumarate, and oxalate. Examples of basic salts include those in which the (remaining) counterions are alkali metals such as sodium and potassium, alkaline earth metals such as calcium salts and potassium salts, and ammonium ions. + N(R')4(wherein R' is C, which can be substituted independently and at will) 1~6 - Alkyl, optionally substituted C 2~6 -These are salts selected from alkenyls, optionally substituted aryls, or optionally substituted heteroaryls.

[0389] The therapeutic portion may be an oligonucleotide. The oligonucleotide may be an antisense compound (AC). Examples of oligonucleotides include, but are not limited to, antisense oligonucleotides, small interfering RNA (siRNA), microRNA (miRNA), ribozymes, immunostimulatory nucleic acids, antagonist mirs, antimirs, microRNA mimes, supermirs, Ul adapters, CRISPR mechanisms, and aptamers. The terms “antisense oligonucleotide” or simply “antisense” mean that the oligonucleotide is complementary to the target polynucleotide sequence. Non-limiting examples of antisense oligonucleotides for the treatment of Duchenne muscular dystrophy can be found in U.S. Patent Application Publication 2019 / 0365918, U.S. Patent Application Publication 2020 / 0040336, U.S. Patent No. 9,499,818, and U.S. Patent No. 9,447,417, each incorporated by reference in whole for any purpose.

[0390] The treatment area includes the following diseases: neuromuscular diseases, Pompe disease, β-thalassemia, dystrophin dystrophin, Duchenne muscular dystrophy, Becker muscular dystrophy, diabetes, Alzheimer's disease, cancer, cystic fibrosis, merosin-deficient congenital muscular dystrophy type 1A (MDC1A), proximal spinal muscular atrophy (SMA), Huntington's disease, Huntington's chorea-like disease (HDL2), myotonic dystrophy, spinocerebellar degeneration, bulbar spinal muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis, frontotemporal type It can be used to treat any one of the following conditions: dementia, fragile X syndrome, fragile X intellectual disability 1 (FMR1), fragile X intellectual disability 2 (FMR2), fragile XE intellectual disability (FRAXE), Friedreich's ataxia (FRDA), fragile X-associated tremor / ataxia syndrome (FXTAS), myoclonus epilepsy, oculopharyngeal muscular dystrophy (OPMD), symptomatic or non-symptomatic X-linked intellectual disability, myotonic dystrophy, myotonic dystrophy type 1, myotonic dystrophy type 2, epilepsy, Dravet syndrome, or Alzheimer's disease. The therapeutic portion can be used to treat cancer selected from glioma, acute myeloid leukemia, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial carcinoma, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, or melanoma. The treatment portion can be used to treat eye diseases. Non-exclusive examples of eye diseases include refractive errors, macular degeneration, cataracts, diabetic retinopathy, glaucoma, amblyopia, or strabismus.

[0391] The therapeutic portion may include a targeting portion. The targeting portion may include, for example, a sequence of amino acids that can target one or more enzyme domains. The targeting portion may include an inhibitor of an enzyme that can play a role in a disease, such as cancer, cystic fibrosis, diabetes, obesity, or a combination thereof. The targeting portion targets one or more of the following genes: FMR1, AFF2, FXN, DMPK, SCA8, PPP2R2B, ATN1, DRPLA, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, ATP7B, HTT, SCN1A, BRCA1, LAMA2, CD33, VEGF, ABCA4, CEP290, RHO, USH2A, OPA1, CNGB3, PRPF31, GYS1, or RPGR. The therapeutic portion may be an antisense compound (AC) described in U.S. Patent Application Publication No. 2019 / 0365918, which is incorporated herein by reference in its entirety. For example, the targeting portion may include any of the sequences listed in Table 7.

[0392] [Table 13] * Fpa,Σ:L-4-fluorophenylalanine; Pip,θ:L-homoproline; Nle,Ω:L-norleucine; Phg,ΨL-phenylglycine; F2Pmp,Λ:L-4-(phosphonodifluoromethyl)phenylalanine; Dap,L-2,3-diaminopropionic acid; Nal,Φ':L-β-naphthylalanine; Pp,θ:L-pipecolic acid; Sar,Ξ:Sarcosine; Tm,trimesic acid.

[0393] The targeting region and the cell-permeable peptide can overlap. That is, residues that form the cell-permeable peptide may also be part of the sequence that forms the targeting region, and vice versa.

[0394] The therapeutic portion can be bound to the cell-permeable peptide by the amino group, carboxylate group, or side chain of any amino acid of the cell-permeable peptide (for example, by the amino group, carboxylate group, side chain, or amino acid of cCPP). The therapeutic portion can be bound to the detectable portion.

[0395] The therapeutic portion may include a targeted portion that can act as an inhibitor of Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, or a combination thereof.

[0396] Ras is a protein encoded by the RAS gene in humans. Normal Ras protein plays an essential role in normal tissue signaling, while mutations in the Ras gene are associated with the development of many cancers. Ras can act as a molecular on / off switch; when switched on, it recruits and activates proteins necessary for the propagation of growth factors and other receptor signals. Mutations in Ras are associated with various cancers, including lung cancer, colon cancer, pancreatic cancer, and various types of leukemia.

[0397] Protein tyrosine phosphatase 1B (PTP1B) is a prototypical member of the PTP superfamily and plays many roles in eukaryotic cell signaling. PTP1B is a negative regulator of the insulin signaling pathway and is considered a promising therapeutic target, particularly for the treatment of type II diabetes. PTP1B is also involved in the development of breast cancer.

[0398] Pin1 is an enzyme that binds to a subset of proteins and plays a post-phosphorylation regulatory role in regulating protein function. Pin1 activity can modulate the outcomes of proline-directed kinase signaling, thereby regulating cell proliferation and cell survival. Deregulation of Pin1 may play a role in various diseases. Upregulation of Pin1 may be involved in certain cancers, and downregulation of Pin1 may be involved in Alzheimer's disease. Inhibitors of Pin1 may have therapeutic implications for cancer and immune disorders.

[0399] Grb2 is an adapter protein involved in signal transduction and cellular communication. The Grb2 protein contains a single SH2 domain that can bind to tyrosine phosphorylation sequences. Grb2 is widely expressed and essential for multiple cellular functions. Inhibition of Grb2 function can impair developmental processes and block transformation and proliferation of various cell types.

[0400] Recently, it has been reported that the activity of cystic fibrosis membrane conductance regulator (CFTR), a mutated chloride ion channel protein in patients with cystic fibrosis (CF), is negatively regulated by CFTR-related ligand (CAL) via its PDZ domain (CAL-PDZ) (Wolde, M et al. Biol. Chem. 2007, 282, 8099). Inhibition of the CFTR / CAL-PDZ interaction has been shown to improve the activity of ΔPhe508-CFTR, the most common form of CFTR mutation (Cheng, SH et al. Cell 1990, 63, 827; Kerem, BS et al. Science 1989, 245, 1073), by reducing its proteasome-mediated degradation (Cushing, PR et al. Angew. J. Chem. Int. Ed. 2010, 49, 9907). Accordingly, a method for treating a subject having cystic fibrosis is disclosed herein by administering an effective amount of the compound or composition disclosed herein. The compound or composition administered to the subject may include a therapeutic portion that may include a targeted portion capable of acting as an inhibitor of CAL PDZ. The composition or composition disclosed herein may also be administered together with a molecule that modifies CFTR function.

[0401] The therapeutic moiety can be attached to a cyclic peptide by an amino group or a carboxylate group, or by the side chain of any of the amino acids of the cyclic peptide (for example, by an amino group or carboxylate group on the side chain of an amino acid of the cyclic peptide). In some examples, the therapeutic moiety can be attached to a detectable moiety.

[0402] Compositions containing the compounds described herein are also disclosed herein.

[0403] Pharmacovigilant salts and prodrugs of the compounds disclosed herein are also disclosed herein. Pharmacovigilant salts include salts of the compounds disclosed herein prepared with acids or bases, depending on the specific substituents found on the compounds. It may be appropriate to administer the compounds as salts under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable, non-toxic acidic or basic salts. Examples of pharmaceutically acceptable base-added salts include sodium, potassium, calcium, ammonium, or magnesium salts. Examples of physiologically acceptable acid-added salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids such as acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, alpha-ketoglutaric acid, alpha-glycophosphate, maleic acid, tosylic acid, and methanesulfonic acid. Accordingly, disclosed herein are hydrochlorides, nitrates, phosphates, carbonates, bicarbonates, sulfates, acetates, propions, benzoates, succinates, fumarates, mandelates, oxalates, citrates, tartrates, malons, ascorbicates, alpha-ketoglutarates, alpha-glycolinates, maleates, tosylates, and mesylates. Pharmacochemically acceptable salts of these compounds can be obtained using standard procedures well known in the art (for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid that yields a physiologically acceptable anion). Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0404] The therapeutic portion may include a therapeutic polypeptide, oligonucleotide, or small molecule. The therapeutic polypeptide may include a peptide inhibitor. The therapeutic polypeptide may include a conjugate reagent that specifically binds to the target of interest. The conjugate reagent may include an antibody or its antigen-binding fragment that specifically binds to the target of interest. The antigen-binding fragment may include a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, an Fv fragment, a mini-body, a dia-body, a nano-body, a single-domain antibody (dAb), a single-strand variable fragment (scFv), or a multispecific antibody.

[0405] Oligonucleotides may contain antisense compounds (ACs). The ACs may contain nucleotide sequences complementary to the target nucleotide sequence encoding the desired protein target.

[0406] The therapeutic portion (TM) can be conjugated to the chemically reactive side chains of amino acids in cCPP. TM can be linked to cCPP using any amino acid side chain on cCPP that can form a covalent bond or can be modified in that way. The amino acids on cCPP may be natural or non-natural amino acids. Chemically reactive side chains may include amine groups, carboxylic acids, amides, hydroxyl groups, sulfhydryl groups, guanidinyl groups, phenol groups, thioether groups, imidazolyl groups, or indolyl groups. The amino acids on cCPP to which TM is conjugated may include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, tryptophan, or their analogues. The amino acids on cCPP used to conjugate TM may be ornithine, 2,3-diaminopropionic acid, or their analogues. The amino acid may be lysine or an analogue thereof. The amino acid may be glutamic acid or an analogue thereof. The amino acid may be aspartic acid or an analogue thereof. The side chain may be bonded to TM or substituted with a linker.

[0407] TM may contain a therapeutic polypeptide, and cCPP may be conjugated to the chemically reactive side chain of an amino acid of the therapeutic polypeptide. cCPP can be linked to TM using any amino acid side chain on TM that can form a covalent bond or can be modified in that way. The amino acids on TM may be natural or non-natural amino acids. The chemically reactive side chain may contain an amine group, carboxylic acid, amide, hydroxyl group, sulfhydryl group, guanidinyl group, phenol group, thioether group, imidazolyl group, or indolyl group. The amino acids of TM to which cCPP is conjugated may include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, tryptophan, or analogs thereof. The amino acids on TM used to conjugate cCPP may be ornithine, 2,3-diaminopropionic acid, or analogs thereof. The amino acid may be lysine or an analogue thereof. The amino acid may be glutamic acid or an analogue thereof. The amino acid may be aspartic acid or an analogue thereof. The side chain of TM may be bonded to cCPP or substituted with a linker.

[0408] TM may be an antisense compound (AC) containing an oligonucleotide in which the 5' or 3' end of the oligonucleotide is conjugated to the chemically reactive side chain of an amino acid of cCPP. AC can be chemically conjugated to cCPP via a portion on the 5' or 3' end of AC. The chemically reactive side chain of cCPP may contain an amine group, carboxylic acid, amide, hydroxyl group, sulfhydryl group, guanidinyl group, phenol group, thioether group, imidazolyl group, or indolyl group. The amino acid of cCPP to which AC is conjugated may contain lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, or tryptophan. The amino acid of cCPP to which AC is conjugated may contain lysine or cysteine.

[0409] oligonucleotides This compound may contain a cyclic cell-permeable peptide (cCPP) conjugated to an antisense compound (AC) as the therapeutic portion. The AC may include antisense oligonucleotides, siRNAs, microRNAs, antagonist miRNAs, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, super miRNAs, miRNA mimetics, miRNA inhibitors, or combinations thereof.

[0410] Antisense oligonucleotides The therapeutic portion may include antisense oligonucleotides. The terms “antisense oligonucleotide” or simply “antisense” refer to oligonucleotides complementary to the target polynucleotide sequence. Antisense oligonucleotides may include a single strand of DNA or RNA complementary to a selected sequence, such as the target gene mRNA.

