Peptide-based delivery agents and methods of making and using same

Abstract

The present disclosure provides a delivery agent that promotes the effective delivery of drugs and other compounds through a lipid layer. The delivery agent disclosed herein is lipid-soluble under selected conditions and water-soluble under other conditions, and can effectively deliver compounds to the cell cytosol. The delivery agent of the present disclosure also avoids adverse interactions with serum, thus providing efficient in vivo delivery of therapeutic agents.

Description

[Technical Field]

[0001] (Technical field) The present disclosure relates to peptide-based delivery agents that facilitate the delivery of compounds into cells.

[0002] (Reference to Electronic Sequence Listing) The electronic sequence listing, created on Aug. 11, 2023, and submitted as XML file 10413-107991-02.xml (341,757 bytes), is hereby incorporated by reference in its entirety.

[0003] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims benefit of and priority to the earlier filing date of U.S. Provisional Patent Application No. 63 / 397,601, filed August 12, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0004] Lipid layers, such as cell membranes (including plasma membranes and vesicle membranes), can constitute barriers to effective drug delivery. For optimal delivery, drugs must be freely soluble in both aqueous compartments of the body and the lipid layers surrounding those compartments. While many low-molecular-weight compounds with low to moderate polarity can pass directly through lipid layers, compounds with higher polarity and / or higher molecular weights (such as nucleic acids and proteins) generally enter eukaryotic cells only via endocytosis or related processes. During endocytosis, compounds are internalized into the cell through progressive invagination of membrane regions, ultimately forming intracellular closed vesicles, or endosomes. Endosomes can then fuse with lysosomes, resulting in entrapment within the vesicle, exposing the internalized compound to degradative enzymes.

[0005] Thus, there is a need for improved agents that facilitate more effective delivery of drugs and other compounds through lipid layers, such as agents that are lipid-soluble under selected conditions and water-soluble under other conditions, and that can effectively deliver compounds to the cell cytosol. Summary of the Invention

[0006] An aspect of the present disclosure is a peptide-based delivery agent having the formula [X 1 Y 1 Y 1 X 1 ] m a lytic peptide group having a structure according to 1 At least one X 1 is a basic amino acid, an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 1 is, independently for each occurrence, a non-polar amino acid or derivative thereof, and m is an integer selected from 2 to 8, 1 Y 1 Y 1 X 1 ] m a lytic peptide group having a structure according to the formula [X 2 Y 2 Y 2 X 2 ] m’ a mask peptide group having a structure according to 2 At least one X 2 is an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 2 is, independently for each occurrence, a non-polar amino acid or derivative thereof, and m' is an integer selected from 2 to 8, 2 Y 2 Y 2 X 2 ] m’ and an anchor group selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group; a targeting group selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule, or a combination thereof.

[0007] In some embodiments of the disclosure, the delivery agent has a structure according to formula IA or formula IB: [N-terminal group]-[X 1 Y 1 Y 1 X 1 ] m -[cleavable linker]-[X 2 Y 2 Y 2 X 2 ] m’ -[anchor / targeting group]-[C-terminal group] (Formula 1A) [N-terminal group]-[X 2 Y 2 Y 2 X 2 ] m’ -[anchor / targeting group]-[cleavable linker]-[X 1 Y 1 Y 1 X 1 ] m -[C-terminal group] (Formula 1B) each X 1 At least one X 1 is a basic amino acid, an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 1 is, independently for each occurrence, a nonpolar amino acid or derivative thereof, and each of m and m' is, independently for each occurrence, an integer selected from 2 to 8; 1 Y 1 Y 1 X 1 ] m and each X 2 At least one X 2 is an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 2is, independently for each occurrence, a nonpolar amino acid or derivative thereof; the cleavable linker is an amino acid sequence that is 2 to 10 amino acids in length; the anchor group, if present, is selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group; the targeting group, if present, is selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule, or a combination thereof; the C-terminal group comprises an amine-terminated glycine moiety; and the N-terminal group comprises a capping group or a fluorophore.

[0008] Aspects of the present disclosure also relate to compositions comprising a peptide-based delivery agent according to the present disclosure and a therapeutic agent.

[0009] Embodiments of the present disclosure also relate to methods comprising contacting a cell with a delivery agent and / or composition according to the present disclosure.Embodiments of the present disclosure also relate to methods comprising administering to a subject a therapeutically effective amount of a delivery agent and / or composition according to the present disclosure.

[0010] Aspects of the present disclosure also relate to a method for identifying a therapeutic compound, the method comprising contacting a cell with a peptide-based delivery agent according to the present disclosure and one or more compounds; determining an effect of the one or more compounds on the contacted cells; and comparing the effect of the one or more compounds on the contacted cells with a control, wherein a differential effect of the one or more compounds on the contacted cells relative to the control indicates that the one or more compounds are therapeutic compounds.

[0011] Aspects of the present disclosure also relate to kits, comprising a container, the container comprising a peptide-based delivery agent or composition according to aspects of the present disclosure, the container being selected from a syringe, a vial, a tube, an ampoule, a capsule, or a bottle.