[0411] Antisense oligonucleotides can modulate one or more aspects of protein transcription, translation, expression, and function through hybridization of the antisense oligonucleotide with a target nucleic acid. Hybridization of an antisense oligonucleotide to its target sequence can suppress the expression of the target protein. Hybridization of an antisense oligonucleotide to its target sequence can suppress the expression of one or more target protein isoforms. Hybridization of an antisense oligonucleotide to its target sequence can upregulate the expression of the target protein. Hybridization of an antisense oligonucleotide to its target sequence can downregulate the expression of the target protein.

[0412] Antisense compounds can inhibit gene expression by binding to complementary mRNA. Binding to a target mRNA can lead to inhibition of gene expression by interfering with the translation of the complementary mRNA strand or by causing degradation of the target mRNA. Antisense DNA can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. Antisense oligonucleotides may contain approximately 10 to 50 nucleotides, approximately 15 to 30 nucleotides, or approximately 20 to 25 nucleotides. This term also encompasses antisense oligonucleotides that do not need to be perfectly complementary to the desired target gene. Therefore, the compounds disclosed herein can be used when non-target-specific activity is found in the antisense, or when an antisense sequence containing one or more mismatches with the target sequence is desired.

[0413] Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis and can therefore be used to specifically inhibit protein synthesis by target genes. The effectiveness of antisense oligonucleotides for inhibiting protein synthesis has been well proven.

[0414] Methods for producing antisense oligonucleotides are publicly known in the art and can be readily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence of interest. Selection of antisense oligonucleotide sequences specific to a given target sequence is based on analysis of the selected target sequence and determination of its secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides may be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or hinder specific binding to the target mRNA in host cells. Target regions of mRNA include the AUG translation start codon or its vicinity, and sequences substantially complementary to the 5' region of mRNA. These secondary structure analyses and target site selections can be performed, for example, using version 4 of the OLIGO primer analysis software (Molecular Biology Insights) and / or the BLASTN 2.0.5 algorithm software (Altschul et al, Nucleic Acids Res. 1997, 25(17):3389-402).

[0415] RNA interference nucleic acid The therapeutic portion may be an RNA interference (RNAi) molecule or a small interfering RNA molecule. RNA interference methods using RNAi or siRNA molecules can be used to inhibit the expression of a target gene or polynucleotide.

[0416] Small interfering RNAs (siRNAs) are typically double-stranded RNA molecules, usually about 16 to 30 nucleotides long, that can associate with cytoplasmic multiprotein complexes known as RNAi-induced silencing complexes (RISCs). Since siRNA-loaded RISCs mediate the degradation of homologous mRNA transcripts, siRNAs can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNAs function through innate mechanisms that have evolved to regulate gene expression via non-coding RNAs. Various RNAi reagents, such as siRNAs targeting clinically relevant targets, are currently under development, as described in de Fougerolles, A. et al, Nature Reviews 6:443-453 (2007).

[0417] The first RNAi molecules described were RNA:RNA hybrids containing both RNA sense and RNA antisense strands, but it has now been demonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrids can also mediate RNAi (Lamberton, J.S. and Christian, AT, (2003) J. Molecular Biotechnology 24:111-119). RNAi molecules containing any of these different types of double-stranded molecules can be used. Furthermore, it is understood that RNAi molecules can be used and introduced into cells in a variety of forms. RNAi molecules can encompass any molecule capable of inducing an RNAi response in a cell, including, but not limited to, double-stranded oligonucleotides containing two separate strands (i.e., a sense strand and an antisense strand) (e.g., small interfering RNA (siRNA)); double-stranded oligonucleotides containing two separate strands linked together by a non-nucleotidyl linker; oligonucleotides containing hairpin loops of complementary sequences that form a double-stranded region (e.g., shRNAi molecules); and expression vectors that express one or more polynucleotides that can form double-stranded polynucleotides, either alone or in combination with other polynucleotides.

[0418] As used herein, "single-stranded siRNA compound" refers to an siRNA compound consisting of a single molecule. It may include a double-stranded region formed by intra-chain pairing, which may be, for example, a hairpin or panhandle structure. A single-stranded siRNA compound may be antisense with respect to a target molecule.

[0419] The single-stranded siRNA compound may be long enough to enter RISC and participate in RISC-mediated cleavage of target mRNA. The single-stranded siRNA compound has a nucleotide length of at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, or up to about 50. The single-stranded siRNA has a nucleotide length of less than about 200, less than about 100, or less than about 60.

[0420] Hairpin siRNA compounds may have a double-stranded region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs, or at least about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotide pairs. The double-stranded region may have a nucleotide pair length of about 200 or less, about 100 or less, or about 50 or less. The range of double-stranded regions is nucleotide pair lengths of about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21. The hairpin may have a single-stranded overhang or an unpaired terminal region. The overhang may be about 2 to about 3 nucleotides long. The overhang may be on the sense side or the antisense side of the hairpin.

[0421] "Double-stranded siRNA compound," as used herein, is an siRNA compound comprising two or more (possibly two) strands on which interstrand hybridization can form a region of a double-stranded structure.

[0422] The antisense strand of a double-stranded siRNA compound may have a nucleotide length of 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 60, or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, or about 60. It may have a nucleotide length of about 200 or less, about 100 or less, or about 50 or less. The range may be about 17 to about 25, about 19 to about 23, and about 19 to about 21 nucleotide lengths. As used herein, the term “antisense strand” means the strand of the siRNA compound that is sufficiently complementary to the target molecule (e.g., target RNA).

[0423] The sense strand of a double-stranded siRNA compound may have a length of at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, or about 60 nucleotides. It may have a nucleotide length of about 200 or less, about 100 or less, or about 50 or less. The range may be nucleotide lengths of about 17 to about 25, about 19 to about 23, and about 19 to about 21.

[0424] The double-stranded portion of a double-stranded siRNA compound may have 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 60 nucleotide pairs, or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 40, or about 60 nucleotide pairs, which may be about 200 or less, about 100 or less, or about 50 nucleotide pairs. The range may be about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21 nucleotide pairs.

[0425] The siRNA compound may be large enough to be cleaved by an endogenous molecule (e.g., by Dicer) to produce smaller siRNA compounds (e.g., siRNA agents).

[0426] The sense and antisense strands can be selected such that the double-stranded siRNA compound contains single-stranded or unpaired regions at one or both ends of the molecule. Thus, the double-stranded siRNA compound may contain sense and antisense strands paired to include overhangs, e.g., one or two 5' or 3' overhangs, or 3' overhangs of one to three nucleotides. Overhangs may result from one strand being longer than the other, or from two strands of equal length being offset. In some embodiments, there may be at least one 3' overhang. In embodiments, both ends of the siRNA molecule may have 3' overhangs. The overhangs may consist of two nucleotides.

[0427] The length of the double-stranded region may range from about 15 to about 30 nucleotides, or about 18, 19, 20, 21, 22, or 23 nucleotides, for example, within the range of the above-mentioned ssiRNA (siRNA with sticky overhang) compounds. ssiRNA compounds may be of similar length, and natural Dicer-treated products can be constructed from longer dsiRNAs. Embodiments in which the two strands of the ssiRNA compound are linked (e.g., covalently bonded) are also included. This includes hairpins, or other single-stranded structures that provide a double-stranded region, and 3' overhangs.

[0428] The siRNA compounds described herein, such as double-stranded siRNA compounds and single-stranded siRNA compounds, can mediate the silencing of target RNA, such as mRNA, or transcripts of protein-coding genes. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Generally, the RNA to be silenced is an endogenous gene.

[0429] As used herein, the phrase "RNAi-mediated" means the ability to silence target RNA in a sequence-specific manner. While we do not wish to be bound by theory, silencing is thought to involve an RNAi mechanism or process and guide RNA, such as an ssiRNA compound of about 21 to about 23 nucleotides.

[0430] An siRNA compound that is "sufficiently complementary" to a target RNA (e.g., target mRNA) can silence the production of the protein encoded by the target mRNA. An siRNA compound that is "sufficiently complementary" to the RNA encoding the protein of interest can silence the production of the protein of interest encoded by the mRNA. An siRNA compound can be "exactly complementary" to the target RNA, for example, by annealing the target RNA and the siRNA compound to form a hybrid consisting only of Watson-Crick base pairs in a strictly complementary region. A "sufficiently complementary" target RNA may contain an internal region (e.g., at least about 10 nucleotides) that is strictly complementary to the target RNA. In embodiments, the siRNA compound specifically identifies a single nucleotide difference. In this case, the siRNA compound mediates RNAi only when exact complementarity is found in the region of the single nucleotide difference (e.g., within 7 nucleotides).

[0431] microRNA The therapeutic component may be a microRNA molecule. MicroRNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA in plant and animal genomes but are not translated into proteins. Processed miRNAs are single-stranded 17-25 nucleotide (nt)RNA molecules that are incorporated into the RNA-induced silencing complex (RISC) and have been identified as important regulators of development, cell proliferation, apoptosis, and differentiation. They are thought to play a role in regulating gene expression by binding to the 3' untranslated region of specific mRNAs. RISCs mediate downregulation of gene expression through translation inhibition, transcriptional cleavage, or both. RISCs are also involved in transcriptional silencing in the nuclei of various eukaryotes.

[0432] Antagomir The therapeutic portion may be an antagonist miRNA. Antagono miRNAs are RNA-like oligonucleotides with various modifications for RNase protection and pharmacological properties (e.g., enhanced tissue and cellular uptake). They differ from normal RNA, for example, with respect to complete 2'-O-methylation of sugars, a phosphorothioate backbone, and, for example, a cholesterol moiety at the 3' terminus. Antagono miRNAs can be used to efficiently silence endogenous miRNAs by forming a double helix containing the antagonist miRNA and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagonisto miRNA-mediated miRNA silencing is the silencing of miR-122 described in Krutzfeldt et al., Nature, 2005, 438:685-689 (the whole of which is expressly incorporated herein by reference). Antagono miRNAs can be synthesized using standard solid-phase oligonucleotide synthesis protocols. See U.S. Patent Applications No. 11 / 502,158 and No. 11 / 657,341 (the disclosures of each of these are incorporated herein by reference).

[0433] Antagomir may comprise a ligand-binding monomer subunit and a monomer for oligonucleotide synthesis. The monomer is described in U.S. Patent Application No. 10 / 916,185, filed on August 10, 2004. Antagomir may have a ZXY structure, such as that described in PCT Application No. PCT / US2004 / 07070, filed on March 8, 2004. Antagomir may be complexed with an amphiphilic moiety. The amphiphilic moiety for use with oligonucleotide agents is described in PCT Application No. PCT / US2004 / 07070, filed on March 8, 2004.

[0434] Aptamer The therapeutic portion may be an aptamer. An aptamer is a nucleic acid or peptide molecule that binds to a specific molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). DNA aptamers or RNA aptamers that bind to many different entities, from large proteins to small organic molecules, have been successfully produced. See Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct. Biol. 9:324-9 (1999), and Hermann and Patel, Science 287:820-5 (2000). Aptamers may be RNA or DNA-based and may contain a riboswitch. Riboswitches are parts of mRNA molecules that can directly bind to small target molecules, and the binding of these targets affects the activity of genes. Therefore, mRNA containing riboswitches is directly involved in regulating its own activity depending on the presence or absence of its target molecule. Generally, aptamers are manipulated through in vitro selection, i.e., iterative rounds of SELEX (phylogenetic evolution of ligands by exponential enrichment), to bind to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers can be prepared by any known method, such as synthesis, recombination, and purification, and can be used alone or in combination with other aptamers specific to the same target. Furthermore, the term “aptamer” also includes “secondary aptamers” containing consensus sequences derived from comparing two or more known aptamers with a given target. Aptamers may be “intracellular aptamers” or “intramers” that specifically recognize intracellular targets. See Famulok et al., Chem Biol. 2001, Oct, 8(10):931-939 and Yoon and Rossi, Adv Drug Deliv Rev. 2018, Sep, 134:22-35 (each incorporated herein by reference).

[0435] Ribozyme The therapeutic component may be a ribozyme. Ribozymes are RNA molecular complexes that have a specific catalytic domain with endonuclease activity (Kim and Cech, Proc Natl Acad Sci US A. 1987 Dec; 84(24): 8788-92; Forster and Symons, Cell. 1987 Apr 24; 49(2): 211-20). For example, many ribozymes accelerate phosphate transfer reactions with high specificity, often cleaving only one of several phosphate esters in an oligonucleotide substrate (Cech et al., Cell. 1981 Dec; 27(3 Pt 2): 487-96; Michel and Westhof, J Mol Biol. 1990 Dec 5; 216(3): 585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14; 357(6374): 173-6). This specificity stems from the requirement that the substrate binds to the internal guide sequence ("IGS") of the ribozyme via a specific base-pairing interaction before the chemical reaction.

[0436] At least six basic variants of naturally occurring enzymatic RNAs are currently known. Each can catalyze the hydrolysis of trans RNA phosphodiester bonds under physiological conditions (and thus cleave other RNA molecules). Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs via a target-binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA via complementary base pairing, and once bound to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA disrupts its ability to direct the synthesis of the encoded protein. After the enzymatic nucleic acid has bound to and cleaved its RNA target, it can be released from that RNA to search for another target and can repeatedly bind to and cleave new targets.

[0437] Enzymatic nucleic acid molecules can be formed, for example, in hammerhead, hairpin, δ-type hepatitis virus, group I intron, or RNaseP RNA (associated with an RNA guide sequence) or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described in Rossi et al. Nucleic Acids Res. 1992 Sep 11;20(17):4559-65. Examples of hairpin motifs are described in Hampel et al. (European Patent Application Publication No. 0360257), Hampel and Tritz, Biochemistry 1989 Jun 13;28(12):4929-33, Hampel et al., Nucleic Acids Res. 1990 Jan 25;18(2):299-304, and U.S. Patent No. 5,631,359. Examples of hepatitis virus motifs are described in Perrotta and Been, Biochemistry. 1992 Dec 1;31(47):11843-52, examples of RNaseP motifs are described in Guerrier-Takada et al, Cell. 1983 Dec;35(3 Pt 2):849-57, Neurospora VS RNA ribozyme motifs are described in Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96, Saville and Collins, Proc Natl Acad Sci US A. 1991 Oct l;88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar 23;32(11):2795-9), and examples of group I introns are described in U.S. Patent No. 4,987,071. Enzyme nucleic acid molecules may have specific substrate-binding sites complementary to one or more target gene DNA or RNA regions, and may have nucleotide sequences within or around these substrate-binding sites that confer RNA cleavage activity to the molecule. Therefore, ribozyme constructs are not limited to the specific motifs mentioned herein.

[0438] Methods for producing ribozymes targeting polynucleotide sequences are known in the art. The ribozymes can be designed and synthesized as described in International Publication No. 93 / 23569 and International Publication No. 94 / 02595, which are specifically incorporated herein by reference, and tested in vitro and in vivo.

[0439] Ribozyme activity can be increased by chemically synthesizing ribozymes that have modifications to alter the length of ribozyme-binding arms or to prevent their degradation by serum ribonucleases (see, for example, International Publication 92 / 07065, International Publication 93 / 15187, International Publication 91 / 03162, European Patent Application Publication 92110298.4, U.S. Patent No. 5,334,711, and U.S. Patent No. 94 / 13688, which describe various chemical modifications that can be made to the sugar portion of enzyme RNA molecules, modifications that enhance their effectiveness in cells, and removal of stem π ​​bases to shorten RNA synthesis time and reduce chemical requirements).

[0440] Immunostimulatory oligonucleotides The therapeutic portion may be an immunostimulatory oligonucleotide. Immunostimulatory oligonucleotides (ISSs; single-stranded or double-stranded) can induce an immune response when administered to mammals or other patients. ISSs include, for example, specific palindromes that result in hairpin secondary structures (see Yamamoto S., et al. (1992) J.Immunol. 148:4072-4076), or CpG motifs, as well as other known ISS features (e.g., multi-G domains, see International Publication No. 96 / 11266).

[0441] The immune response can be either an innate or adaptive immune response. The immune system is divided into the more innate immune system and the acquired and adaptive immune system of vertebrates, the latter of which is further divided into humoral cellular components. The immune response can be mucosal.

[0442] Immunostimulating nucleic acids are considered non-sequence-specific if they do not require the specific binding of target polynucleotides to induce an immune response and the reduction of their expression. Therefore, certain immunostimulating nucleic acids may contain sequences corresponding to naturally occurring gene or mRNA regions, but they can still be considered non-sequence-specific immunostimulating nucleic acids.

[0443] An immunostimulatory nucleic acid or oligonucleotide may contain at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be methylated or not. An immunostimulatory nucleic acid may contain at least one CpG dinucleotide having methylated cytosine. A nucleic acid may contain a single CpG dinucleotide in which the cytosine is methylated. A nucleic acid may contain the sequence 5'TAACGTTGAGGG'CAT 3'. A nucleic acid may contain at least two CpG dinucleotides in which at least one cytosine is methylated. Each cytosine in a CpG dinucleotide present in the sequence can be methylated. A nucleic acid may contain multiple CpG dinucleotides in which at least one of the CpG dinucleotides contains methylated cytosine.

[0444] Further specific nucleic acid sequences of oligonucleotides (ODNs) suitable for use in this composition and method are described in Raney et al, Journal of Pharmacology and Experimental Therapeutics, 298:1185-1192 (2001). The ODNs used in this composition and method may have a phosphodiester ("PO") or phosphorothioate ("PS") skeleton and / or at least one methylated cytosine residue in a CpG motif.

[0445] Decoy oligonucleotides The therapeutic portion may be a decoy oligonucleotide. Since transcription factors recognize their relatively short binding sequences, short oligonucleotides possessing the consensus binding sequence of a particular transcription factor can be used as tools to manipulate gene expression in living cells, even in the absence of surrounding genomic DNA. This strategy involves intracellular delivery of such “decoy oligonucleotides” that are then recognized and bound by the target factor. Once the DNA binding site of the transcription factor is occupied by the decoy, the transcription factor is subsequently unable to bind to the promoter region of the target gene. The decoy can be used as a therapeutic agent for either inhibiting the expression of a gene activated by the transcription factor, or upregulating a gene that is repressed by the binding of the transcription factor. An example of the use of decoy oligonucleotides can be found in Mann et al., J. Clin. Invest, 2000, 106:1071-1075, which is expressly incorporated herein by reference in its entirety.

[0446] Supermir The therapeutic portion may be a supermir. A supermir refers to a single-stranded, double-stranded, or partially double-stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both, or a modified thereof, that has a nucleotide sequence substantially identical to miRNA and antisense against its target. This term includes oligonucleotides that consist of naturally occurring nucleic acid bases, sugars, and covalent nucleoside (skeletal) bonds and contain at least one non-naturally occurring moiety that functions similarly. Such modified or substituted oligonucleotides have desirable properties, such as enhanced cellular uptake, increased affinity for nucleic acid targets, and increased stability in the presence of nucleases. Supermirs may not contain a sense strand. Supermirs may not self-hybridize to a considerable extent. Supermirs may have a secondary structure, but are substantially single-stranded under physiological conditions. A supermir that is substantially single-stranded is single-stranded to such an extent that less than 50% of the supermir (e.g., less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%) is double-stranded with itself. A supermir may contain hairpin segments (e.g., sequences) that can self-hybridize, for example, at the "3" terminus to form a double-stranded region (e.g., a double-stranded region consisting of at least about 1, about 2, about 3, or about 4 nucleotides, or less than about 8, about 7, less than 6, or less than 5 nucleotides, or about 5 nucleotides). The double-stranded region may be linked by linkers, for example, nucleotide linkers, for example, about 3, about 4, about 5, or about 6 dTs, for example, modified dTs. Supermir can be doubled with shorter oligonucleotides, for example, about 5, 6, 7, 8, 9, or 10 nucleotides in length, for example, at one or both of the 3' and 5' ends, or at one end of the supermir and the non-terminal or central end.

[0447] miRNA mimetic The therapeutic portion may be a miRNA mime. A miRNA mime represents a class of molecules that can be used to mimic the gene-silencing ability of one or more miRNAs. Thus, the term “microRNA mime” refers to synthetic non-coding RNA (i.e., miRNA not obtained by purification from an endogenous miRNA source) that can enter the RNAi pathway and regulate gene expression. A miRNA mime can be designed as a mature molecule (e.g., single-stranded) or a mimetic precursor (e.g., a previous pre-miRNA). A miRNA mime may include, but is not limited to, nucleic acids (modified or modified nucleic acids) such as RNA, modified RNA, DNA, modified DNA, locked nucleic acid, or 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), or oligonucleotides including any combination of the above (including DNA-RNA hybrids). In addition, a miRNA mime may include conjugates that may affect delivery, intracellular compartmentalization, stability, specificity, functionality, strand use, and / or potency. In one design, the miRNA mime is a double-stranded molecule (e.g., having a double-stranded region of about 16 to about 31 nucleotides in length) and contains one or more sequences that are identical to the mature strand of a given miRNA. Modifications may include 2' modifications (including 2'-0 methyl and 2'F modifications) on one or both strands of the molecule, as well as internucleotide modifications (e.g., phosphorothioate modifications) that enhance nucleic acid stability and / or specificity. In addition, the miRNA mime may contain overhangs. Overhangs may contain about 1 to about 6 nucleotides at either the "3" or 5' end of either strand and may be modified to enhance stability or functionality. The miRNA mime may contain a double-stranded region consisting of about 16 to about 31 nucleotides and may contain one or more of the following chemical modification patterns. In other words, the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide), as well as all of C and U, while the antisense strand modification may include 2'F modifications of all of C and U, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide bonds associated with the 2-nucleotide 3' overhang.

[0448] miRNA inhibitors The therapeutic component may be a miRNA inhibitor. The terms “anti-mir,” “microRNA inhibitor,” “miR inhibitor,” or “miRNA inhibitor” are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the ability of a particular miRNA. Generally, inhibitors are natural nucleic acids or modified nucleic acids, such as oligonucleotides including RNA, modified RNA, DNA, modified DNA, locked nucleic acid (LNA), or any combination of the above.

[0449] Modifications include 2' modifications (including 2'-0 alkyl and 2'F modifications) and internucleotide modifications (e.g., phosphorothioate modifications) that may affect delivery, stability, specificity, intracellular compartmentalization, or efficacy. In addition, miRNA inhibitors may contain conjugates that may affect delivery, intracellular compartmentalization, stability, and / or efficacy. Inhibitors can employ various configurations, such as single-stranded, double-stranded (RNA / RNA or RNA / DNA double-stranded), and hairpin designs. Generally, microRNA inhibitors contain one or more sequences or sequences that are complementary or partially complementary to the (one or more) mature strands of the targeted miRNA. Furthermore, miRNA inhibitors may also contain additional sequences located at 5' and 3' relative to the sequence that is the reverse complement of the mature miRNA. The additional sequence may be the reverse complement of a sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA originates, or the additional sequence may be any sequence (having a mixture of A, G, C, or U). One or both of the additional sequences may be any sequences capable of forming a hairpin. Sequences that are the reverse complement of the miRNA may be adjacent to the hairpin structure at the 5' and 3' ends. MicroRNA inhibitors, in the case of double-stranded sequences, may contain mismatches between nucleotides on opposite strands. Furthermore, microRNA inhibitors may be ligated to a conjugate portion to facilitate the uptake of the inhibitor into cells. For example, a microRNA inhibitor may be ligated to cholesteryl 5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypentylcarbamate), which enables passive uptake of the microRNA inhibitor into cells. MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13:723-730 (2007), and in International Publications 2007 / 095387 and 2008 / 036825, respectively, which are incorporated herein by reference in their entirety.Those skilled in the art can select sequences from a database for a desired miRNA and design inhibitors useful for the methods disclosed herein.

[0450] Antisense compounds (AC) The therapeutic portion comprises an antisense compound (AC) capable of altering one or more aspects of the translation or expression of a target gene. The principle behind antisense technology is that an antisense compound hybridizing to a target nucleic acid modulates gene expression activity, such as translation, through one of several antisense mechanisms. Antisense technology is an effective means of altering the expression of one or more specific gene products and therefore may prove useful in several therapeutic, diagnostic, and research applications.

[0451] The compounds described herein may contain one or more chiral centers, thereby resulting in enantiomers, diastereomers, and other stereoisomers that can be defined with respect to absolute stereochemistry as (R) or (S), α or β, or D or L. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0452] Antisense compound hybridization site The antisense mechanism relies on the hybridization of the antisense compound to the target nucleic acid.

[0453] AC can hybridize with sequences of approximately 5 to approximately 50 nucleic acid lengths, which can also be considered the length of AC. AC may have nucleic acid lengths of approximately 5 to approximately 10, approximately 10 to approximately 15, approximately 15 to approximately 20, approximately 20 to approximately 25, approximately 25 to approximately 30, approximately 30 to approximately 35, approximately 35 to approximately 40, approximately 40 to approximately 45, or approximately 45 to approximately 50. AC may have a nucleic acid length of approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. AC may have a nucleic acid length of approximately 10. AC may have a nucleic acid length of approximately 15. AC may have a nucleic acid length of approximately 20. AC may have a nucleic acid length of approximately 25. AC may have a nucleic acid length of approximately 30.