[0012] The above and other objects and features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram illustrating how peptide-based delivery agents and therapeutic agents disclosed herein enter the cytosol of eukaryotic cells by endocytic uptake. [Figure 2] Figure 2A shows a helical wheel diagram of an exemplary EXXE motif-based lytic peptide group and how the distribution of acidic residues affects the faces of polar and non-polar regions. Figure 2B shows how the distribution of acidic residues affects the faces of polar and non-polar regions. A schematic diagram showing the interaction between an exemplary lytic peptide group backbone and an exemplary mask peptide group backbone. [Figure 3] Figure 3A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 23-6 and 23-7 discussed herein in combination with a control morpholino compound, and Figure 3B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-6 and 23-7 discussed herein in combination with a control morpholino compound. [Figure 4] 1 is a graph of the average luciferase activity (LUC) per protein in 1% serum for delivery agents 23-10, 23-16, 23-20, and a positive control discussed herein. [Figure 5] Figure 5A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 30-2, 30-4, 30-6, 30-10, 30-14, 30-16, 30-18, and 30-22 discussed herein. Figure 5B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 30-2, 30-4, 30-6, 30-10, 30-14, 30-16, 30-18, and 30-22 discussed herein. [Figure 6]Figure 6A is a graph showing the average luciferase activity (LUC) per protein in 80% serum using 12 μM delivery agent for delivery agents 40-1 and 40-46 discussed herein. Figure 6B is a graph showing the average luciferase activity (LUC) per protein in 80% serum using 6 μM delivery agent for delivery agents 42-9 and 42-39 discussed herein. [Figure 7] 1 is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 40-1, 40-46, 42-9, and 42-39 discussed herein. [Figure 8] Figure 8A shows the spiral wheel diagram of delivery agents 23-26. Figure 8B shows the spiral wheel diagram of delivery agents 50-22. Figure 8C shows the average luciferase activity (LUC) per protein in 80% serum of delivery agents. [Figure 9] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum using delivery agents 23-26, 51-11, 51-12, 51-13, 51-14, 51-15, 51-16, 51-17, 51-18 discussed herein and a morpholino-only control. [Figure 10] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum using delivery agents 23-26, 51-24, 51-25, 51-26, 51-27, 51-28, 51-29, 51-30, 51-31, 50-23 discussed herein and a morpholino only control. [Figure 11] 1 is a graph showing the average luciferase activity (LUC) per protein in 1% serum using different concentrations of delivery agents 56-3, 56-5, 23-26, and a control discussed herein. [Figure 12]Figure 12A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 60-16, 60-17, 60-4, 62-9, and 23-26 discussed herein. Figure 12B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 60-16, 60-17, 60-4, 62-9, and 23-26 discussed herein. [Figure 13] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum using control and delivery agents 23-26, 88-8, 88-9, 88-10, 88-11, and 88-12 discussed herein. [Figure 14] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum using untreated cells and delivery agents 104-3+4, 109-7, 114-1, 114-3, 114-12, 114-30, and 114-33 discussed herein. [Figure 15] Figure 15A shows a spiral wheel diagram for delivery agent 114-12. Figure 15B shows a spiral wheel diagram for delivery agent 114-6. Figure 15C shows a spiral wheel diagram for delivery agent 114-9. Figure 15D shows the average luciferase activity (LUC) per protein in 80% serum for the above delivery agents and compound 104-3+4. [Figure 16] 1 is a graph showing the percentage of compound bound to human serum albumin as a function of the number of amino acids in the lytic peptide group. [Figure 17] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum using untreated cells or cells treated with delivery agents 104-3+4, 114-12, 114-18, 114-21, 114-24, or 114-27 discussed herein. [Figure 18]Figure 18A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-11 and 19-1 discussed herein. Figure 18B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-11 and 19-1 discussed herein. [Figure 19] Figure 19A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 36-47 and 36-48 discussed herein. Figure 19B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 36-47 and 36-48 discussed herein. [Figure 20] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 96-1, 96-4, 96-5, 96-6, and 96-7 described herein. [Figure 21] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 26-17, 26-2, and a negative control discussed herein. [Figure 22] Figure 22A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 19-1, 19-3, 19-5, and two controls discussed herein. Figure 22B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 19-1, 19-3, 19-5, and two controls discussed herein. [Figure 23] Figure 23A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 23-12, 23-13, and 23-14 discussed herein. Figure 23B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-12, 23-13, and 23-14 discussed herein. [Figure 24]Figure 24A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 21-12, 21-13, and 19-1 with different anchor group tail lengths, as discussed herein. Figure 24B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-12, 21-13, and 19-1 with different anchor group tail lengths, as discussed herein. [Figure 25] 1 is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 23-15 and 19-1 discussed herein. [Figure 26] Figure 26A is a graph showing the average luciferase activity (LUC) per protein in 1% serum at 3 μM for delivery agents 19-1 and 21-8 discussed herein. Figure 26B is a graph showing the average luciferase activity (LUC) per protein in 80% serum at 9 μM for delivery agents 19-1 and 21-8 discussed herein. Figure 26C is a graph showing the average luciferase activity (LUC) per protein in 1% serum at 2 μM for delivery agents 19-1 and 21-8 discussed herein. Figure 26D is a graph showing the average luciferase activity (LUC) per protein in 80% serum at 9 μM for delivery agents 19-1 and 21-8 discussed herein. [Figure 27] 1 is a graph showing the mean luciferase activity (LUC) per protein in 80% serum for delivery agents 40-31 through 40-46 described herein. [Figure 28] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-26 and 53-6 through 53-15 discussed herein, along with a no-treatment control. [Figure 29]Figure 29A is a graph showing the average luciferase activity (LUC) per 1% serum protein at 1 or 3 μM for delivery agents 96-10, 23-26, and a control, discussed herein, with different DMSO concentrations. Figure 29B is a graph showing the average luciferase activity (LUC) per 80% serum protein at 12 μM for delivery agents 96-10, 23-26, and a control, discussed herein. Figure 29C is a graph showing the average luciferase activity (LUC) per 80% serum protein at 9 or 18 μM for delivery agent 26-13A, discussed herein, with or without DMSO. [Figure 30] Figure 30A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-1, 21-4, and 21-6 discussed herein, as well as a control. Figure 30B is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 21-1, 21-4, and 21-6 discussed herein, as well as a control. [Figure 31] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for different concentrations of delivery agents 19-1, 21-4, and 21-6 discussed herein, and a control. [Figure 32] Figure 32A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-27 and 23-4 discussed herein. Figure 32B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-27 and 23-4 discussed herein. [Figure 33] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 19-1, 23-4, and 23-5 discussed herein. [Figure 34]Figure 34A is a graph showing the average luciferase activity (LUC) per protein in 80% serum at 18 μM for delivery agents 26-7a / a' and 26-7b / b' discussed herein. Figure 34B is a graph showing the average luciferase activity (LUC) per protein in 80% serum at 9 μM for delivery agents 26-14A and 26-14B with different anchor group tail lengths discussed herein. [Figure 35] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum at different concentrations of delivery agents 26-14A-D with different anchor group tail lengths, as discussed herein. [Figure 36] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 26-15A / B with different anchor tail lengths at different concentrations and for a control, as discussed herein. [Figure 37] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 26-2A and 26-11 (no anchor group), delivery agents 26-2B and 26-13, and a control discussed herein. [Figure 38] 1 is a graph showing the average luciferase activity per protein (LUC) in 80% serum for delivery agents 33-24 and 23-26 discussed herein, as well as control and untreated. [Figure 39] Figure 39A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 40-23A, 40-46, and a control, discussed herein. Figure 39B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 40-23A, 40-23B, and 40-46, discussed herein. [Figure 40] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-4a, 21-4b, 21-4b', 21-6a, 21-6b, and 21-7a with different anchor group tail lengths, as discussed herein. [Figure 41] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 60-1b, 60-1a, and 23-26 discussed herein, and controls. [Figure 42] 1 is a graph showing the average luciferase activity per protein (LUC) in 80% serum for delivery agents 104-3+4, 109-1, and 109-8 discussed herein, as well as controls. [Figure 43] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-26, 78-1, and 78-2 discussed herein, and a control. [Figure 44] Figure 44A is a graph showing the average luciferase activity (LUC) per protein in 80% serum of delivery agents 23-26, 85-2C, and 85-2P discussed herein, and a control. Figure 44B is a graph showing the average luciferase activity (LUC) per protein in 80% serum of delivery agents 23-26, 85-2C, 85-2P, 86-1C, 86-1P, 86-4C, and 86-4P discussed herein, and a control (Figure 44B). [Figure 45] 1 is a graph showing the average luciferase activity per protein (LUC) in 80% serum for delivery agents 23-26, 85-AC, and 86-5A P discussed herein, as well as controls. [Figure 46] 1 is a graph showing the average luciferase activity per protein (LUC) in 80% serum for delivery agents 23-26, 50-23, 50-24, and 50-26 discussed herein, and a control. [Figure 47]Figure 47A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 23-26, 30-3, 30-5, 30-7, 30-9, 30-11, 30-15, 30-17, and 30-22 discussed herein. Figure 47B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-26, 30-3, 30-5, 30-7, 30-9, 30-11, 30-15, 30-17, and 30-22 discussed herein. [Figure 48] Figure 48A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 19-1, 19-17A, and 19-17C discussed herein, as well as two controls. Figure 48B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 19-1, 19-17A, and 19-17C discussed herein, as well as two controls. [Figure 49] Figure 49A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 21-4 and 21-5 discussed herein, and a control. Figure 49B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-4 and 21-5 discussed herein, and a control. [Figure 50] Figure 50A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 19-1 and 22-11 discussed herein, as well as a control. Figure 50B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 19-1 and 22-11 discussed herein, as well as a control. [Figure 51]Figure 51A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 24-1, 23-27, 23-1, and 23-4 discussed herein. Figure 51B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 24-1, 23-27, 23-1, and 23-4 discussed herein. [Figure 52] Figure 52A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 23-1 to 23-5 discussed herein. Figure 52B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-1 to 23-5 discussed herein. [Figure 53] Figure 53A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for delivery agents 21-4, 22-1, and 22-12 discussed herein, as well as a control. Figure 53B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-4, 22-1, and 22-12 discussed herein, as well as a control. [Figure 54] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 21-1, 33-26a, 33-26b, 33-26c, 34-32, 34-33, and 34-34 discussed herein. [Figure 55] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 104-3+4 and 109-2 discussed herein. [Figure 56]Figure 56A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for the delivery agents 23-26, MO+23-26, Vivo-MO+23-26, Vivo-MO, and no treatment discussed herein. Figure 56B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for the delivery agents MO+23-26, Vivo-MO, and Vivo-MO+23-26 at different concentrations. [Figure 57] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-26+a and 23-26+b discussed herein, and for no treatment. [Figure 58] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 23-26+a and 23-26+b discussed herein, and 23-26 without added morpholino. [Figure 59] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for the delivery agents 102-3 and 98-1-MO conjugates discussed herein, and for no treatment. [Figure 60] 1 is a graph showing the average luciferase activity (LUC) per protein in 80% serum for the compounds discussed herein in Example 59 and controls. [Figure 61] 1 is a graph showing the average luciferase activity per protein (LUC) in 80% serum for the delivery agents 104-3+4P+MO and 107-8-MO conjugates discussed herein, as well as controls. [Figure 62] Figure 62A is a graph showing the cell viability results of compound 104-3+4. Figure 62B is a graph showing the results of saporin delivery when saporin toxin is combined with compound 104-3+4. [Figure 63] 1 is an image showing mRNA (isolated from muscle tissue) amplified using RT-PCR and then analyzed using gel electrophoresis. [Figure 64]1 is an image showing mRNA (isolated from muscle tissue) amplified using RT-PCR and then analyzed using gel electrophoresis. [Figure 65] Figure 65A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for compound 16-6 and two controls. Figure 65B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for compound 16-6 and two controls. [Figure 66] Figure 66A is a graph showing the average luciferase activity (LUC) per protein in 1% serum for compound 19-1 and two controls. Figure 66B is a graph showing the average luciferase activity (LUC) per protein in 80% serum for compound 19-1 and two controls. [Figure 67] Figure 67A is a graph showing the average luciferase activity (LUC) per protein in 80% serum for delivery agents 215-2 DBCO, 216-A DBCO, and 216-B DBCO. Figure 67B is a graph showing the average luciferase activity (LUC) per protein in 10% serum for delivery agents 215-2 DBCO, 216-A DBCO, and 216-B DBCO. [Figure 68] Graph showing the average luciferase activity (LUC) per protein in 10% serum for delivery agents 215-2 FA, 216-A FA, 216-B FA, 215-2 DBCO, 216-A DBCO, and 216-B DBCO, where "FA" is free amine. [Figure 69] 1 is a graph showing the average luciferase activity (LUC) per protein in 10% serum for delivery agents 218-1, 218-2, 219-1, and 219-2. [Figure 70]Figure 70A is a graph showing the results of evaluation of different delivery agents disclosed herein, showing GFP expression in 10% serum for the control and delivery agents 215-2 Ab, 216-2 Ab, 216-1 Ab, 215-2 PEG12-BCN, and 216-1 PEG12-BCN. Figure 70B is a graph showing the results of evaluation of different delivery agents disclosed herein, showing the average luciferase activity (LUC) in 10% serum of HeLa Luc / 705 cells transfected with the transferrin receptor (TFRC). Figure 70C is a graph showing the results of evaluation of different delivery agents disclosed herein, showing the average luciferase activity (LUC) in 10% serum of untransfected HeLa Luc / 705 cells. [Figure 71] The mean luciferase activity (LUC) in 80% serum for delivery agents 224-2, 225-2, and 216-2 is shown. [Figure 72] The mean luciferase activity (LUC) in 80% serum of delivery agents 223-2 and 89-5 is shown. [Sequence Table]

[0014] The nucleic acid and amino acid sequences set forth in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases and amino acids as defined in 37 CFR § 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that any reference to the displayed strand also includes the complementary strand.