[0454] AC may have less than approximately 100 percent complementarity to the target nucleic acid sequence. As used herein, the term “percentage of complementarity” refers to the number of nucleic acid bases of AC that have nucleic acid base complementarity with the corresponding nucleic acid bases of the oligomer compound or nucleic acid, divided by the total length of AC (number of nucleic acid bases). Those skilled in the art will understand that the inclusion of mismatches is possible without precluding the activity of the antisense compound. AC may contain up to approximately 20% of nucleotides that inhibit base pairing of AC with the target nucleic acid. AC may contain or contain approximately 15% or less, approximately 10% or less, or 5% or less of mismatches. AC may have at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100% complementarity to the target nucleic acid. The percentage of complementarity 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 of an oligonucleotide region is calculated by dividing the number of complementary nucleic acid bases in the region by the total number of nucleic acid base regions.

[0455] The incorporation of nucleotide affinity modifications can allow for a greater number of mismatches compared to unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences. Those skilled in the art can determine an appropriate number of mismatches between oligonucleotides, or between an oligonucleotide and a target nucleic acid, for example, by determining the melting temperature (Tm). Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, the technique described by Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allows those skilled in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA double strands.

[0456] Antisense mechanism The ACs described herein can regulate one or more aspects of protein transcription, translation, and expression.

[0457] ACs can regulate transcription, translation, or protein expression through steric blocking. The following review, namely Roberts et al. Nature Reviews Drug Discovery (2020) 19:673-694, describes the mechanisms and applications of steric blocking and is incorporated herein by reference in their entirety.

[0458] The antisense mechanism functions through hybridization between an antisense compound and a target nucleic acid. An AC can hybridize to its target sequence and downregulate the expression of the target protein. An AC can hybridize to its target sequence and downregulate the expression of one or more target protein isomers. An AC can hybridize to its target sequence and upregulate the expression of the target protein. An AC can hybridize to its target sequence and increase the expression of one or more target protein isomers.

[0459] The effectiveness of antisense compounds (ACs) can be assessed by evaluating the antisense activity induced by their administration. As used herein, the term “antisense activity” refers to any detectable and / or measurable activity resulting from the hybridization of an antisense compound to its target nucleic acid. Such detection and / or measurement may be direct or indirect. In embodiments, antisense activity is assessed by detecting and / or measuring the amount of the target protein. Antisense activity can also be assessed by detecting and / or measuring the amount of the target nucleic acid.

[0460] Design of antisense compounds The design of ACs according to this disclosure is dependent on the target sequence. Targeting an AC to a specific target nucleic acid molecule can be a multi-step process. This process typically begins with the identification of the target nucleic acid whose expression is regulated. As used herein, the terms “target nucleic acid” and “nucleic acid encoding a target gene” also encompass the DNA encoding the selected target gene, the RNA transcribed from such DNA (such as pre-mRNA and mRNA), and the cDNA derived from such RNA.

[0461] Those skilled in the art can design, synthesize, and screen antisense compounds for different nucleic acid sequences to identify sequences that produce antisense activity. For example, antisense compounds that inhibit the expression of a target protein can be designed. Methods for designing, synthesizing, and screening antisense compounds for 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 in whole by reference for any purpose.

[0462] Antisense compounds containing approximately 8 to 30 linked nucleosides are provided. The antisense compounds may include modified nucleosides, inter-modified nucleoside bonds, and / or conjugate groups.

[0463] Antisense compounds may also be "tricyclo-DNA (tc-DNA)," which refers to a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring, limiting the conformational flexibility of the backbone and enhancing the skeletal shape with a torsion angle γ. Homobasic adenine and thymine-containing tc-DNA form highly stable AT base pairs with complementary RNA.

[0464] Nucleoside Antisense compounds may contain linked nucleosides. Some or all of the nucleosides may be modified nucleosides. One or more nucleosides may contain modified nucleic acid bases. One or more nucleosides may contain modified sugars. Chemically modified nucleosides are routinely incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for target RNA. Non-limiting examples of nucleosides are provided in Khvorova et al. Nature Biotechnology (2017) 35:238-248, which is incorporated herein by reference in its entirety.

[0465] 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 mimics known to those skilled in the art are suitable for the compounds described herein. The terms modified nucleic acid base and nucleic acid base mimic may overlap, but generally, a modified nucleic acid base refers to a nucleic acid base whose structure is quite similar to a parent nucleic acid base, such as 7-deazapurine, 5-methylcytosine, or G-clamp, while a nucleic acid base mimic includes more complex structures, such as tricyclic phenoxazine nucleic acid base mimics. Methods for preparing the above modified nucleic acid bases are well known to those skilled in the art.

[0466] AC may comprise one or more nucleosides having a modified sugar moiety. The furanosyl sugar ring of natural nucleosides can be modified in several ways, but are not limited to, by the addition of substituents, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA), and substitution of atoms or groups such as -S-, -N(R)- or -C(R1)(R2) of the 4'-position ring oxygen. Modified sugar moieties are well known and can be used to alter (typically increase) the affinity of an antisense compound for its target and / or to increase nuclease resistance. A representative list of modified sugars includes, but is not limited to, non-bicyclic substituted sugars, particularly non-bicyclic 2'-substituted sugars having 2'-F, 2'-OCH3 or 2'-O(CH2)2-OCH3 substituents, and 4'-thio-modified sugars. Sugars can also be substituted with sugar mimetic groups, among other things. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications teaching the preparation of such modified sugars include U.S. Patents No. 4,981,957, No. 5,118,800, No. 5,319,080, No. 5,359,044, No. 5,393,878, No. 5,446,137, No. 5,466,786, No. 5,514,785, No. 5,519,134, No. 5,567,811, No. 5,576,427, and others. Examples include, but are not limited to, publications No. 5,591,722, No. 5,597,909, No. 5,610,300, No. 5,627,053, No. 5,639,873, No. 5,646,265, No. 5,658,873, No. 5,670,633, No. 5,792,747, No. 5,700,920, and No. 6,600,032, as well as International Publication No. 2005 / 121371.

[0467] Nucleosides may include 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). Specific such BNAs have been prepared and disclosed in patent and scientific literature (e.g., Srivastava, et al. J.Am.Chem.Soc.2007, ACS Advanced online). publication,10.1021 / ja071106y,Albaek et al.J.Org.Chem.,2006,71,7731-7740,Fluiter,et al.Chembiochem 2005,6,1104-1109,Singh et al.,Chem.Commun.,1998,4,455-456,Koshkin et al. al., Tetrahedron,1998,54,3607-3630, Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638, Kumar et al. See al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222, International Publication No. 94 / 14226, International Publication No. 2005 / 021570, Singh et al., J. Org. Chem., 1998, 63, 10035-10039, International Publication No. 2007 / 090071). Examples of issued U.S. patents and published applications disclosing BNA include, for example, U.S. Patents No. 7,053,207, No. 6,268,490, No. 6,770,748, No. 6,794,4 Examples include Patent No. 99, No. 7,034,133, and No. 6,525,191, as well as U.S. Published Patent Publications 2004-0171570, 2004-0219565, 2004-0014959, 2003-0207841, 2004-0143114, and 20030082807.

[0468] Locked nucleic acids (LNAs) are also provided herein, in which the 2'-hydroxyl group of a ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage and creating a bicyclic sugar moiety (referred to in Eayadi et al. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Patents 6,268,490 and 6,670,461). The bond can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom. The term LNA is used for the bicyclic portion, and the term ENA (trademark) is used for the ethylene group at this position (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 very high double-strand thermal stability (Tm = +3 to +10°C) against complementary DNA and RNA, stability against 3'-exonuclease degradation, and good solubility. Strong, non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al. Natl.Acad.Sci.USA, 2000, 97, 5633-5638).

[0469] A similarly studied isomer of LNA is alpha-L-LNA, which has been shown to have excellent 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).

[0470] 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 International Publication Nos. 98 / 39352 and 99 / 14226.

[0471] LNA analogs, phosphorothioate-LNA and 2'-thio-LNA, have also been prepared (Kumar et al. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide doubles as substrates for nucleic acid polymerases has also been described (Wengel et al., International Publication No. 99 / 14226). Furthermore, the synthesis of 2'-amino-LNA, a novel structurally restricted high-affinity oligonucleotide analog, has been described in the art (Singh et al. Chem., 1998, 63, 10035-10039). In addition, 2'-amino and 2'-methylamino-LNA have been prepared, and the thermal stability of their double helix with complementary RNA and DNA strands has been previously reported.

[0472] Nucleoside internucleoside bonding This specification describes internucleoside linking groups that link nucleosides or otherwise modified monomer units together to form antisense compounds. Two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside links include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Representative non-phosphorus-containing internucleoside links 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 compounds containing non-phosphorus nucleoside bonds are called oligonucleosides. Modified nucleoside bonds can be used to alter (typically increase) the nuclease resistance of antisense compounds compared to natural phosphodiester bonds. Nucleoside bonds containing chiral atoms can be prepared as racemic, chiral, or mixtures. Typical chiral nucleoside bonds include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known to those skilled in the art.

[0473] Phosphate groups can be linked to the 2', 3', or 5' hydroxyl portions of sugars. In oligonucleotide formation, phosphate groups covalently bond adjacent nucleosides to each other, forming linear polymer compounds. Within oligonucleotides, phosphate groups are generally considered to form the internucleoside skeleton of the oligonucleotide. The usual bond or skeleton of RNA and DNA is the 3'-5' phosphodiester bond.

[0474] conjugated group Cargo can be modified by covalent bonding of one or more conjugate groups. Generally, conjugate groups modify one or more properties of the cargo, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in the chemical field and are linked to the parent compound directly or via an optional linkage or linkage group. Examples of 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. The conjugated group may include polyethylene glycol (PEG). PEG can be conjugated to either the cargo or cCPP. The cargo may include peptides, oligonucleotides, or small molecules.

[0475] The conjugate group can be a lipid moiety, e.g., cholesterol moiety (Letsinger et al. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, 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), or aliphatic chain, e.g., dodecanediol or undecyl residue (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 It may contain the palmityl moiety (Lett., 1995, 36, 3651), the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or the octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0476] Linking groups or difunctional linking moieties, such as those known in the art, are suitable for the compounds provided herein. Linking groups are useful for attaching chemical functional groups, conjugate groups, reporter groups, and other groups to selective sites in a parent compound, such as AC. Generally, a difunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One functional group is selected to attach to the parent molecule or compound of interest, and the other is selected to attach to essentially any selected group, such as a chemical functional group or a conjugated group. Any of the linkers described herein may be used. Linkers may include chain structures or oligomers of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups conventionally used in difunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophiles. Difunctional linking moieties may include amino, hydroxyl, carboxylic acid, thiol, and unsaturated (e.g., double or triple bonds) groups. 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, though not limited to these, include substituted C1-C10 alkyl groups and substituted or unsubstituted C2-C10 groups. 10 Alkenyl or substituted or unsubstituted C2-C 10 Alkynnyls are examples, and an unrestricted list of substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0477] AC can be approximately 5 to approximately 50 nucleotides long. AC can be approximately 5 to approximately 10 nucleotides long. AC can be approximately 10 to approximately 15 nucleotides long. AC can be approximately 15 to approximately 20 nucleotides long. AC can be approximately 20 to approximately 25 nucleotides long. AC can be approximately 25 to approximately 30 nucleotides long. AC can be approximately 30 to approximately 35 nucleotides long. AC can be approximately 35 to approximately 40 nucleotides long. AC may be approximately 40 to approximately 45 nucleotides long. AC can be approximately 45 to approximately 50 nucleotides long.

[0478] Clustered and regularly arranged short palindromic sequence repeats (CRISPR) gene editing mechanism This compound may comprise one or more cCPPs (or cCPPs) conjugated to a CRISPR gene editing mechanism. As used herein, “CRISPR gene editing mechanism” refers to a protein, nucleic acid, or combination thereof that can be used to edit a genome. Non-limiting examples of gene editing mechanisms include gRNAs, nucleases, nuclease inhibitors, and combinations and complexes thereof. Each of the aforementioned patent documents describing the CRISPR gene editing mechanism, namely U.S. Patent Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233, 8,999,641, U.S. Patent Application No. 14 / 704,551, and U.S. Patent Application No. 13 / 842,859, is incorporated herein by reference in its entirety.

[0479] The linker can conjugate cCPP to a CRISPR gene editing mechanism. Any linker described in this disclosure or known to those skilled in the art may be used.