[0015] SEQ ID NO:1 is the amino acid sequence of the lytic peptide group. SEQ ID NO:2 is the amino acid sequence of the lytic peptide group. SEQ ID NO:3 is the amino acid sequence of the cleavable linker. SEQ ID NO:4 is the amino acid sequence of the masked peptide group. SEQ ID NO:5 is the amino acid sequence of the C-terminal portion. SEQ ID NO:6 is the amino acid sequence of the lytic peptide group, the cleavable linker, and the mask peptide group of the delivery agent. SEQ ID NO:7 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, and the amino acid portion of the two anchor groups of the delivery agent. SEQ ID NO:8 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portions of the two anchor groups, and the C-terminal portion of the delivery agent. SEQ ID NO:9 is the amino acid sequence of the lytic peptide group, the cleavable linker, and the mask peptide group of the delivery agent. SEQ ID NO:10 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, and the amino acid portion of the two anchor groups of the delivery agent. SEQ ID NO:11 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portions of the two anchor groups, and the C-terminal portion of the delivery agent. SEQ ID NO:12 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portions of the two anchor groups, and the C-terminal portion of the delivery agent. SEQ ID NO:13 is the amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and C-terminal portion of a delivery agent, such as those contained in delivery agents 21-4, 40-46, 42-39, 23-4, and 23-26. SEQ ID NO:14 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portions of the two anchor groups, and the C-terminal portion of the delivery agent. SEQ ID NO:15 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portions of the two anchor groups, and the C-terminal portion of the delivery agent. SEQ ID NO:16 is the amino acid sequence of the lytic peptide group. SEQ ID NO:17 is the amino acid sequence of the lytic peptide group. SEQ ID NO:18 is the amino acid sequence of delivery agent 23-6. SEQ ID NO:19 is the amino acid sequence of delivery agent 23-7. SEQ ID NO:20 is the amino acid sequence of the lytic peptide group. SEQ ID NO:21 is the amino acid sequence of delivery agent 23-10. SEQ ID NO:22 is the amino acid sequence of delivery agent 23-16. SEQ ID NO:23 is the amino acid sequence of delivery agent 23-20. SEQ ID NO:24 is the amino acid sequence of the lytic peptide group. SEQ ID NO:25 is the amino acid sequence of delivery agent 30-2. SEQ ID NO:26 is the amino acid sequence of the lytic peptide group. SEQ ID NO:27 is the amino acid sequence of delivery agent 30-4. SEQ ID NO:28 is the amino acid sequence of the lytic peptide group. SEQ ID NO:29 is the amino acid sequence of delivery agent 30-6. SEQ ID NO:30 is the amino acid sequence of the lytic peptide group. SEQ ID NO:31 is the amino acid sequence of delivery agent 30-10. SEQ ID NO:32 is the amino acid sequence of the lytic peptide group. SEQ ID NO:33 is the amino acid sequence of delivery agent 30-14. SEQ ID NO:34 is the amino acid sequence of the lytic peptide group. SEQ ID NO:35 is the amino acid sequence of delivery agent 30-16. SEQ ID NO:36 is the amino acid sequence of the lytic peptide group. SEQ ID NO:37 is the amino acid sequence of delivery agent 30-18. SEQ ID NO:38 is the amino acid sequence of the lytic peptide group. SEQ ID NO:39 is the amino acid sequence of delivery agent 40-1. SEQ ID NO:40 is the amino acid sequence of the lytic peptide group. SEQ ID NO:41 is the amino acid sequence of the masked peptide group. SEQ ID NO:42 is the amino acid sequence of delivery agent 50-22. SEQ ID NO:43 is the amino acid sequence of the lytic peptide group. SEQ ID NO:44 is the amino acid sequence of the lytic peptide group. SEQ ID NO:45 is the amino acid sequence of the lytic peptide group. SEQ ID NO:46 is the amino acid sequence of the lytic peptide group. SEQ ID NO:47 is the amino acid sequence of the lytic peptide group. SEQ ID NO:48 is the amino acid sequence of the lytic peptide group. SEQ ID NO:49 is the amino acid sequence of the lytic peptide group. SEQ ID NO:50 is the amino acid sequence of the lytic peptide group. SEQ ID NO:51 is the amino acid sequence of the masked peptide group. SEQ ID NO:52 is the amino acid sequence of delivery agent 51-11. SEQ ID NO:53 is the amino acid sequence of delivery agent 51-12. SEQ ID NO:54 is the amino acid sequence of delivery agent 51-13. SEQ ID NO:55 is the amino acid sequence of delivery agent 51-14. SEQ ID NO:56 is the amino acid sequence of delivery agent 51-15. SEQ ID NO:57 is the amino acid sequence of delivery agent 51-16. SEQ ID NO:58 is the amino acid sequence of delivery agent 51-17. SEQ ID NO:59 is the amino acid sequence of delivery agent 51-18. SEQ ID NO:60 is the amino acid sequence of the lytic peptide group. SEQ ID NO:61 is the amino acid sequence of the lytic peptide group. SEQ ID NO:62 is the amino acid sequence of the lytic peptide group. SEQ ID NO:63 is the amino acid sequence of the lytic peptide group. SEQ ID NO:64 is the amino acid sequence of the lytic peptide group. SEQ ID NO:65 is the amino acid sequence of the lytic peptide group. SEQ ID NO:66 is the amino acid sequence of the lytic peptide group. SEQ ID NO:67 is the amino acid sequence of the lytic peptide group. SEQ ID NO:68 is the amino acid sequence of the lytic peptide group. SEQ ID NO:69 is the amino acid sequence of delivery agent 51-24. SEQ ID NO:70 is the amino acid sequence of delivery agent 51-25. SEQ ID NO:71 is the amino acid sequence of delivery agent 51-26. SEQ ID NO:72 is the amino acid sequence of delivery agent 51-27. SEQ ID NO:73 is the amino acid sequence of delivery agent 51-28. SEQ ID NO:74 is the amino acid sequence of delivery agent 51-29. SEQ ID NO:75 is the amino acid sequence of delivery agent 51-30. SEQ ID NO:76 is the amino acid sequence of delivery agent 51-31. SEQ ID NO:77 is the amino acid sequence of delivery agent 51-23. SEQ ID NO:78 is the amino acid sequence of the lytic peptide group. SEQ ID NO:79 is the amino acid sequence of delivery agent 56-5. SEQ ID NO:80 is the amino acid sequence of the lytic peptide group. SEQ ID NO:81 is the amino acid sequence of the masked peptide group. SEQ ID NO:82 is the amino acid sequence of delivery agent 60-16. SEQ ID NO:83 is the amino acid sequence of delivery agent 60-17. SEQ ID NO:84 is the amino acid sequence of the lytic peptide group. SEQ ID NO:85 is the amino acid sequence of the lytic peptide group. SEQ ID NO:86 is the amino acid sequence of the lytic peptide group. SEQ ID NO:87 is the amino acid sequence of delivery agent 88-8. SEQ ID NO:88 is the amino acid sequence of delivery agent 88-9. SEQ ID NO:89 is the amino acid sequence of delivery agent 88-10. SEQ ID NO:90 is the amino acid sequence of delivery agent 88-11. SEQ ID NO:91 is the amino acid sequence of delivery agent 88-12. SEQ ID NO:92 is the amino acid sequence of the lytic peptide group. SEQ ID NO:93 is the amino acid sequence of the lytic peptide group. SEQ ID NO:94 is the amino acid sequence of the lytic peptide group. SEQ ID NO:95 is the amino acid sequence of the lytic peptide group. SEQ ID NO:96 is the amino acid sequence of the lytic peptide group. SEQ ID NO:97 is the amino acid sequence of the lytic peptide group. SEQ ID NO:98 is the amino acid sequence of the masked peptide group. SEQ ID NO:99 is the amino acid sequence of the masked peptide group. SEQ ID NO:100 is the amino acid sequence of the masked peptide group. SEQ ID NO:101 is the amino acid sequence of the masked peptide group. SEQ ID NO:102 is the amino acid sequence of delivery agent 109-7. SEQ ID NO:103 is the amino acid sequence of delivery agent 114-1. SEQ ID NO:104 is the amino acid sequence of delivery agent 114-3. SEQ ID NO:105 is the amino acid sequence of delivery agent 114-12. SEQ ID NO:106 is the amino acid sequence of delivery agent 114-30. SEQ ID NO:107 is the amino acid sequence of delivery agent 114-33. SEQ ID NO:108 is the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portions of the two anchor groups, and the C-terminal portion of the delivery agent. SEQ ID NO:109 is the amino acid sequence of the lytic peptide group. SEQ ID NO:110 is the amino acid sequence of the lytic peptide group. SEQ ID NO:111 is the amino acid sequence of the masked peptide group. SEQ ID NO:112 is the amino acid sequence of the masked peptide group. SEQ ID NO:113 is the amino acid sequence of delivery agent 114-6. SEQ ID NO:114 is the amino acid sequence of delivery agent 114-9. SEQ ID NO:115 is the amino acid sequence of the lytic peptide group. SEQ ID NO:116 is the amino acid sequence of the lytic peptide group. SEQ ID NO:117 is the amino acid sequence of the lytic peptide group. SEQ ID NO:118 is the amino acid sequence of delivery agent 97-1. SEQ ID NO:119 is the amino acid sequence of delivery agent 97-2. SEQ ID NO:120 is the amino acid sequence of delivery agent 97-3. SEQ ID NO:121 is the amino acid sequence of delivery agent 97-5. SEQ ID NO:122 is the amino acid sequence of delivery agent 97-7. SEQ ID NO:123 is the amino acid sequence of delivery agent 91-6. SEQ ID NO:124 is the amino acid sequence of delivery agent 114-18. SEQ ID NO:125 is the amino acid sequence of delivery agent 114-21. SEQ ID NO:126 is the amino acid sequence of delivery agent 114-24. SEQ ID NO:127 is the amino acid sequence of delivery agent 114-27. SEQ ID NO:128 is the amino acid sequence of delivery agent 21-11. SEQ ID NO:129 is the amino acid sequence of delivery agents 19-1, 36-47, 26-2, 21-1, 23-27, and 26-2B. SEQ ID NO:130 is the amino acid sequence of delivery agent 36-48. SEQ ID NO:131 is the amino acid sequence of delivery agent 96-1. SEQ ID NO:132 is the amino acid sequence of delivery agent 96-4. SEQ ID NO:133 is the amino acid sequence of delivery agent 96-5. SEQ ID NO:134 is the amino acid sequence of the masked peptide group. SEQ ID NO:135 is the amino acid sequence of delivery agent 26-17. SEQ ID NO:136 is the amino acid sequence of the masked peptide group. SEQ ID NO:137 is the amino acid sequence of the masked peptide group. SEQ ID NO:138 is the amino acid sequence of delivery agent 19-3. SEQ ID NO:139 is the amino acid sequence of delivery agent 19-5. SEQ ID NO:140 is the amino acid sequence of the masked peptide group. SEQ ID NO:141 is the amino acid sequence of the masked peptide group. SEQ ID NO:142 is the amino acid sequence of delivery agent 23-12. SEQ ID NO:143 is the amino acid sequence of delivery agent 23-13. SEQ ID NO:144 is the amino acid sequence of delivery agent 23-14. SEQ ID NO:145 is the amino acid sequence of the masked peptide group. SEQ ID NO:146 is the amino acid sequence of delivery agent 21-13. SEQ ID NO:147 is the amino acid sequence of delivery agents 23-15 and 21-12. SEQ ID NO:148 is the amino acid sequence of the masked peptide group. SEQ ID NO:149 is the amino acid sequence of delivery agent 21-8. SEQ ID NO:150 is the amino acid sequence of the lytic peptide group. SEQ ID NO:151 is the amino acid sequence of the lytic peptide group. SEQ ID NO:152 is the amino acid sequence of the lytic peptide group. SEQ ID NO:153 is the amino acid sequence of the lytic peptide group and / or the mask peptide group. SEQ ID NO:154 is the amino acid sequence of the masked peptide group. SEQ ID NO:155 is the amino acid sequence of the masked peptide group. SEQ ID NO:156 is the amino acid sequence of the masked peptide group. SEQ ID NO:157 is the amino acid sequence of the masked peptide group. SEQ ID NO:158 is the amino acid sequence of the masked peptide group. SEQ ID NO:159 is the amino acid sequence of delivery agent 40-31. SEQ ID NO:160 is the amino acid sequence of delivery agent 40-32. SEQ ID NO:161 is the amino acid sequence of delivery agent 40-33. SEQ ID NO:162 is the amino acid sequence of delivery agent 40-34. SEQ ID NO:163 is the amino acid sequence of delivery agent 40-35. SEQ ID NO:164 is the amino acid sequence of delivery agent 40-36. SEQ ID NO:165 is the amino acid sequence of delivery agent 40-37. SEQ ID NO:166 is the amino acid sequence of delivery agent 40-38. SEQ ID NO:167 is the amino acid sequence of delivery agent 40-39. SEQ ID NO:168 is the amino acid sequence of delivery agent 40-40. SEQ ID NO:169 is the amino acid sequence of delivery agent 40-41. SEQ ID NO:170 is the amino acid sequence of delivery agent 40-42. SEQ ID NO:171 is the amino acid sequence of delivery agent 40-43. SEQ ID NO:172 is the amino acid sequence of the masked peptide group. SEQ ID NO:173 is the amino acid sequence of the masked peptide group. SEQ ID NO:174 is the amino acid sequence of the masked peptide group. SEQ ID NO:175 is the amino acid sequence of the masked peptide group. SEQ ID NO:176 is the amino acid sequence of the masked peptide group. SEQ ID NO:177 is the amino acid sequence of the masked peptide group. SEQ ID NO:178 is the amino acid sequence of the masked peptide group. SEQ ID NO:179 is the amino acid sequence of the masked peptide group. SEQ ID NO:180 is the amino acid sequence of the masked peptide group. SEQ ID NO:181 is the amino acid sequence of the masked peptide group. SEQ ID NO:182 is the amino acid sequence of delivery agent 53-6. SEQ ID NO:183 is the amino acid sequence of delivery agent 53-7. SEQ ID NO:184 is the amino acid sequence of delivery agent 53-8. SEQ ID NO:185 is the amino acid sequence of delivery agent 53-9. SEQ ID NO:186 is the amino acid sequence of delivery agent 53-10. SEQ ID NO:187 is the amino acid sequence of delivery agent 53-11. SEQ ID NO:188 is the amino acid sequence of delivery agent 53-12. SEQ ID NO:189 is the amino acid sequence of delivery agent 53-13. SEQ ID NO:190 is the amino acid sequence of delivery agent 53-14. SEQ ID NO:191 is the amino acid sequence of delivery agent 53-15. SEQ ID NO:192 is the amino acid sequence of delivery agent 96-10. SEQ ID NO:193 is the amino acid sequence of delivery agent 21-6 and / or 23-5. SEQ ID NO:194 is the amino acid sequence of delivery agent 26-13. SEQ ID NO:195 is the amino acid sequence of delivery agent 21-7b. SEQ ID NO:196 is the amino acid sequence of delivery agent 109-8. SEQ ID NO:197 is the amino acid sequence of delivery agent 78-2. SEQ ID NO:198 is the amino acid sequence of delivery agent 86-5. SEQ ID NO:199 is the amino acid sequence of delivery agent 50-23. SEQ ID NO:200 is the amino acid sequence of delivery agent 50-24. SEQ ID NO:201 is the amino acid sequence of delivery agent 50-25. SEQ ID NO:202 is the amino acid sequence of delivery agent 50-26. SEQ ID NO:203 is the amino acid sequence of the masked peptide group. SEQ ID NO:204 is the amino acid sequence of the masked peptide group. SEQ ID NO:205 is the amino acid sequence of the masked peptide group. SEQ ID NO:206 is the amino acid sequence of the masked peptide group. SEQ ID NO:207 is the amino acid sequence of the masked peptide group. SEQ ID NO:208 is the amino acid sequence of delivery agent 30-3. SEQ ID NO:209 is the amino acid sequence of delivery agent 30-5. SEQ ID NO:210 is the amino acid sequence of delivery agent 30-7. SEQ ID NO:211 is the amino acid sequence of delivery agent 30-9. SEQ ID NO:212 is the amino acid sequence of delivery agent 30-11. SEQ ID NO:213 is the amino acid sequence of delivery agent 30-15. SEQ ID NO:214 is the amino acid sequence of delivery agent 30-17. SEQ ID NO:215 is the amino acid sequence of delivery agent 19-17C. SEQ ID NO:216 is the amino acid sequence of delivery agent 21-5. SEQ ID NO:217 is the amino acid sequence of delivery agent 22-11. SEQ ID NO:218 is the amino acid sequence of delivery agent 23-1. SEQ ID NO:219 is the amino acid sequence of delivery agent 23-2. SEQ ID NO:220 is the amino acid sequence of delivery agent 23-3. SEQ ID NO:221 is the amino acid sequence of delivery agent 22-1. SEQ ID NO:222 is the amino acid sequence of delivery agent 22-12. SEQ ID NO:223 is the amino acid sequence of delivery agent 34-32. SEQ ID NO:224 is the amino acid sequence of delivery agent 34-33. SEQ ID NO:225 is the amino acid sequence of delivery agent 34-34. SEQ ID NO:226 is the amino acid sequence of delivery agent 109-2. SEQ ID NO:227 is the amino acid sequence of the cleavable linker. SEQ ID NO:228 is the amino acid sequence of delivery agent 33-3. SEQ ID NO:229 is the nucleic acid sequence of Morpholino 1. SEQ ID NO:230 is the nucleic acid sequence of Morpholino 2. SEQ ID NO:231 is the amino acid sequence of delivery agent 96-6. SEQ ID NO:232 is the amino acid sequence of delivery agent 16-6. SEQ ID NO:233 is the amino acid sequence of the cleavable linker. SEQ ID NO:234 ​​is the amino acid sequence of the cleavable linker. SEQ ID NO:235 is the amino acid sequence of delivery agent 5. SEQ ID NO:236 is the amino acid sequence of delivery agent 7. SEQ ID NO:238 is the amino acid sequence of delivery agent 8. SEQ ID NO:238 is the nucleic acid sequence of a forward DNA primer spanning dystrophin exon 21 and exon 24. SEQ ID NO:239 is the nucleic acid sequence of a reverse DNA primer spanning dystrophin exon 21 and exon 24. SEQ ID NO:240 is the nucleic acid sequence of the morpholino. SEQ ID NO:241 is the nucleic acid sequence of the morpholino. SEQ ID NO:242 is the nucleic acid sequence of the morpholino. SEQ ID NO:243 is the nucleic acid sequence of the morpholino. SEQ ID NO:244 is the nucleic acid sequence of the morpholino. SEQ ID NO:245 is the branched amino acid sequence of delivery agent 22-1. SEQ ID NO:246 is the amino acid sequence of delivery agents 30-22, 21-4, 40-46, 42-39, 23-4, and 23-26. SEQ ID NO:247 is the amino acid sequence of delivery agent 62-9. SEQ ID NO:248 is the amino acid sequence of delivery agent 60-4. SEQ ID NO:249 is the amino acid sequence of delivery agents 96-7, 86-4, and 102-3. SEQ ID NO:250 is the amino acid sequence of delivery agent 26-13A. SEQ ID NO:251 is the amino acid sequence of delivery agent 26-15A. SEQ ID NO:252 is the amino acid sequence of delivery agent 26-7a'. SEQ ID NO:253 is the amino acid sequence of delivery agent 26-7b. SEQ ID NO:254 is the amino acid sequence of delivery agent 26-7b'. SEQ ID NO:255 is the amino acid sequence of delivery agent 26-14B. SEQ ID NO:256 is the amino acid sequence of delivery agent 26-14D. SEQ ID NO:257 is the amino acid sequence of delivery agent 26-2A. SEQ ID NO:258 is the amino acid sequence of delivery agent 26-11. SEQ ID NO:259 is the amino acid sequence of delivery agent 40-23B. SEQ ID NO:260 is the amino acid sequence of delivery agent 21-4b. SEQ ID NO:261 is the amino acid sequence of delivery agent 21-4b'. SEQ ID NO:262 is the amino acid sequence of delivery agent 21-6b. SEQ ID NO:263 is the amino acid sequence of delivery agent 60-1b. SEQ ID NO:264 is the amino acid sequence of delivery agent 78-1. SEQ ID NO:265 is the amino acid sequence of delivery agent 19-17A. SEQ ID NO:266 is the amino acid sequence of a branch of delivery agent 22-12. SEQ ID NO:267 is the amino acid sequence of delivery agent 33-26a. SEQ ID NO:268 is the amino acid sequence of delivery agent 33-26b. SEQ ID NO:269 is the amino acid sequence of delivery agent 33-26c. SEQ ID NO:270 is the amino acid sequence of delivery agent 6. SEQ ID NO:271 is the amino acid sequence of delivery agent 40-23A. SEQ ID NO:272 is the amino acid sequence of delivery agents 215-2 DBCO, 215-2 FA, 215-2 PEG12-BCN, and 215-2 Ab. SEQ ID NO:273 is the amino acid sequence of delivery agents 216-A DBCO, 216-A FA, 216-1 PEG12-BCN, and 216-1 Ab. SEQ ID NO:274 is the amino acid sequence of delivery agents 216-B DBCO, 216-B FA, and 217-2. SEQ ID NO:275 is the amino acid sequence of delivery agent 218-2. SEQ ID NO:276 is the amino acid sequence of delivery agent 219-1. SEQ ID NO:277 is the amino acid sequence of delivery agent 219-2. SEQ ID NO:278 is the amino acid sequence of delivery agent 224-2 without the masked peptide group. SEQ ID NO:279 is the amino acid sequence of delivery agent 225-2 without the masked peptide group. SEQ ID NO:280 is the amino acid sequence of delivery agent 223-2. DETAILED DESCRIPTION OF THE INVENTION