[0480] SHRNA This compound may contain cCPP conjugated to gRNA. The gRNA targets genomic loci in prokaryotic or eukaryotic cells.

[0481] A gRNA may be a single-molecule guide RNA (sgRNA). An sgRNA includes a spacer sequence and a scaffold sequence. The spacer sequence is a short nucleic acid sequence used to target a nuclease (e.g., Cas9 nuclease) to a specific nucleotide region of interest (e.g., the genomic DNA sequence to be cleaved). The spacer may be about 17–24 base pairs long, for example, about 20 base pairs long. The spacer may be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 base pairs long. The spacer may be at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 base pairs long. The spacer may be approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs long. The spacer sequence may have a GC content of approximately 40% to 80%.

[0482] The spacer can target the site immediately preceding the 5' protospacer adjacent motif (PAM). The PAM sequence can be selected based on the desired nuclease. For example, the PAM sequence may be one of the PAM sequences shown in the table below, where N represents any nucleic acid, R represents A or G, Y represents C or T, W represents A or T, and V represents A, C, or G.

[0483] [Table 14]

[0484] Spacers can target sequences of mammalian genes, such as human genes. Spacers can target mutant genes. Spacers can target coding sequences.

[0485] The scaffold sequence is a sequence within sgRNA involved in nuclease (e.g., Cas9) binding. The scaffold sequence does not contain spacer / targeting sequences. In embodiments, the scaffold may be about 1 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides long. The scaffolding is approximately 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 , approximately 36, approximately 37, approximately 38, approximately 39, approximately 40, approximately 41, approximately 42, approximately 43, approximately 44, approximately 45, approximately 46, approximately 47, approximately 48, approximately 49, approximately 50, approximately 51, approximately 52, approximately 53, approximately 54, approximately 55, approximately 56, approximately 57, approximately 58, approximately 59, approximately 60, approximately 60, approximately 61, approximately 62, approximately 63, approximately 64, approximately 65, approximately 66, approximately 67, approximately 6 8, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99, approximately 100, approximately 101 The scaffold may be approximately 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or approximately 125 nucleotides long. The scaffold may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, or at least 125 nucleotides long.

[0486] gRNA can be a bimolecular guide RNA, such as crRNA and tracrRNA. gRNA may further contain a poly(A) tail.

[0487] The compound contains cCPP conjugated to a nucleic acid containing gRNA. The nucleic acid may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 gRNAs. The gRNAs may recognize the same target. The gRNAs may recognize different targets. The nucleic acid containing gRNAs contains a sequence encoding a promoter, which drives the expression of the gRNAs.

[0488] Nuclease This compound may contain a cyclic cell-permeable peptide (cCPP) conjugated to a nuclease. The nuclease may be a type II, type VA, type VB, type VC, type VU, or type VI-B nuclease. The nuclease may be a transcription, activator-like effector nuclease (TALEN), meganuclease, or zinc finger nuclease. The nuclease may be a Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. The nuclease may be a Cas9 nuclease or a Cpf1 nuclease.

[0489] Nucleases may be modified forms or variants of Cas9, Cas12a(Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a(C2c2), Cas13b, or Cas14 nucleases. Nucleases may also be modified forms or variants of TAL nucleases, meganucleases, or zinc finger nucleases. "Modified" or "mutant" nucleases may be, for example, cleaved, fused to another protein (such as another nuclease), or catalytically inactivated. The nuclease may have at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or at least approximately 100% sequence identity with naturally occurring Cas9, Cas12a(Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a(C2c2), Cas13b, Cas14 nucleases, or TALEN, meganuclease, or zinc finger nuclease. The nuclease may be a Cas9 nuclease derived from S. pyogenes (SpCas9). The nuclease may have at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with a Cas9 nuclease derived from S. pyogenes (SpCas9). The nuclease may be Cas9 (SaCas9) derived from Staphylococcus aureus. The nuclease may have at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with Cas9 (SaCas9) derived from Staphylococcus aureus. Cpf1 may be the Cpf1 enzyme derived from Tillandsia pallidum (species BV3L6, UniProt accession number U2UMQ6). The nuclease may have at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the Cpf1 enzyme derived from Tillandsia pallidum (species BV3L6, UniProt accession number U2UMQ6).

[0490] Cpf1 may be a Cpf1 enzyme derived from the family Lachnospiraceae (species ND2006, UniProt accession number A0A182DWE3). The nuclease may have at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with the Cpf1 enzyme derived from Lachnospiraceae. The sequence encoding the nuclease can be codon-optimized for expression in mammalian cells. The sequence encoding the nuclease can be codon-optimized for expression in human or mouse cells.

[0491] This compound may contain cCPP conjugated to a nuclease. The nuclease may be a soluble protein.

[0492] This compound may contain cCPP conjugated to a nucleic acid encoding a nuclease. The nucleic acid encoding the nuclease may contain a sequence encoding a promoter, which drives the expression of the nuclease.

[0493] gRNA and nuclease combination The compound may contain one or more cCPPs conjugated to gRNA and / or nucleases. One or more cCPPs may be conjugated to nucleic acids encoding gRNA and / or nucleases. The nucleic acids encoding nuclease and gRNA may contain sequences encoding promoters, which drive the expression of nuclease and gRNA. The nucleic acids encoding nuclease and gRNA may contain two promoters, the first promoter controlling nuclease expression and the second promoter controlling gRNA expression. The nucleic acids encoding gRNA and nucleases may encode about 1 to about 20 gRNAs, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19 gRNAs up to about 20 gRNAs. gRNAs may recognize different targets. gRNAs may recognize the same target.

[0494] This compound may contain a circular cell-permeable peptide (or cCPP) conjugated to a ribonucleoprotein (RNP) containing gRNA and a nuclease.

[0495] A composition comprising (a) cCPP conjugated to gRNA and (b) a nuclease can be delivered to cells.

[0496] A composition comprising (a) a first cCPP conjugated to gRNA and (b) a second cCPP conjugated to a nuclease can be delivered to cells. The first and second cCPPs may be the same. The first and second cCPPs may be different.

[0497] Target gene element This compound may contain a circular cell-permeable peptide (cCPP) conjugated to a target gene element. The target gene element can replace a genomic DNA sequence cleaved by a nuclease. Non-limiting examples of the target gene element include genes, single nucleotide polymorphisms, promoters, or terminators.

[0498] Nuclease inhibitors The compound may include a cyclic cell-permeable peptide (cCPP) conjugated to a nuclease inhibitor (e.g., a Cas9 inhibitor). A limitation of gene editing is potential off-target editing. Delivery of nuclease inhibitors can limit off-target editing. Nuclease inhibitors may be polypeptides, polynucleotides, or small molecules. Nuclease inhibitors are described in U.S. Patent Publication No. 2020 / 087354, International Publication No. 2018 / 085288, U.S. Patent Publication No. 2018 / 0382741, International Publication No. 2019 / 089761, International Publication No. 2020 / 068304, International Publication No. 2020 / 041384, and International Publication No. 2019 / 076651, the whole of which is incorporated herein by reference.

[0499] Therapeutic polypeptides antibody The therapeutic portion may include an antibody or an antigen-binding fragment. The antibody and antigen-binding fragment may be derived from any suitable source, such as humans, mice, camelids (e.g., camels, alpacas, llamas), rats, ungulates, or non-human primates (e.g., monkeys, rhesus monkeys).

[0500] The term “antibody” refers to an immunoglobulin (Ig) molecule that can bind to a specific target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, this term encompasses intact polyclonal or monoclonal antibodies and their antigen-binding fragments. Natural immunoglobulin molecules generally consist of two heavy-chain polypeptides and two light-chain polypeptides. Each heavy-chain polypeptide associates with a light-chain polypeptide by an interchain disulfide bond between the heavy-chain polypeptide and the light-chain polypeptide to form two heterodimeric proteins or polypeptides (i.e., proteins consisting of two heterogeneous polypeptide chains). The two heterodimeric proteins then associate by further interchain disulfide bonds between the heavy-chain polypeptides to form an immunoglobulin protein or polypeptide.

[0501] As used herein, the term “antigen-binding fragment” refers to a polypeptide fragment containing at least one complementarity-determining region (CDR) of an immunoglobulin heavy chain and / or light chain that binds to at least one epitope of the antigen of interest. An antigen-binding fragment may contain one, two, or three CDRs of antibody-derived variable heavy chain (VH) sequences that specifically bind to the target molecule. An antigen-binding fragment may contain one, two, or three CDRs of antibody-derived variable light chain (VL) sequences that specifically bind to the target molecule. An antigen-binding fragment may contain all six CDRs of antibody-derived variable heavy chain (VH) and variable light chain (VL) sequences that specifically bind to the target molecule. Antigen-binding fragments include proteins that contain a portion of a full-length antibody, generally its antigen-binding region or variable region, such as Fab, F(ab')2, Fab', Fv fragments, mini-bodies, diabodies, single-domain antibodies (dAb), single-chain variable fragments (scFv), nanobodies, multispecific antibodies formed from antibody fragments, and any other modified configuration of an immunoglobulin molecule that may contain an antigen-binding site or a fragment of the desired specificity.

[0502] The term "F(ab)" refers to two protein fragments resulting from the proteolytic cleavage of an IgG molecule by the enzyme papain. Each F(ab) may contain a covalent heterodimer consisting of a VH chain and a VL chain, and may contain an intact antigen-binding site. Each F(ab) may be a monovalent antigen-binding fragment. The term "Fab'" refers to a fragment derived from F(ab')2, and may contain a small portion of Fc. Each Fab' fragment may be a monovalent antigen-binding fragment.

[0503] The term "F(ab')2" refers to a protein fragment of IgG produced by proteolytic cleavage by the enzyme pepsin. Each F(ab')2 fragment may contain two F(ab') fragments and is therefore a bivalent antigen-binding fragment.

[0504] "Fv fragment" refers to a non-covalent VH:VL heterodimer that retains much of the antigen recognition and binding ability of a natural antibody molecule but lacks the CH1 and CL domains contained within the Fab and contains an antigen-binding site. (Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662, Hochman et al. (1976) Biochem 15:2706-2710, and Ehrlich et al. (1980) Biochem 19:4091-4096.)

[0505] A bispecific antibody (BsAb) is an antibody that can simultaneously bind to two distinct, unique antigens (or different epitopes of the same antigen). The therapeutic portion may contain a bispecific antibody that can simultaneously bind to two different targets of interest. BsAbs can redirect cytotoxic immune effector cells to enhance tumor cell death by antibody-dependent cell-mediated cytotoxicity (ADCC) and other cytotoxic mechanisms mediated by effector cells.

[0506] Recombinant antibody manipulation has made it possible to produce recombinant bispecific antibody fragments containing the variable heavy chain (VH) domain and variable light chain (VL) domain of the parent monoclonal antibody (mab). Non-limiting examples include scFv (single-stranded variable fragment), BsDb (bispecific diabody), scBsDb (single-stranded bispecific diabody), scBsTaFv (single-stranded bispecific tandem variable domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (single-domain antibody), and BssdAb (bispecific single-domain antibody).

[0507] BsAbs with an Fc region are useful for performing Fc-mediated effector functions such as ADCC and CDC. They have the half-life of normal IgG. On the other hand, BsAbs without an Fc region (bispecific fragments) rely solely on their antigen-binding ability to achieve therapeutic activity. Due to their smaller size, these fragments have better solid tumor permeability. BsAb fragments do not require glycosylation and can be produced in bacterial cells. Size, valency, flexibility, and half-life of BsAbs suitable for application.

[0508] Using recombinant DNA technology, bispecific IgG antibodies can be constructed from two different heavy and light chains expressed in the same cell line. Random assembly of the different chains leads to the formation of non-functional molecules and undesirable HC homodimers. To address this problem, a second binding site (e.g., a single-chain variable fragment) can be fused to the N-terminus or C-terminus of the H or L chain to obtain a tetravalent BsAb containing two binding sites for each antigen. Further methods to address LC-HC mispairing and HC homodimerization are described below.

[0509] Knob-into-hole BsAb IgG H chain heterodimerization is forced by introducing different mutations into two CH3 domains, resulting in an asymmetric antibody. Specifically, a "knob" mutation is introduced into one HC and a "hole" mutation into the other HC to promote heterodimerization.

[0510] Direct addition of a novel antigen-binding moiety to a full-length IgG Ig-scFv fusion results in a tetravalent fusion protein. Examples include IgG C-terminal scFv fusions and IgG N-terminal scFv fusions.

[0511] Diabody-Fc fusion: This involves replacing the Fab fragment of IgG with a bispecific diabody (a derivative of scFv).

[0512] Dual variable domain-IgG (DVD-IgG) was formed by fusing the VL and VH domains of IgG with one specificity to the N-terminuses of the VL and VH domains of IgG with different specificities, respectively, via linker sequences.

[0513] The term "diabody" refers to a bispecific antibody in which the VH and VL domains are expressed in a single polypeptide chain using a linker that is too short to allow pairing between two domains on the same chain, thereby pairing the domains with complementary domains on another chain and creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121-23 (1994)). A diabody can be designed to bind to two different antigens and is a bispecific antigen-binding construct.

[0514] The terms "nanobodies" or "single-domain antibodies" refer to antigen-binding fragments of a single monomer variable antibody domain containing one variable domain (VH) of a heavy-chain antibody. They offer several advantages over traditional monoclonal antibodies (mAbs), including smaller size (15 kD), stability in the reducing intracellular environment, and ease of production in bacterial systems (Schumacher et al., (2018) Nanobodies: Chemical Functionalization Strategies and Intracellular Applications. Angew. Chem. Int. Ed. 57, 2314; Siontorou, (2013) Nanobodies as novel agents for disease diagnosis and therapy. International Journal of Nanomedicine, 8, 4215-27). These features make nanobodies modifiable for genetic and chemical modifications (Schumacher et al., (2018) Nanobodies: Chemical Functionalization Strategies and Intracellular Applications. Angew. Chem. Int. Ed. 57, 2314), facilitating their application as research tools and therapeutic agents (Bannas et al., (2017) Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Frontiers in Immunology, 8, 1603).Over the past decade, nanobodies have been used for protein immobilization (Rothbauer et al., (2008) A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins. Mol.Cell.Proteomics, 7, 282-289), imaging (Traenkle et al., (2015) Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells. Mol.Cell.Proteomics, 14, 707-723), and detection of protein-protein interactions (Herce et al., (2013) Visualization and targeted disruption of protein interactions in living cells. Nat.Commun., 4, 2660; Massa et al., (2014) Site-Specific Labeling of Cysteine-Tagged Camelid Single-Domain Antibody-Fragments for Use in Molecular Imaging. Bioconjugate) It has been used for purposes such as Chem, 25, 979-988, and as a polymer inhibitor (Truttmann et al., (2015) HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation. J. Biol. Chem. 290, 9087-9100).

[0515] The therapeutic portion may be an antigen-binding fragment that binds to the target of interest. The antigen-binding fragment that binds to the target of interest may contain one, two, or three CDRs of antibody-derived variable heavy chain (VH) sequences that specifically bind to the target of interest. The antigen-binding fragment that binds to the target of interest may contain one, two, or three CDRs of antibody-derived variable light chain (VL) sequences that specifically bind to the target of interest. The antigen-binding fragment that binds to the target of interest may contain all one, two, three, four, five, or six CDRs of antibody-derived variable heavy chain (VH) and / or variable light chain (VL) sequences that specifically bind to the target of interest. The antigen-binding fragment that binds to the target may be a portion of a full-length antibody, such as a multispecific antibody formed from a Fab, F(ab')2, Fab', Fv fragment, minibody, diabody, single-domain antibody (dAb), single-chain variable fragment (scFv), nanobody, antibody fragment, or any other modified structure of an immunoglobulin molecule containing an antigen-binding site or fragment of the desired specificity.

[0516] The therapeutic portion may contain bispecific antibodies. A bispecific antibody (BsAb) is an antibody that can simultaneously bind to two distinct, unique antigens (or different epitopes of the same antigen).

[0517] The treatment area may include the "diabody."

[0518] The therapeutic portion may include nanobodies or single-domain antibodies (which may be referred to herein as sdAb or VHH).

[0519] The therapeutic portion may include a "mini-body." A mini-body (Mb) contains a CH3 domain fused to or ligated to an antigen-binding fragment (e.g., a CH3 domain fused to or ligated to an scFv, domain antibody, etc.). The term "Mb" may refer to a single CH3 domain. A CH3 domain may refer to a mini-body. (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, E. et al., Nature 341, 544-546 (1989), Bird et al., Science, 242, 423-426, 1988, Huston et al., PNAS USA, 85, 5879-5883, 1988), International application PCT / US92 / 09965, International publication 94 / 13804, P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993, Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996, S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0520] The therapeutic portion may include a "monobody." The term "monobody" refers to a synthetic binding protein constructed using a fibronectin type III domain (FN3) as a molecular scaffold.

[0521] The therapeutic component may be an antibody mimetic. Antibody mimetics are compounds that, like antibodies, can specifically bind to antigens but are structurally unrelated to antibodies. They are typically artificial peptides or proteins with a molar mass of approximately 3 to 20 kDa (compared to the molar mass of an antibody of approximately 150 kDa). Examples of antibody mimetic molecules include affibody molecules (constructed on a scaffold of protein A domains, see Nygren (June 2008). FEBS J. 275(11): 2668-76), affilins (constructed on a scaffold of gamma-B crystalline or ubiquitin, see Ebersbach H et al. (September 2007). J. Mol. Biol. 372(1): 172-85), affimers (constructed on a crystallin scaffold, see Johnson A et al., (Aug 7, 2012). Anal. Chem. 84(15): 6553-60), and afitins (constructed on Sac7d derived from the S. acidocardarius scaffold, see Krehenbrink M et al., (November See J.Mol.Biol.383(5):1058-68 (2008), alpha body (constructed on a triple helix coiled-coil scaffold, see Desmet, J et al., (5 Feb 2014). Nature Communications.5:5237), antikarin (constructed on a lipokarin scaffold, see Skerra A (June 2008). FEBS J.275(11):2677-83), avimer (constructed on scaffolds of various membrane receptors, see Silverman J et al. (December 2005). Nat. Biotechnol.23(12):1556-61), DARPin (constructed on an ankyrin repeat motif scaffold, see Stumpp et al., (August 2008). Drug See Discov.Today.13(15-16):695-701), finomer (constructed on a scaffold of Fyn's SH3 domain, Grabulovski et al., (2007). J Biol Chem.Examples include Knitz domain peptides (see 282(5):3196-3204), Knitz domain peptides (constructed on the scaffold of Knitz domains of various protease inhibitors; see Nixon et al (March 2006). Curr Opin Drug Discov Dev. 9(2):261-8), and monobodies (constructed on the scaffold of the type III domain of fibronectin; see Koide et al (2007). Methods Mol. Biol. 352:95-109).

[0522] The therapeutic portion may include "engineered ankyrin repeats" or "DARPin." DARPin is derived from the natural ankyrin protein, which consists of at least three repeat motif proteins, and is typically composed of four or five repeats.

[0523] The therapeutic portion may include "bivariable domain-IgG" or "DVD-IgG." DVD-IgG is generated from two parental monoclonal antibodies by fusing the VL and VH domains of one specific IgG to the N-terminuses of the VL and VH domains of IgG with different specificities via a linker sequence.

[0524] The treated area may contain F(ab) fragments.

[0525] The treated area may contain the F(ab')2 fragment.

[0526] The treated area may contain Fv fragments.

[0527] Antigen-binding fragments may include "single-chain variable fragments" or "scFv." scFv refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, linked by a short linker peptide of 10 to approximately 25 amino acids. (Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883). The linker can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Numerous methods have been described for identifying the chemical structures necessary to convert naturally aggregated but chemically separated light and heavy polypeptide chains derived from the antibody V region into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patents 5,091,513 and 5,132,405 by Huston et al., and U.S. Patent 4,946,778 by Ladner et al.

[0528] Antigen-binding constructs may contain two or more antigen-binding moieties. Antigen-binding constructs can bind to two distinct, unique antigens, or to different epitopes of the same antigen. Knob-into-hole BsAb IgG H chain heterodimerization is forced by introducing different mutations into two CH3 domains, resulting in an asymmetric antibody. Specifically, a "knob" mutation is introduced into one HC and a "hole" mutation into the other HC to promote heterodimerization.

[0529] Peptide inhibitors The therapeutic portion may contain peptides. These peptides can act as agonists, increasing the activity of target proteins. They can also act as antagonists, decreasing the activity of target proteins. These peptides may be configured to inhibit protein-protein interactions (PPIs). Protein-protein interactions (PPIs) are crucial in many biochemical processes, including nucleic acid transcription and various post-translational modifications of translated proteins. PPIs can be experimentally determined by biophysical techniques such as X-ray crystallography, NMR spectroscopy, surface plasma resonance (SPR), biolayer interferometry (BLI), isothermal titration calorimetry (ITC), radioligand binding, spectrophotometric assays, and fluorescence spectroscopy. Peptides that inhibit protein-protein interactions are sometimes called peptide inhibitors.

[0530] The therapeutic portion may include a peptide inhibitor. The peptide inhibitor may contain approximately 5 to 100 amino acids, approximately 5 to 50 amino acids, approximately 15 to 30 amino acids, or approximately 20 to 40 amino acids. The peptide inhibitor may include one or more chemical modifications, for example, to reduce proteolysis and / or to improve the in vivo half-life. The peptide inhibitor may include one or more synthetic amino acids and / or skeletal modifications. The peptide inhibitor may have an α-helical structure.

[0531] Peptide inhibitors can target the dimerization domain of the desired homodimer or heterodimer target protein.

[0532] small molecule The therapeutic portion may contain small molecules. The therapeutic portion may contain small molecule kinase inhibitors. The therapeutic portion may contain small molecules that inhibit kinases that phosphorylate the target of interest. By inhibiting the phosphorylation of the target of interest, nuclear translocation of the target of interest can be blocked. The therapeutic portion may contain small molecule inhibitors of MyD88.

[0533] composition A composition comprising the compounds described herein is provided.

[0534] Provided are pharmaceutically acceptable salts and / or prodrugs of the compounds of the Disclosure. A pharmaceutically acceptable salt includes a salt of the compound of the Disclosure prepared with an acid or base, depending on the specific substituent found on the compound. It may be appropriate to administer the compound as a salt under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable, non-toxic acidic or basic salts. Examples of pharmaceutically acceptable base-added salts include sodium, potassium, calcium, ammonium, or magnesium salts. Examples of physiologically acceptable acid-added salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids such as acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, alpha-ketoglutaric acid, alpha-glycophosphate, maleic acid, tosylic acid, and methanesulfonic acid. Accordingly, disclosed herein are hydrochlorides, nitrates, phosphates, carbonates, bicarbonates, sulfates, acetates, propions, benzoates, succinates, fumarates, mandelates, oxalates, citrates, tartrates, malons, ascorbicates, alpha-ketoglutarates, alpha-glycolinates, maleates, tosylates, and mesylates. Pharmacochemically acceptable salts of these compounds can be obtained using standard procedures well known in the art (for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid that yields a physiologically acceptable anion). Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0535] Mechanism of oligonucleotide therapeutic agents and target molecules Many types of oligonucleotides can regulate gene transcription, translation, and / or protein function in cells. Non-exclusive examples of such oligonucleotides include, for example, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense, antagonist mirs, antimirs, microRNA mimes, supermirs, Ul adapters, and aptamers. Further examples include DNA targeting, triple-stranding oligonucleotides, strand-entry oligonucleotides, and synthetic guide strands for CRISPR / Cas. These nucleic acids act through a variety of mechanisms. See Smith and Zain, Annu Rev Pharmacol Toxicol. 2019, 59:605-630, incorporated herein by reference.

[0536] Splice-switching antisense oligonucleotides are short, synthetic antisense-modified nucleic acids that disrupt the normal splicing repertoire of a transcript by forming base pairs with pre-mRNA and blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing mechanism and pre-mRNA. Splicing of pre-mRNA is necessary for the proper expression of the vast majority of protein-coding genes, and therefore, targeting the process provides a means of manipulating protein production from genes. Splicing regulation is particularly valuable in cases of diseases caused by mutations that result in inhibition of normal splicing, or where disrupting the normal splicing process of a gene transcript may be therapeutic. Such antisense oligonucleotides provide an effective and specific method for targeting and modifying splicing in a therapeutic manner. See Havens and Hastings, Nucleic Acids Res. 2016 Aug 19;44(14):6549-6563, incorporated herein by reference.

[0537] In the case of siRNA or miRNA, these nucleic acids can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi). After introduction of siRNA or miRNA into the cytoplasm, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of the siRNA or miRNA is substituted from the RISC complex, providing a template within RISC that can recognize and bind to mRNA having a sequence complementary to the sequence of the bound siRNA or miRNA. Upon binding to the complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strand. RNAi can enable downregulation of specific proteins by targeting the specific disruption of the corresponding mRNA encoding protein synthesis.