[0016] (I. Terminology and Overview) The following explanations of terms are provided to better explain the present disclosure and to guide those skilled in the art in practicing the disclosure. As used herein, "comprising" means "including," and the singular forms "a" or "an" or "the" include the plural forms unless the context clearly dictates otherwise. The term "or" refers to a single element of listed alternative elements or a combination of two or more elements unless the context clearly dictates otherwise.

[0017] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Methods and materials similar or equivalent to those described herein can be used to practice or test this disclosure, and suitable methods and materials are described below. Materials, methods, and examples are for illustrative purposes only and are not limiting unless otherwise specified. Other features of the present disclosure will be apparent from the following detailed description and claims.

[0018] Where a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, and between any other stated or intervening value in that presented range, to the nearest tenth of the lower limit, is encompassed within the scope of the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the scope of the disclosure, subject to any expressly excluded limit in the presented range. When a presented range includes one or both limits, ranges excluding either or both of the included limits are also included in the disclosure.

[0019] Unless otherwise indicated, all numerical values ​​expressing amounts, molecular weights, percentages of ingredients, temperatures, times, and the like used in the specification or claims are understood to be modified by the term "about." Thus, unless expressly or implicitly indicated otherwise, the numerical parameters described are approximations that may depend on the desired properties sought and / or the limits of detection under standard test conditions / methods. When directly and explicitly distinguishing an embodiment from the prior art being discussed, the numerical values ​​of the embodiments are not approximations unless the word "about" is present. Furthermore, not all alternatives described herein are equivalent. Also, within any numerical range, the specifically recited endpoints are considered part of the range.

[0020] The compounds disclosed herein may contain one or more asymmetric elements, such as asymmetric centers, chiral axes, or asymmetric carbon atoms, and therefore, chemical conjugates may exist in different stereoisomeric forms. These compounds may be, for example, racemic or optically active. In the case of compounds with two or more asymmetric elements, these compounds may also be mixtures of diastereomers. In the case of compounds with asymmetric centers, all optical isomers in pure form and mixtures thereof are included. In these situations, single enantiomers, i.e., optically active forms, can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or resolution of the racemates. Resolution of the racemates can also be achieved by conventional methods, such as crystallization in the presence of a resolving agent or chromatography using, for example, a chiral HPLC column. All forms are contemplated herein, regardless of the method used to obtain them.

[0021] The definitions and rules of stereochemistry used herein generally follow those in S.P. Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994), John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule's chiral centers. The prefixes d and l or (+) and (-) are used to specify the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory.

[0022] All forms of the probe (eg, solvates, optical isomers, enantiomeric forms, polymorphs, free compounds and salts) may be used alone or in combination.

[0023] To facilitate review of the various aspects of the disclosure, the following explanations of certain terms are provided. Certain functional group terms include a "-" symbol at the beginning of the functional group formula. This symbol is not part of the functional group, but indicates how the functional group is connected to the formulas described herein. For example, the formula "-OC(O)R b The " functional group is attached to an atom of the functionalized compound by the oxygen atom of the functional group next to the "-" symbol.

[0024] Administer, administering, administration: As used herein, administering a delivery agent and / or therapeutic agent (e.g., a delivery agent described herein and a therapeutic agent such as a morpholino or other therapeutic agent described herein) to a subject means applying, giving, or carrying the agent to a subject by any effective route. Administration can be achieved by a variety of routes, including, for example, intravenous, intratumoral, topical, oral, subcutaneous, transdermal, intrathecal, intramuscular, intraperitoneal, intranasal, and similar routes, or combinations thereof. Exemplary routes of administration are described herein.

[0025] Aliphatic: Substantially hydrocarbon-based compounds, including alkanes, alkenes, alkynes, and cyclic versions thereof, further including straight-chain and branched-chain arrangements, and all stereoisomers and positional isomers, or their free radicals (e.g., CH for the hexane free radical) 13). Unless explicitly stated otherwise, aliphatic groups contain 1 to 25 carbon atoms, e.g., 1 to 15, 1 to 10, 1 to 6, or 1 to 4 carbon atoms. The term "lower aliphatic" refers to an aliphatic group containing 1 to 10 carbon atoms. The aliphatic chain can be substituted or unsubstituted. Unless explicitly stated as "unsubstituted aliphatic," an aliphatic group can be either unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (up to two substituents for each methylene carbon in the aliphatic chain, or up to one substituent for each carbon of a C=C double bond in the aliphatic chain, or up to one substituent for the carbon of the terminal methine group). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamido, sulfhydryl, thioalkoxy, or other functional groups. In some examples, the substituted aliphatic group comprises at least one sp bond attached through a double bond. 3 Hybridized carbon or two sp 2 It contains a hybridized carbon or at least two sp hybridized carbons joined by a triple bond.

[0026] Alkyl: A hydrocarbon group having a saturated carbon chain. The chain can be cyclic (such as cycloalkyl), branched, or unbranched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like. The term lower alkyl means that the chain contains 1 to 10 carbon atoms. The terms alkenyl and alkynyl refer to hydrocarbon groups having a carbon chain containing one or more double or triple bonds, respectively. In some examples, substituted alkyl groups have at least one sp 3 Contains hybrid carbon.

[0027] Aliphatic Aryl: An aryl group that is or can be conjugated to a compound disclosed herein, where the aryl group is or becomes conjugated via an aliphatic group.

[0028] Aliphatic heteroaryl: A heteroaryl group that can be conjugated or attached to the compounds disclosed herein, where the heteroaryl group becomes conjugated or attached via an aliphatic group.

[0029] Amine: -NR b R c , R b and R c is independently selected from hydrogen, aliphatic, aryl, heteroaliphatic, aliphatic aryl, heteroaryl, aliphatic heteroaryl, heteroaliphatic aryl, heteroaliphatic heteroaryl, and any combination thereof.

[0030] Amino acid: an organic acid that contains both a basic amino group (e.g., -NH2) and an acidic carboxyl group (e.g., -COOH). The amino acids that are the building blocks of proteins are α-amino acids, in which the -NH2 group is attached to the carbon atom adjacent to the -COOH group.

[0031] Antibody: A polypeptide ligand (such as an immunoglobulin, an antigen-binding fragment, or a derivative thereof) with at least one variable region that recognizes and binds to (e.g., specifically recognizes and binds to) an epitope of an antigen. As used herein, the term "antibody" is used in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), targeted antibodies, and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity. Antibodies specifically react with antigens in some demonstrable manner. Therapeutic antibodies recognize and bind to antigen receptors and activate or inhibit a series of biological processes, such as blocking the growth of cancer cells and / or triggering an immune system response.

[0032] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and antigen-binding fragments thereof that retain binding affinity to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, dsFv, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and ds-scFv), and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of whole antibodies or antigen-binding fragments synthesized de novo using recombinant DNA methodology (see, e.g., Kontermann and Duebel (eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer-Verlag, 2010).

[0033] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). Antibodies also include defucosylated forms of the disclosed antibodies.

[0034] An antibody may have two or more binding sites. If there are multiple binding sites, the binding sites may be identical to one another or may be different. For example, a natural immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

[0035] Mammalian immunoglobulin molecules are composed of heavy (H) and light (L) chains, each of which has a variable region, called a variable heavy chain (V H ) region and the variable light chain (V L ) region. V H Area and V LThe α and β regions together are responsible for binding to the antigen recognized by the antibody. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the primary antibody produced by birds and reptiles and shares some functional similarities with mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0036] The variable region of an antibody contains "framework" regions and hypervariable regions called "complementarity-determining regions" or "CDRs." The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, "Kabat" numbering scheme), Chothia et al. (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989), Al-Lazikani et al. (JMB 273, 927-948, 1997; see "Chothia" numbering scheme), and the ImMunoGeneTics (IMGT) database (Lefranc, Nucleic Acids Res 29:207-9, 2001; see "IMGT" numbering scheme). The Kabat and IMGT databases are maintained online.

[0037] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell transfected with a single antibody coding sequence. Monoclonal antibodies include humanized monoclonal antibodies.

[0038] Cancer: Cancer is characterized by abnormal or uncontrolled cell growth (malignant cells). Other features often associated with malignant tumors include metastasis, interference with the normal function of nearby cells, release of abnormal levels of cytokines or other secretory products, suppression or exacerbation of inflammatory or immune responses, and invasion of surrounding or distant tissues or organs, such as lymph nodes. "Metastatic disease" refers to cancer cells leaving the original cancer site and traveling to other parts of the body, such as via the bloodstream or lymphatic system.

[0039] A subject's "cancer burden" can be measured as the number, volume, and / or weight of one or more tumors. Tumors that do not metastasize are called "benign." Tumors that invade and / or metastasize to surrounding tissues are cancerous (and called "malignant"). In some examples, the cancer is a blood cancer, such as leukemia (including acute leukemia (such as 11q23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia) or chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia)), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, etc. In specific, non-limiting examples, the lymphoid malignancy can be adult T-cell leukemia, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, Hodgkin's lymphoma, or diffuse large B-cell lymphoma.

[0040] Examples of solid cancers, such as sarcomas and cell tumors, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, bile duct carcinoma (especially intrahepatic cholangiocarcinoma), osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer (e.g., colorectal cancer), lymphoid malignancies, pancreatic cancer, breast cancer (including basal breast cancer, ocular melanoma, ductal and lobular breast cancer, and triple-negative breast cancer), uterine / endometrial cancer, neuroendocrine cancer, lung cancer (including non-small cell lung cancer), ovarian cancer, prostate cancer, hepatocellular carcinoma, angiosarcoma, and squamous cell carcinoma. Cancers include epithelial carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, gallbladder carcinoma, esophageal carcinoma, kidney carcinoma (such as renal cell carcinoma), melanoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, brain cancer, pleural carcinoma, bronchial carcinoma, testicular cancer, seminoma, bladder carcinoma, and central nervous system cancer (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoma can be a solid cancer in some cases. Particularly, cancers include, but are not limited to, colon cancer, kidney cancer, and melanoma.

[0041] Cathepsin B: A lysosomal cysteine ​​protease with endopeptidase activity (cleaving internal bonds) favored at neutral / alkaline pH and exopeptidase (carboxydipeptidase) activity favored at acidic pH, which may be involved in protein metabolism. Cathepsin B can hydrolyze proteins with broad specificity. However, its cleavage specificity favors, but is not limited to, basic amino acids at the P1 position and hydrophobic or arginine residues at the P2 position. Mature cathepsin B consists of a 25-26 kDa heavy chain and a 5 kDa light chain linked by a disulfide dimer.

[0042] Chemical linker: A molecule or atomic group located between two moieties. For example, the delivery agents described herein may include a chemical linker between a component of the delivery agent (e.g., an anchor group, a targeting group, a C-terminal group, etc.) and a therapeutic agent. Typically, the chemical linker is bifunctional, and thus the chemical linker has a functional group at each end, which is used to attach the linker to the delivery agent and the therapeutic agent. The two functional groups can be the same (called a homobifunctional linker) or different (called a heterobifunctional linker).

[0043] Chemotherapeutic agent: A chemical or biological agent that is therapeutically effective in treating diseases characterized by abnormal cell proliferation. For example, a chemotherapeutic agent may be effective in treating solid cancers such as sarcoma, carcinoma, lymphoma, colon cancer, or skin cancer. Specific examples of chemotherapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In one embodiment, the chemotherapeutic agent is a radioactive compound. Other chemotherapeutic agents that may be used are provided in Sausville and Longo, "Principles of Cancer Treatment," Chapter 69 of Harrison's Principles of Internal Medicine (20th ed.), McGraw-Hill, 2018; Niederhuber et al., "Cancer Pharmacology," Chapter 25 of Abeloff's Clinical Oncology (6th ed.), Elsevier, 2019; Gullatte et al., "Clinical Guide to Antineoplastic Therapy: A Chemotherapy Handbook (4th ed.)," Oncology Nursing Society, 2020; Chabner and Longo, "Cancer Chemotherapy, Immunotherapy and Biotherapy: Principles and Practice (6th ed.)," Lippincott Williams & Wilkins, 2018; and Skeel, "Handbook of Cancer Chemotherapy (9th ed.)," Lippincott Williams & Wilkins, 2016. Combination chemotherapy is the administration of two or more chemotherapy agents to treat cancer.

[0044] Cleavable linker: A chemical group and / or peptide sequence located between two moieties. For example, the delivery agents described herein may include a cleavable linker between the lytic peptide group and the masking peptide group. In some embodiments of the present disclosure, the cleavable linker comprises a peptide sequence that can be cleaved to separate the lytic peptide group from the masking peptide group. In some embodiments of the present disclosure, such a cleavable linker is cleaved by an enzyme. Suitable cleavable linkers are described herein.

[0045] Combination, Combination Therapy: A therapy combining two or more therapeutic components, such as a delivery agent disclosed herein and one or more therapeutic agents, for the treatment of a condition or disease, such as a genetic disease, a rare disease, cancer, an immune disease, or an infectious disease. As used herein, the therapeutic components provided in combination (such as a disclosed delivery agent, one or more therapeutic agents, or a conjugate formed from these two therapeutic components) can be contacted with a cell and / or administered to a subject substantially simultaneously or sequentially in any order, at two or more different times, or a combination thereof. The one or more therapeutic agents of the combination can characteristically target multiple biological pathways in a synergistic or additive manner to treat a condition or disease. The combination therapies disclosed herein can be useful for treating one type of condition or disease, such as cancer (e.g., melanoma), or two or more different conditions or diseases, such as two or more types of cancer (e.g., kidney cancer and colon cancer).

[0046] Conservative variant: A "conservative" amino acid substitution is one that does not substantially affect or reduce the affinity or activity of a protein, such as an antibody or peptide (such as a lytic peptide group, a masking peptide group, or a cleavable linker), as disclosed herein. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the variant retains activity. A non-conservative substitution is one that reduces the activity of a protein.

[0047] Conservative amino acid substitution tables exist that provide functionally similar amino acids. The following six groups are examples of amino acids that are considered conservative substitutions for one another: (1) Alanine (A), serine (S), threonine (T), (2) Aspartic acid (D), glutamic acid (E), (3) Asparagine (N), Glutamine (Q), (4) Arginine (R), Lysine (K), (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), (6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0048] Contacting: Placement in direct physical association, including both solid and liquid forms. In one example, contacting involves association of a therapeutic component (such as a delivery agent and / or a therapeutic agent disclosed herein) with one or more cells (such as cells of a subject or cells in culture) in a liquid medium. Contacting can occur in vitro with isolated cells or tissues, or in vivo by administration to a subject.

[0049] Derivative: A compound derived from a similar compound, or a compound that could be considered to arise from another compound, for example, when one atom is replaced by another atom or group of atoms.

[0050] Directly or indirectly conjugated: As used herein, the phrase "directly conjugated" means that the referenced groups or compounds are chemically conjugated to each other with nothing intervening between them. As used herein, the phrase "indirectly conjugated" means that the referenced groups are chemically conjugated to each other through another moiety (e.g., a functional group, a linker, or a combination thereof).

[0051] Effective amount, therapeutically effective amount: The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent (such as one or more delivery agents provided herein, alone, in combination, or potentially in combination with other therapeutic agents) sufficient to induce a desired biological result. The result may be the introduction of a therapeutic agent into the cytosol of a cell and / or the amelioration or alleviation of the signs, symptoms, or causes of a disease (such as a reduction in the cancer burden in a subject), or other desired modification of a biological system. The effective amount may vary depending on the condition being treated, the stage of progression of the condition, and the type and concentration of the formulation applied. In some embodiments of the present disclosure, an effective amount of a combination of therapeutic agents disclosed herein is an amount sufficient to elicit a detectable therapeutic response when administered to a subject. Such a response may include, for example, a reduction in the burden of a genetic disease, a rare disease, cancer, an immune disease, or an infectious disease. In any case, the appropriate amount will be readily apparent or can be determined by routine experimentation, such as administering a combination of therapeutic agents (such as a combination therapy for the treatment of a genetic disease, a rare disease, cancer, an immune disease, or an infectious disease) and observing the subject's response.

[0052] In one embodiment, a therapeutically effective amount is the amount necessary to eliminate a tumor, reduce the size of a tumor, or prevent tumor metastasis, e.g., by reducing the size and / or volume of a tumor by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% and / or reducing the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% compared to the size / volume / number before treatment.

[0053] Fluorescent dye, fluorescent pigment, or fluorophore: A molecular component that causes a molecule to be fluorescent. This component can be a functional group of a molecule that absorbs energy at a specific wavelength and re-emits the energy at a different (but equally specific) wavelength. In some aspects of the present disclosure, a fluorophore can fluoresce when the compound (or a sample or composition containing the compound) is exposed to an excitation source or after being cleaved from an embodiment of the compound. In some aspects of the present disclosure, the molecular component that causes a molecule to be fluorescent is a dye. In some such aspects, the amount and wavelength of energy emitted depends on both the dye and the chemical environment of the dye. Many dyes are available, including Fmoc-Lys(Mca)-OH, Fmoc-Asp(EDANS)-OH, Fmoc-Glu(EDANS)-OH, Fmoc-Lys(Dabcyl)-OH, Fmoc-Lys(Dnp)-OH, p-cyanophenylalanine, 5-cyanotryptophan, 4-cyanotryptophan, 2-cyanophenylalanine, 7-azatryptophan, 7-cyanotryptophan, β-(1-azulenyl)-L-alanine, acridon-2-ylalanine, L-leucine 7-amido-4-methylcoumarin, trans-4-hydroxy-L-proline 7-amido-4-methylcoumarin, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 633, Alexa 647, Atto 465, Atto 488, and Atto 532, Atto 550, Atto 565, Atto 647N, Atto 655, BODIPY-TMR, Cy3, Sulfo-Cy3, Cy3B, Sulfo-Cy5, Dyomics 654, Oregon Green 488, Oregon Green 514, Sulforhodamine B, Texas Red, Tetramethylrhodamine, Fluorescein isothiocyanate (FITC), R-Phycoerythrin (PE), PE-Texas Red tandem, PE-Cy5 tandem, Propidium iodem, EGFP, EYGP, ECF, DsRed, Allophycocyanin (APC), PerCp, SYTOX Green, Coumarin, AlexaFluor (350, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, 750), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Hoechst 33342, DAPI, Hoechst 33258, SYTOX Blue, chromomycin A3, mithramycin, YOYO-1, SYTOX Orange, ethidium bromide, 7-AAD, acridine orange, TOTO-1, TO-PRO-1, thiazole orange, TOTO-3, TO-PRO-3, thiazole orange, propidium iodide (PI), LDS 751, Indo-1, Fluo-3, DCFH, DHR, SNARF, Y66F, Y66H, EBFP, GFPuv, ECFP, GFP, AmCyanl, Y77W, S65A, S65C, S65L, S65T, ZsGreenl, ZsYellowl, DsRed2, DsRed monomer, AsRed2, mRFP1, HcRedl, monochlorobimane, calcein, DyLight Fluor, cyanine, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Lucifer Yellow, NBD, PE-Cy5 conjugate, PE-Cy7 conjugate, APC-Cy7 conjugate, Red 613, fluorescein, FluorX, BODIDY-FL, TRITC, X-rhodamine, Lissamine rhodamine B, TruRed, and derivatives thereof.

[0054] Haloaliphatic: An aliphatic group in which one or more hydrogen atoms, for example, 1 to 10 hydrogen atoms, are independently replaced with a halogen atom, such as fluorine, bromine, chlorine, or iodine.

[0055] Haloaliphatic Aryl: An aryl group that is or can be conjugated to the compounds disclosed herein, where the aryl group is or becomes conjugated via a haloaliphatic group.

[0056] Haloaliphatic heteroaryl: A heteroaryl group that is or can be conjugated to the compounds disclosed herein, where the heteroaryl group is or becomes conjugated via a haloaliphatic group.

[0057] Haloheteroaliphatic: an aliphatic group in which one or more hydrogen atoms, such as 1 to 10 hydrogen atoms, are independently replaced with a halogen atom, such as fluorine, bromine, chlorine, or iodine.

[0058] Heteroaliphatic: An aliphatic group comprising at least 1 to 20 heteroatoms, such as 1 to 15 heteroatoms, or 1 to 5 heteroatoms, which may be selected from, but are not limited to, oxygen, nitrogen, sulfur, selenium, phosphorus, and oxidized forms thereof within the group. Exemplary heteroaliphatic groups include, but are not limited to, aliphatic groups containing any of the following: ether, thioether, ester, amine, carboxy, carbonyl, or amide.

[0059] Heteroaliphatic Aryl: An aryl group that is or can be conjugated to a compound disclosed herein, where the aryl group is or becomes conjugated via a heteroaliphatic group.