[0538] Because siRNA and miRNA constructs can be synthesized using any nucleotide sequence for the target protein, the therapeutic applications of RNAi are extremely broad. To date, siRNA constructs have demonstrated the ability to specifically downregulate target proteins in both in vitro and in vivo models, as well as in clinical studies.

[0539] Antisense oligonucleotides and ribozymes can also inhibit the translation of mRNA into protein. In the case of antisense constructs, these single-stranded deoxynucleic acids have a sequence complementary to the sequence of the target protein mRNA and can bind to the mRNA by Watson-Crick base pairing. This binding interferes with the translation of the target mRNA and / or causes RNase H degradation of the mRNA transcript, and as a result, antisense oligonucleotides have great potential for specificity of action (i.e., downregulation of specific disease-related proteins). To date, these compounds have been shown to be promising in several in vitro and in vivo models, such as models of inflammatory diseases, cancer, and HIV (seen in Agrawal, Trends in Biotech. 14:376-387 (1996)). Antisense can also influence cellular activity by specifically hybridizing with chromosomal DNA.

[0540] Immunostimulating nucleic acids include deoxyribonucleic acid and ribonucleic acid. In the case of deoxyribonucleic acid, specific sequences or motifs are known to induce immunostimulation in mammals. These sequences or motifs include CpG motifs, pyrimidine-rich sequences, and palindromic sequences. CpG motifs in deoxyribonucleic acid are specifically recognized by the endosomal receptor, Toll-like receptor 9 (TLR-9), which is thought to then induce both innate and acquired immunostimulatory pathways. Specific immunostimulating ribonucleic acid sequences have also been reported. These RNA sequences are thought to induce immune activation by binding to Toll-like receptors 6 and 7 (TLR-6 and TLR-7). Furthermore, double-stranded RNA has also been reported to be immunostimulant and is thought to be activated via binding to TLR-3.

[0541] Tables 9A and 9B show non-limiting examples of mechanisms and targets of antisense oligonucleotides (ASOs) for regulating gene transcription, translation, and / or protein function.

[0542] [Table 15]

[0543] [Table 16]

[0544] Clustered and regularly arranged short palindromic sequence repeats (CRISPR) and associated Cas proteins constitute the CRISPR-Cas system. CRISPR-Cas is a gene editing mechanism. An RNA-guided (e.g., gRNA) Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner. The Cas9 endonuclease can be replaced with any nuclease of this disclosure. The gRNA targets the nuclease (e.g., Cas9 nuclease) to a specific nucleotide region of interest (e.g., the genomic DNA sequence to be cleaved), and cleaves the genomic DNA. The genomic DNA can then be replaced with the desired genetic element.

[0545] Methods for regulating tissue distribution and / or retention This specification provides compounds and methods for modulating the tissue distribution and / or retention of therapeutic agents in a subject. Compounds that modulate the tissue degradation of therapeutic agents may include cyclic cell-permeable peptides (cCCPs) and extracyclic peptides (EPs). Methods for modulating tissue distribution may include administering a compound comprising cyclic cell-permeable peptides (cCPPs) and extracyclic peptides (EPs) to a subject. Modulation of the tissue distribution or retention of a compound can be evaluated by measuring the amount, expression, function, or activity of the compound in vivo in different tissues. The tissues may be different tissues of the same biological system, e.g., different types of muscle tissue or different tissues within the central nervous system. The tissue may be muscle tissue, and there may be modulation of the distribution or retention of the compound in myocardial tissue compared to at least one other type of muscle tissue (e.g., skeletal muscle including but not limited to the diaphragm, tibialis anterior and triceps muscles, or smooth muscle). The tissue may be CNS tissue, and there may be modulation of the distribution or retention of the compound in at least one CNS tissue compared to at least one other type of CNS tissue.

[0546] Any of the EPs described herein are suitable for inclusion in the compounds used in this method. EP may be PKKKRKV. EP may also be KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, and PKKKRKG. EP is KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, K Can be KKKK, KKKRK, KBKBK, KKKRKV, PGKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV and PKKKRKG.

[0547] EP may contain PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry. EP may be PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

[0548] EP may include amino acid sequences identified in the art as nuclear localization sequences (NLS). EP may consist of amino acid sequences identified in the art as nuclear localization sequences (NLS). EP may include an NLS containing the amino acid sequence PKKKRKV. EP may consist of an NLS containing the amino acid sequence PKKKRKV. EP may include an NLS containing amino acids selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK. EP may consist of NLS containing amino acids selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK.

[0549] The amount, expression, function, or activity of this compound may increase by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue compared to a second tissue.

[0550] The amount, expression, function, or activity of this compound may be reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue compared to a second tissue.

[0551] The amount or expression of this compound can be evaluated in different tissue types by methods known in the art (but not limited to the methodologies described in the examples). Tissues can be prepared by standard methods. The amount or expression of this compound in different tissues can be measured by well-established methods in the art, for example, by LC-MS / MS, Western blot analysis, or ELISA. The function or activity of this compound in different tissues can be measured by established methods for evaluating the relevant function or activity, for example, by using RT-PCR to evaluate the activity of oligonucleotide-based therapeutic moieties. For example, for antisense compounds (ACs) used as therapeutic moieties (TMs) that induce exon skipping in target mRNA of interest, RT-PCR can be used to quantify the level of exon skipping in different tissues.

[0552] The tissue distribution and / or retention of therapeutic agents in central nervous system (CNS) tissues can be modulated with compounds containing cyclic cell-permeable peptides (cCPPs) and extracyclic peptides (EPs). These compounds can be administered intrathecally to the target and can modulate the tissue distribution and / or retention of therapeutic agents in CNS tissues. Non-limiting examples of CNS tissues include the cerebellum, cortex, hippocampus, olfactory bulb, spinal cord, dorsal root ganglia (DRGs), and cerebrospinal fluid (CSF). Compounds containing cCPPs and EPs can be administered intrathecally, and the level of expression, activity, or function of the therapeutic agent may be higher in at least one CNS tissue compared to another CNS tissue. Compounds containing cCPPs and EPs can be administered intrathecally, and the level of expression, activity, or function of the therapeutic agent may be lower in at least one CNS tissue compared to another CNS tissue. The therapeutic agent may include a CD33-targeted therapeutic agent (e.g., a CD33-targeted antisense compound), which is administered intrathecally. This compound, containing cCPP and EP, can be administered intrathecally at doses of at least 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 50 mg / kg.

[0553] Methods for regulating the tissue distribution or retention of therapeutic agents in the target central nervous system (CNS) are: (a) Circular cell-permeable peptide (cCPP), (b) The therapeutic portion (TM) containing the above therapeutic agent, (c) A method comprising intrathecal administration of a compound comprising an extracyclic peptide (EP) containing at least one positively charged amino acid residue, wherein the amount, expression, function, or activity of the therapeutic agent is regulated by at least 10% in at least one tissue of the target CNS compared to a second tissue of the target CNS.

[0554] The amount, expression, function, or activity of the therapeutic agent may be regulated by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue of the target CNS compared to a second tissue of the target CNS.

[0555] Any of the therapeutic agents described herein for CNS-related diseases or disorders are suitable for inclusion in the compounds used in this method. The therapeutic agent may include a CD33-targeted therapeutic agent, for example, any of the CD33-targeted antisense compounds described herein.

[0556] Any of the CPPs described herein are suitable for inclusion in the compounds used in this method. CPPs may be cyclic CPPs (cCPPs).

[0557] This compound can be used to treat subjects with central nervous system diseases or disorders or neuroinflammatory diseases or disorders. In this embodiment, the subject has Alzheimer's disease or Parkinson's disease.

[0558] The tissue distribution and / or retention of the therapeutic agent can be regulated in different types of muscle tissue. Non-limiting examples of muscle tissue include the diaphragm, cardiac muscle, tibialis anterior, triceps, other skeletal muscles, and smooth muscle. Compounds containing cCPP, EP, and the therapeutic agent can be administered, and the level of expression, activity, or function of the therapeutic agent may be higher in at least one muscle tissue compared to another. Compounds containing cCPP, EP, and the therapeutic agent can be administered, and the level of expression, activity, or function of the therapeutic agent may be lower in at least one muscle tissue compared to another. The therapeutic agent may be a dystofin-targeted therapeutic agent (e.g., a DMD-targeted antisense compound). This compound can be administered in doses of at least 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 50 mg / kg.

[0559] A method for regulating the tissue distribution or retention of a therapeutic agent in the target muscle system, (a) Circular cell-permeable peptide (cCPP), (b) The therapeutic portion (TM) containing the above therapeutic agent, (c) A method comprising administering a compound comprising an extracyclic peptide (EP) containing at least one positively charged amino acid residue to a target, wherein the amount, expression, function, or activity of the therapeutic agent is regulated by at least 10% in at least one tissue of the target muscle system compared to a second tissue of the target muscle system.

[0560] The amount, expression, function, or activity of the therapeutic agent may be regulated by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in at least one tissue of the muscular system of interest, compared to a second tissue of the muscular system of interest.

[0561] Any of the therapeutic agents described herein for diseases or disorders related to the muscular system are suitable for inclusion in the compound used in this method. The therapeutic agent may be a DMD-targeted therapeutic agent, such as a DMD-targeted antisense compound.

[0562] Any of the CPPs described herein are suitable for inclusion in the compound used in this method. In the embodiments, the CPP is a cyclic CPP (cCPP).

[0563] In the embodiment, the subject has a neuromuscular disorder or a musculoskeletal disorder. In the embodiment, the subject has Duchenne muscular dystrophy.

[0564] Diseases and exemplary target genes associated with abnormal splicing The human genome contains over 40,000 genes, approximately half of which correspond to protein-coding genes. However, the number of human protein species is predicted to be orders of magnitude greater due to single-amino acid polymorphisms, post-translational modifications, and, importantly, alternative splicing. RNA splicing, which generally occurs in the nucleus, is a process in which precursor messenger RNA (pre-mRNA) is converted into mature messenger RNA (mRNA) by removing non-coding regions (introns) and joining the remaining coding regions (exons). The resulting mRNA can then be transported out of the nucleus and translated into proteins. Alternative splicing is a regulated process in gene expression that results in a single gene encoding multiple proteins. In this process, specific exons of a gene may be included in or excluded from the final processed mRNA produced from that gene. Alternative splicing is a normal phenomenon in eukaryotes and contributes to the biodiversity of proteins encoded by the genome, but abnormal mutations in splicing are significantly associated with disease. The majority of human genetic disorders are caused by splicing mutations. Abnormal splicing mutations contribute to the development of cancer, and splicing factor genes frequently mutate in different types of cancer.

[0565] Approximately 10% of the roughly 80,000 mutations reported in the Human Genetic Variation Database (HGMD) affect splice sites. In HGMD, there are 3,390 disease-causing mutations occurring at the +1 donor splice site. These mutations affect 2,754 exons in 901 genes. The prevalence is even higher in neuromuscular disorders (NMD), due to the abnormally large size and multi-exon structure of genes encoding muscle structural proteins, further highlighting the importance of these mutations in NMD.

[0566] Previously, the correction of point mutations, such as splice site mutations, has been attempted via homologous recombination repair (HDR) pathways, but this is extremely inefficient in postmittal tissues such as skeletal muscle, hindering its therapeutic usefulness in NMD. Furthermore, standard gene therapy approaches for reintroducing corrected coding regions into the genome are hindered by large-sized genes encoding muscle structural proteins, for example. Moreover, many existing therapies rely on the inefficient delivery of therapeutic compounds to diseased cells, resulting in impractical in vivo treatments and higher toxicity.

[0567] The target gene in this disclosure may be any eukaryotic gene containing one or more introns and one or more exons. The target gene may be a mammalian gene. Mammals may include humans, mice, cattle, rats, pigs, horses, chickens, sheep, etc. The target gene may be a human gene.

[0568] A target gene may be a gene containing a mutation that results in abnormal splicing. A target gene may be a gene containing one or more mutations. A target gene may be a gene containing one or more mutations such that the transcription and translation of the target gene do not result in a functional protein. A target gene may be a gene containing one or more mutations such that the transcription and translation of the target gene result in a target protein that is less active or non-functional than the wild-type target protein.

[0569] The target gene may be a gene underlying a genetic disorder. The target gene may have abnormal gene expression in the central nervous system. The target gene may be a gene involved in the pathogenesis of neuromuscular disorders (NMD). The target gene may be a gene involved in the pathogenesis of musculoskeletal disorders (NMD). The neuromuscular disorder may be Pompe disease, and the target gene may be GYS1.

[0570] Antisense compounds can be used to target genes containing mutations that result in abnormal splicing underlying genetic disorders, in order to redirect splicing and obtain the desired splice product (Kole, Acta Biochimica Polonica, 1997, 44, 231-238).