[0060] Heteroaryl: An aryl group containing at least one to six heteroatoms, e.g., one to four heteroatoms, within the ring, which may be selected from, but are not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorus, and their oxidized forms. Such heteroaryl groups may have a single ring or multiple fused rings, which may or may not be aromatic and / or may contain heteroatoms, provided that the point of attachment is through an atom of the aromatic heteroaryl group.

[0061] Heteroatom: An atom other than carbon or hydrogen, such as, but not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorus. In particularly disclosed embodiments, heteroatom does not include halogen atoms, such as when valence constraints do not permit.

[0062] Immune disease or condition: A disorder or condition, such as an autoimmune disorder or disease, in which the immune system produces an immune response (e.g., a B cell or T cell response) to an endogenous antigen, resulting in tissue damage. Damage can be localized to a specific organ, such as thyroiditis, or can affect specific tissues in different locations, such as Goodpasture's syndrome, or can be systemic, such as systemic lupus erythematosus.

[0063] In some instances, the autoimmune disease is systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, type 1 diabetes, Wegener's granulomatosis, inflammatory bowel disease, polymyositis, dermatomyositis, polyendocrine deficiency syndrome, Schmidt's syndrome, autoimmune uveitis, achalasia, autoimmune encephalitis, Addison's disease, adrenalitis, Graves' disease, eosinophilic granulomatosis with polyangiitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, multiple sclerosis (including Barrow's disease), subacute cutaneous lupus erythematosus, Todes, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, autoimmune vasculitis, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, autoimmune hemolytic anemia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyly, telangiectasia), adult-onset diabetes mellitus (type 2 diabetes), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, Hurst's disease, Crohn's disease, mixed connective tissue disease, polyarteritis, systemic necrotizing hematoma ulcerative colitis, juvenile-onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent pregnancy loss, antiphospholipid syndrome, farmer's lung disease, erythema multiforme, postpericardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird fancier's lung, allergic diseases, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, inclusion body myositis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reactions, leprosy, malaria, leishmaniasis, trypanosomiasis, Nosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sumpter's syndrome, ocular cicatricial pemphigoid, eczema, lymphomatoid granulomatosis, Behçet's disease, Kaplan's syndrome, Kawasaki disease, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum and persistent erythema, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Schulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuchs' cyclitis, IgA nephropathy, Henoch-Schönlein purpura, paroxysmal nocturnal hemoglobinuria,Autoimmune diseases include glomerulonephritis, graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, post-vaccination syndrome, congenital rubella infection, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, juvenile idiopathic arthritis, Waldenström macroglobulinemia, Berger's disease, rubulavirus infection, and Evans syndrome. Diseases that can cause or contribute to autoimmune diseases include Alzheimer's disease, parvovirus infection, rubella virus infection, dengue virus infection, Epstein-Barr virus infection, Hodgkin's and non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, and malignant melanoma.

[0064] Infectious Disease: An infectious disease, also known as a contagious disease or a communicable disease, is a disease resulting from infection. Infections are caused by infectious agents such as viruses, virus-like entities, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, fleas, and lice, fungi such as ringworm, and other macroparasites such as tapeworms and other helminths. The host fights the infection using its immune system, including an innate response involving inflammation (e.g., in mammals), followed by an adaptive response. Medications used to treat infections include antibiotics, antivirals, antifungals, antiprotozoal agents, anthelmintics, and the like. Specific, non-limiting examples of infectious diseases include human immunodeficiency syndrome (HIV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HBV), tuberculosis (TB), malaria, and the like.

[0065] Genetic disease: A condition caused by one or more germline mutations inherited from one or both parents. Such conditions may be monogenic and classified as Mendelian or monogenic, or complex (e.g., multifactorial) and non-Mendelian. Mutations associated with genetic diseases (also known as genetic conditions or disorders, or congenital conditions, diseases, or disorders) may include substitutions, insertions, inversions, point mutations, deletions, mismatches, copy number variations, and / or translocations. Exemplary genetic diseases that can be treated or inhibited using the disclosed delivery agents include those described herein and those described in The Online Metabolic and Molecular Bases of Inherited Disease (Valle et al., (Eds.), 2019, McGraw Hill, https: / / ommbid.mhmedical.com / content.aspx?bookid=2709§ionid=2-3 69235).

[0066] Suppression or treatment of a symptom or disease: For example, preventing a disease or condition from progressing fully in a subject suffering from or at risk of suffering from a genetic disease, rare disease, cancer, immune disease, or infectious disease. "Treatment" refers to a therapeutic intervention that alleviates the signs or symptoms of a disease or condition, such as cancer, after it has begun to progress (e.g., reducing the cancer burden in a subject). The term "alleviation" with respect to a disease or condition refers to an observable beneficial effect of treatment. A beneficial effect may be evidenced, for example, by a delay in the onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of the disease, a delay in disease progression, an improvement in the subject's overall health or well-being, or other parameters specific to a particular disease, such as an increased survival rate in subjects with a genetic disease, rare disease, cancer, immune disease, or infectious disease. Treatment may be assessed by objective or subjective parameters, including, but not limited to, physical examinations, imaging diagnostics, blood test results, etc. A "prophylactic" treatment is a treatment administered to a subject who shows no signs of disease or only early signs of disease, with the intent of reducing the risk of developing a disease, such as preventing the onset or recurrence of cancer, an immune disorder, or an infectious disease.

[0067] Moiety: A moiety is a fragment or part of a conjugate of a molecule. Morpholino: A type of oligomeric molecule used to modify gene expression, such as reducing or preventing gene expression. The molecular structure of a morpholino may contain DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to a small (usually about 25 bases) specific sequence on the base-pairing surface of RNA, such as by binding to complementary sequences of RNA (or single-stranded DNA) through standard nucleobase pairing.

[0068] Muscular dystrophy: A term used to refer to a group of genetic disorders that cause progressive muscle weakness. Muscular dystrophies can result in skeletal muscle weakness and defects in skeletal muscle proteins, leading to a variety of physiological dysfunctions. Existing treatments typically focus on reducing the impact of the disease and improving patients' quality of life, such as through the provision of physical therapy and orthopedic devices.

[0069] Mutated genes associated with muscular dystrophies are responsible for encoding numerous proteins involved in the costameric protein network, including laminin-2, collagen, dystroglycan, integrins, caveolin-3, ankyrin, dystrophin, α-dystrobrevin, vinculin, plectin, BPAG1b, muscle LIM proteins, desmin, actinin-related LIM proteins, α-actin, titin, telethonin, cypher, myotilin, and the sarcoglycan / sarcospan complex.

[0070] The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD), which affects 1 in 3,500 live male births. DMD is an X-linked recessive disorder characterized by mutations in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein approximately 430 kDa in size. This protein functions to connect the cell's cytoskeleton to the extracellular matrix. Loss of dystrophin in DMD patients leads to loss of muscle fiber attachment to the extracellular matrix during contraction, ultimately leading to progressive fiber damage, membrane leakage, and loss of muscle function. Most patients die before the age of 30 due to respiratory or heart failure.

[0071] Becker muscular dystrophy (also known as benign pseudohypertrophic muscular dystrophy) is related to DMD in that both result from mutations in the dystrophin gene; however, the lack of functional dystrophin in DMD makes DMD much more severe than BMD. BMD is an X-linked recessive genetic disorder characterized by slowly progressive muscle weakness in the legs and pelvis. BMD is a type of dystrophinopathy, encompassing a range of muscle disorders in which insufficient production of dystrophin in muscle cells results in structural instability of the muscle cell membrane. This is caused by mutations in the dystrophin gene, which encodes the dystrophin protein. The symptomatic manifestations of BMD are similar to those of DMD, but progress more slowly and at a much slower rate.

[0072] Congenital muscular dystrophies are caused by genetic mutations. Fukuyama congenital muscular dystrophy (FCMD) and congenital muscular dystrophy type 1A (MDC1A) are examples of congenital muscular dystrophies. MDC1A is a congenital muscular dystrophy caused by a genetic mutation in the LAMA2 gene, resulting in a deficiency or complete loss of laminin-α2 protein. The loss of laminin-α2 leads to a lack of laminin-211 / 221. Laminin-211 / 221 is a major component of the extracellular matrix and plays an important role in muscle cell development. During muscle cell differentiation, laminin binds to α7β1 integrin. In the absence of laminin-α2, muscle fibers cannot adhere to the basement membrane, and myotubes undergo apoptosis. Muscle regeneration also fails, leading to a loss of muscle repair and increased muscle fibrosis and inflammation. This chronic tissue damage is the primary cause of morbidity and mortality in MDC1A.

[0073] Congenital muscular dystrophy (CMD) and limb-girdle muscular dystrophy (LGMD) are common forms of muscular dystrophy that are significantly different and can be distinguished by the age of onset. In CMD, symptoms begin at birth or within the first six months of life, whereas in LGMD, symptoms begin in late childhood, adolescence, or adulthood. LGMD is inherited in an autosomal dominant (LGMD type 1) or autosomal recessive (LGMD type 2) manner, whereas CMD is recessively inherited. CMD and LGMD can overlap clinically and genetically.

[0074] MDC1A is a progressive muscle-wasting disease that results in children becoming wheelchair-bound, requiring ventilator support to breathe, and early death. Symptoms are detected at birth as low muscle tone and "hypotonic" baby syndrome. DMD, BMD, and LGMD are progressive muscle-degenerative diseases that are usually diagnosed between the ages of 3 and 5, when children exhibit developmental delays, such as the ability to walk and climb stairs. Because the disease is progressive, children typically become wheelchair-bound and require ventilator support by their teenage years.

[0075] FCMD is a genetic disorder that primarily affects the muscles, brain, and eyes. Congenital muscular dystrophies are a group of genetic disorders that cause muscle weakness and muscle wasting (atrophy) beginning in early childhood. Fukuyama congenital muscular dystrophy affects skeletal muscles, the muscles the body uses for movement. The first signs of the disorder appear in early infancy and include a weak cry, poor feeding, and weak muscle tone (hypotonia). Facial muscle weakness often includes drooping eyelids (ptosis) and an open mouth, leading to a distinctive facial appearance. During childhood, muscle weakness and joint deformities (contractures) limit movement and interfere with the development of motor skills such as sitting, standing, and walking. Fukuyama congenital muscular dystrophy also adversely affects brain development. Patients with this condition have a brain abnormality called cobblestone lissencephaly, which causes the brain's surface to have a bumpy, irregular appearance (like cobblestones). These changes in brain structure result in significant delays in the development of speech and motor skills and moderate to severe intellectual disability. Impairments in social skills are less severe. Most children with Fukuyama congenital muscular dystrophy are unable to stand or walk, although some can sit unsupported and may slide across the floor while seated. More than half of affected children also experience seizures. Other signs and symptoms of Fukuyama congenital muscular dystrophy include vision loss, other eye abnormalities, and slowly progressive heart problems after age 10. As the disease progresses, patients develop difficulty swallowing and may develop a bacterial lung infection called aspiration pneumonia. Due to the serious medical problems associated with Fukuyama congenital muscular dystrophy, most patients with the disorder survive only into late childhood or adolescence.