[0571] The CRISPR gene editing mechanism can be used to target abnormal genes for removal or to regulate gene transcription and translation.

[0572] Diseases may include β-thalassemia (Dominski and Kole, Proc. Natl. Acad. Sci. USA, 1993, 90, 8673-8677; Sierakowska et al., Nucleosides & Nucleotides, 1997, 16, 1173-1182; Sierakowska et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 12840-44; Lacerra et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 9591-9596).

[0573] Diseases may include dystrophin-associated dystrophin (Takeshima et al., J. Clin. Invest., 1995, 95, 515-520).

[0574] The disease may include Duchenne muscular dystrophy (Dunckley et al. Nucleosides & Nucleotides, 1997, 16, 1665-1668; Dunckley et al. Human Mol. Genetics, 1998, 5, 1083-90). The target gene may be the DMD gene, which encodes dystrophin. The protein consists of an N-terminal domain that binds to actin filaments, a central rod domain, and a C-terminal cysteine-rich domain that binds to the dystrophin-glycoprotein complex (Hoffman et al. 1987; Koenig et al. 1988; Yoshida and Ozawa 1990). Mutations in the DMD gene that disrupt the leading frame result in complete loss of dystrophin function, which causes severe Duchenne muscular dystrophy (DMD) [MIM ​​310200]. On the other hand, milder Becker muscular dystrophy (BMD [MIM300376]) is the result of mutations in the same gene that are not frameshifts, resulting in a dystrophin that is internally deleted but retains its partially functional N- and C-terminuses (Koenig et al. 1989, Di Blasi et al. 1996). More than two-thirds of patients with DMD and BMD have deletions of two or more exons (den Dun-nen et al. 1989). Notably, patients exhibiting very mild BMD with up to 67% of the central rod domain have been described (England et al. 1990, Winnard et al. 1993, Mirabella et al. 1998). This suggests that, despite large deletions, a partially functional dystrophin can be produced, provided that the deletion inframes the transcript. These observations led to the idea of ​​using AC to modify splicing so that open reading frames are recovered and severe DMD phenotypes are converted to milder BMD phenotypes.Several studies have demonstrated therapeutic AC-induced single-exon skipping in cells derived from the mdx mouse model (Dunckley et al. 1998, Wilton et al. 1999, Mann et al. 2001, 2002, Lu et al. 2003) and in various DMD patients (Takeshima et al. 2001, van Deutekom et al. 2001, Aartsma-Rus et al. 2002, 2003, De Angelis et al. 2002). AC can be used to skip one or more exons selected from exons 2, 8, 11, 17, 19, 23, 29, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 55, and 59 of DMD. See Aartsma-Rus et al. 2002, incorporated herein by reference. AC can be used to skip one or more exons selected from exons 8, 11, 43, 44, 45, 50, 51, 53, and 55 of DMD. Approximately 75% of all patients with DMD arise from skipping these exons. Skipping an exon adjacent to an out-of-frame deletion or an in-frame exon containing a nonsense mutation can restore the leading frame and induce the synthesis of BMD-like dystrophin in treated cells (van Deutekom et al. 2001, Aartsma-Rus et al. 2003). AC hybridizing to its target sequence within the target DMD pre-mRNA can induce skipping of one or more exons. AC can induce the expression of a respliced ​​target protein containing the active fragment of dystrophin. A non-limiting example of AC for exon 52 is described in U.S. Patent Application Publication 2019 / 0365918, which states that compounds incorporated by reference in whole for any purpose may include cargo targeting the EP, cCPP, and DMD genes.

[0575] Circular cell-permeable peptide (cCPP) conjugated in the cargo portion Circular cell-permeable peptides (cCPPs) can be conjugated to the cargo portion.

[0576] The cargo portion may be conjugated to cCPP via a linker. The cargo portion may contain a therapeutic portion. The therapeutic portion may contain an oligonucleotide, a peptide, or a small molecule. The oligonucleotide may contain an antisense oligonucleotide. The cargo portion is conjugated to the linker at a terminal carbonyl group to form the following structure:

[0577] [ka] It can provide, in the formula, EP is an extracyclic peptide, M, AA SC Cargo, x', y, and z' are defined above, * is AA SC This is a connection point to . x' can be 1. y can be 4. z' can be 11. -(OCH2CH-2) x’ -and / or-(OCH2CH-2) z’ - can be independently replaced with one or more amino acids, such as glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0578] The endosomal escape vehicle (EEV) may contain a cyclic cell-permeable peptide (cCPP), an extracyclic peptide (EP), and a linker, which are conjugated to the cargo to form the structure of formula (C):

[0579] [ka] Alternatively, it can form an EEV-conjugate that includes its protonated form. During the ceremony, R1, R2, and R3 may each be an amino acid residue having a side chain containing H or an aromatic group. R4 is either H or an amino acid side chain. EP is an extracyclic peptide as defined herein, Cargo is a part as defined herein, Each m is an independent integer between 0 and 3. n is an integer between 0 and 2. x' is an integer between 2 and 20. y is an integer between 1 and 5. q is an integer between 1 and 4. z' is an integer between 2 and 20.

[0580] R1, R2, R3, R4, EP, Cargo, m, n, x', y, q, and z' are as defined herein.

[0581] EEV can be conjugated into cargo, and the EEV conjugate has the structure of formula (Ca) or (Cb):

[0582] [ka] Or it may include its protonated form, where EP, m, and z are as previously defined in formula (C).

[0583] EEV can be conjugated into cargo, and EEV-conjugate is given by formula (Cc):

[0584] [ka] The formula may include the structure or its protonated form, where EP, R 1 , R 2 , R 3 , R 4 , and m are as previously defined in formula (III), AA may be an amino acid as defined herein, n may be an integer from 0 to 2, x may be an integer from 1 to 10, y may be an integer from 1 to 5, and z may be an integer from 1 to 10.

[0585] EEV can be conjugated into oligonucleotide cargoes, and the EEV-oligonucleotide conjugate has the structure of formula (C-1), (C-2), (C-3), or (C-4):

[0586] [ka]

[0587] [ka] It may include.

[0588] EEV can be conjugated to oligonucleotide cargo, and the EEV conjugate has the following structure:

[0589] [ka] It may include.

[0590] Cytoplasmic delivery efficiency Modification of cyclic cell-permeable peptides (cCPPs) can improve cytoplasmic delivery efficiency. Improved cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of cCPPs with modified sequences to that of a control sequence. The control sequence is otherwise identical to the modified sequence, except that it does not contain specific substituted amino acid residues (but is not limited to, arginine, phenylalanine, and / or glycine).

[0591] As used herein, cytoplasmic delivery efficiency refers to the ability of cCPPs to cross the cell membrane and enter the cytoplasm of a cell. The cytoplasmic delivery efficiency of cCPPs is not necessarily dependent on the receptor or cell type. Cytoplasmic delivery efficiency may refer to absolute or relative cytoplasmic delivery efficiency.

[0592] Absolute cytoplasmic delivery efficiency is the ratio of the cytosolic concentration of cCPP (or cCPP-cargo conjugate) to the concentration of cCPP (or cCPP-cargo conjugate) in the growth medium. Relative cytoplasmic delivery efficiency refers to the concentration of cCPP in the cytosol compared to the concentration of control cCPP in the cytosol. Quantification can be achieved by fluorescently labeling cCPP (e.g., with FITC dye) and measuring the fluorescence intensity using techniques well known in the art.

[0593] The relative cytoplasmic delivery efficiency is determined by comparing (i) the amount of cCPP of the present invention internalized by a cell type (e.g., HeLa cells) with (ii) the amount of control cCPP internalized by the same cell type. To measure the relative cytoplasmic delivery efficiency, the cell type may be incubated in the presence of cCPP for a specific period (e.g., 30 minutes, 1 hour, 2 hours, etc.), after which the amount of cCPP internalized by the cells is quantified using a method known in the art, such as fluorescence microscopy. Separately, the same concentration of control cCPP is incubated in the presence of the cell type for the same period, and the amount of control cCPP internalized by the cells is quantified.

[0594] The relative cytoplasmic delivery efficiency is the IC of cCPPs with modified sequences targeting intracellular targets. 50 Measure the IC of cCPP having a modified sequence. 50 This can be determined by comparing it with a control sequence (as described herein).

[0595] The relative cytoplasmic delivery efficiency of cCPP is in the range of approximately 50% to 450% compared to cyclo(FfΦRrRrQ), for example, approximately 60%, 70%, 80%, 90%, 100%, 200%, 110%, 210%, 120%, 220%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 300%, 310%, 320%, 230%, 330%, 240%, 250%, 260%, 270%, and 2 The values ​​may range from 80%, approximately 290%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, approximately 500%, approximately 510%, approximately 520%, approximately 530%, approximately 540%, approximately 550%, approximately 560%, approximately 580%, approximately 570%, approximately 580%, or approximately 590% (including all values ​​and partial ranges in between). The relative cytoplasmic delivery efficiency of cCPP can be improved by more than approximately 600% compared to cyclic peptides including cyclic (FfφRrRrQ).

[0596] The absolute cytoplasmic delivery efficiency ranges from approximately 40% to approximately 100%, for example, approximately 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% (including all values ​​and partial ranges between these).

[0597] The cCPPs of this disclosure, compared to otherwise identical sequences, improve cytoplasmic delivery efficiency by approximately 1.1 to 30 times, for example, approximately 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 10 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times, 12.5 times, 13.0 times, 13.5 times, and 14 times. It may improve by 0.0 times, approximately 14.5 times, approximately 15.0 times, approximately 15.5 times, approximately 16.0 times, approximately 16.5 times, approximately 17.0 times, approximately 17.5 times, approximately 18.0 times, approximately 18.5 times, approximately 19.0 times, approximately 19.5 times, approximately 20 times, approximately 20.5 times, approximately 21.0 times, approximately 21.5 times, approximately 22.0 times, approximately 22.5 times, approximately 23.0 times, approximately 23.5 times, approximately 24.0 times, approximately 24.5 times, approximately 25.0 times, approximately 25.5 times, approximately 26.0 times, approximately 26.5 times, approximately 27.0 times, approximately 27.5 times, approximately 28.0 times, approximately 28.5 times, approximately 29.0 times, or approximately 29.5 times (including all values ​​and subranges in between).

[0598] Manufacturing method The compounds described herein 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. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but those skilled in the art can determine such conditions.

[0599] Modifications of the compounds described herein include the addition, removal, or movement of various components, as described for each compound. Similarly, if one or more chiral cent...

Claims

1. Ac-PKKKRK-!!! ２ -Lyss(シクロIL. The １２ -Lyss(ｮ ３ )-82 ２ ; Ac-PKKKRKV-Lys(cyclo[Phe-phe-Nal-Gly-arg-Gly-arg-Gln])-PEG １２ -Lys(N) ３ )-NH ２ ; Ac-PKKKRKV-AEEA-Lys(cyclo[Phe-phe-Nal-Gly-arg-Gly-arg-Gln])-PEG １２ -OH; and Ac-PKKKRKV-PEG ２ -K(Cyclo[Phe-phe-Nal-Gly-arg-Gly-arg-Gln])-PEG ２ -Lys(N ３ )-NH ２ A compound selected from the following.

2. Ac-PKKKRKV-PEG ２ -Lys(cyclo[Phe-phe-Nal-Cit-arg-Cit-arg-Gln]-PEG １２ -Lys(N) ３ )-NH ２ The compound according to claim 1.

3. Ac-PKKKRKV-Lys(cyclo[Phe-phe-Nal-Gly-arg-Gly-arg-Gln])-PEG １２ -Lys(N) ３ )-NH ２ The compound according to claim 1.

4. Ac-PKKKRKV-AEEA-Lys(cyclo[Phe-phe-Nal-Gly-arg-Gly-arg-Gln])-PEG １２ The compound according to claim 1, wherein it is -OH.

5. Ac-PKKKRKV-PEG ２ -K(cyclo[Phe-phe-Nal-Gly-arg-Gly-arg-Gln])-PEG ２ -Lys(N) ３ )-NH ２ The compound according to claim 1.

6. A compound according to any one of claims 1 to 5, conjugated to a cargo portion containing an antisense compound (AC).

7. The compound according to claim 6, wherein the antisense compound is a phosphorodiamidate morpholino oligomer (PMO).

8. A pharmaceutical composition for treating a disease or condition requiring treatment, comprising an effective amount of the compound described in claim 6 or 7, and administered to the subject.

9. The pharmaceutical composition according to claim 8, wherein the administration includes parenteral administration.

10. The pharmaceutical composition according to claim 9, wherein the parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, or intrasternal administration.

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