[0076] FCMD is found almost exclusively in Japan and is the second most common childhood muscular dystrophy (after Duchenne muscular dystrophy). The incidence of Fukuyama congenital muscular dystrophy is estimated to be 2-4 per 100,000 infants in Japan.

[0077] FCMD is caused by mutations in the FKTN gene, which encodes fukutin. The most common mutation in the FKTN gene reduces the amount of fukutin produced in cells. A lack of fukutin can prevent the normal modification of α-dystroglycan, impairing the protein's normal function. Without functional α-dystroglycan to stabilize muscle cells, muscle fibers become damaged as they repeatedly contract and relax during use. Damaged fibers weaken and die over time, leading to the progressive decline and atrophy of skeletal muscle.

[0078] Defective α-dystroglycan can also affect neuronal migration during early brain development. Instead of stopping when they reach their intended destination, some neurons migrate across the brain's surface into the fluid-filled spaces surrounding it. Because FCMD involves dysfunction of α-dystroglycan, the condition is called a dystroglycanopathy.

[0079] Facioscapulohumeral muscular dystrophy (FHMD) is a type of muscular dystrophy characterized by progressive muscle weakness and loss of muscle tissue. Unlike DMD and BMD, which primarily affect the lower extremities, FSHD primarily affects the upper extremities, including the face, shoulders, and upper arm muscles. However, it can also affect muscles around the pelvis, hips, and lower extremities. FSHD symptoms often do not appear until age 10–26, although they can appear much later. In some cases, symptoms may not progress at all. Symptoms are usually mild and worsen very slowly. Facial weakness is common and may include drooping eyelids, difficulty whistling, reduced facial expression, depressed or angry facial expressions, difficulty articulating words, shoulder muscle weakness (leading to deformities such as prominent scapulae (winging scapulae) and sloping shoulders), lower extremity weakness, hearing loss, and possible heart disease.

[0080] Peptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Peptide" is used interchangeably with protein or polypeptide and is used herein to refer to a polymer of amino acid residues. "Peptide" applies to amino acid polymers, including natural and unnatural amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are unnatural amino acids, such as artificial chemical mimics of the corresponding natural amino acids. "Residue" refers to an amino acid or amino acid mimic incorporated into a polypeptide by an amide bond or amide bond mimic. Peptides have an amino terminus (N-terminus) and a carboxy terminus (C-terminus).

[0081] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers useful in embodiments of the present disclosure are conventional. Remington: The Science and Practice of Pharmacy (23rd Edition), by Adeboye Adejare, Academic Press (2020) describes compositions and formulations suitable for pharmaceutical delivery of the therapeutic agents and delivery agents disclosed herein.

[0082] Generally, the nature of the carrier will vary depending on the mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable fluids as a vehicle, such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0083] Polar: Polar compounds are compounds in which electrons are not shared equally between atoms, resulting in persistent separation of regions of positive and negative charge. Polar compounds are usually soluble in water (hydrophilic). Examples of polar molecules include polar amino acids such as arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, threonine, and tyrosine.

[0084] Mental illness: Also known as mental health illness, mental illness refers to a variety of mental health conditions that can affect mood, thoughts, and behavior. Examples of mental illnesses include mood disorders, psychotic disorders, anxiety disorders, personality disorders, eating disorders, dementia-related disorders, and addictive behaviors. A specific example of a mental disorder disclosed herein is depression. Depression is a mood disorder characterized by symptoms such as low mood, loss of interest and pleasure, and / or low energy.

[0085] Nonpolar: Nonpolar compounds are compounds in which electrons are shared equally or nearly equally between atoms. Nonpolar compounds are usually insoluble in water (hydrophobic). Examples of nonpolar molecules are nonpolar amino acids such as alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0086] Rare disease: A disease or condition that affects fewer than 200,000 people in the United States.

[0087] Specific binding: A binding reaction in which, under specified conditions, an antibody binds preferentially to a specific target protein, peptide, or polysaccharide (such as a tumor-associated antigen) and does not bind in significant amounts to other proteins or polysaccharides present in a sample or subject (such as a cell surface). Specific binding also occurs between streptavidin and biotin, avidin and biotin, or neutravidin and biotin. Specific binding can be determined by an appropriate method.

[0088] For antibody-antigen complexes, the specific binding between the antigen and the antibody is 10 -7Less than a mole, e.g., 10 -8 Less than 10 moles -9 Less than 10 moles -10 Submolar K D As an example, the affinity of streptavidin for biotin is described below.

[0089] K D refers to the dissociation constant of a particular interaction, such as a polypeptide-ligand interaction or an antibody-antigen interaction. For example, in the case of a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0090] The antibodies used in the methods disclosed herein specifically bind to a specific target, such as a tumor-associated antigen on a cell surface. Thus, an antibody that specifically binds to an epitope on a tumor-associated antigen is an antibody that substantially binds to the tumor-associated antigen, including cells or tissues that express the tumor-associated antigen. Streptavidin, avidin, and neutravidin substantially bind to biotin. Of course, it is recognized that some degree of nonspecific interaction may occur between a complex containing an antibody or conjugate (such as an antibody that specifically binds to an antigen of interest or a conjugate containing such an antibody) and a non-target (such as a cell that does not express the antigen). Typically, specific binding is much stronger between an antibody and a protein or cell that has the antigen than between an antibody and a protein or cell that lacks the antigen. Similarly, specific binding of streptavidin, avidin, or neutravidin refers to binding between streptavidin, avidin, or neutravidin and a biotin-labeled molecule (such as an antibody or aptamer), but not binding between a molecule labeled with another marker (such as an antibody or aptamer). Specific binding typically results in a 2-fold or greater, e.g., 5-fold or greater, 10-fold or greater, or 100-fold or greater increase in the amount of antibody bound (per unit time) to a protein containing the epitope (or a protein labeled with biotin) or to cells or tissues expressing the target epitope, compared to a protein lacking this epitope (or a protein lacking biotin).

[0091] Subject: A living organism, such as a vertebrate, e.g., a mammal, e.g., a human. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sport animals, pets, etc. In one example, the subject is a non-human mammalian subject, such as a monkey or other non-human primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, horse, or cow. In some examples, the subject is a reptile, amphibian, fish, or bird. The subject can serve as a source of a sample to be analyzed using the disclosed methods and devices.

[0092] Sugar: A compound that can be used to target a specific tissue within the context of the present disclosure. For example, a sugar can be used in combination with a delivery agent according to the present disclosure (e.g., by being conjugated to the delivery agent) to facilitate delivery of the delivery agent to a specific tissue. In some embodiments of the present disclosure, a sugar can be used as a targeting group of the delivery agent. By way of example only, the sugar GalNAc can be used to target liver tissue, thereby allowing the delivery agent to be used to treat liver disease. Other sugars include, but are not limited to, glucose (e.g., for targeting the GLUT1 receptor on the blood-brain barrier), mannose (e.g., for targeting the mannose-6-phosphate receptor in the lung, brain, or immune cells), and the like.

[0093] Therapeutic Agent: Therapeutic agents include therapeutic agents, prophylactic agents, and supplemental agents. Thus, a therapeutic agent can be any substance or combination of substances useful for achieving a purpose or result, such as improving a specific set of conditions in a subject with a disease or disorder, for example, a substance or combination of substances useful for inhibiting the growth or metastasis of cancer in a subject (such as a combination therapy for treating a genetic disease, a rare disease, cancer, an immune disease, or an infectious disease). Therapeutic agents include proteins, nucleic acid molecules, chemical compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Exemplary therapeutic agents include nucleic acid-based therapeutic agents (oligonucleotides, nucleic acid analogs, morpholinos, etc.), protein-based therapeutic agents, and their derivatives. By way of example, a therapeutic agent as used herein can be a chemotherapeutic agent, a therapeutic antibody, an immunotherapeutic agent, an antidepressant, an antiviral agent, or an antibiotic. Exemplary therapeutic agents further include saporin, cisplatin, methotrexate, fluorouracil, doxorubicin, cyclophosphamide, chlorambucil, vinblastine, vincristine, docetaxel, or paclitaxel, chlorhexidine, triclosan, xylitol, or octadecen-1-amine hydrofluoride, 1-hexadecylamine hydrofluoride, or combinations thereof.

[0094] (II. Introduction) Various therapeutic molecules must achieve intracellular delivery into the cell cytosol for their activity. Functional delivery of larger, more polar therapeutic molecules into the cell cytosol remains a challenge because such molecules typically cannot directly cross the cell membrane and are instead internalized via endocytosis or similar mechanisms. Endocytosis is the process by which extracellular material enters cells through invaginations in the cell membrane, which then close to form intracellular vesicles known as endosomes. Endocytosis can be receptor-mediated, in which extracellular compounds bind to specific receptors on the cell surface, or nonspecifically, in which extracellular compounds are internalized by their presence near the cell membrane. The latter process is also known as fluid-phase endocytosis or pinocytosis. A related process, potocytosis, internalizes compounds into cells via vesicles known as caveolae near the cell surface. In each of these processes, the vesicles surrounding the extracellular compounds become increasingly acidic after vesicle formation.

[0095] While peptide compositions for endosomal delivery exist in the art, they exhibit significant drawbacks that adversely affect their in vivo use / activity. For example, such peptide compositions interact with biological components, such as serum, and are excreted from the body through the kidneys and other channels. These interactions can reduce the free active concentration of the drug and reduce its suitability for clinical and other in vivo use. However, the delivery agents of the present disclosure can avoid such problems related to binding to biological components. Without being limited to a single theory, it is currently believed that the delivery agents of the present disclosure exhibit better in vivo activity by using a specially designed masking peptide group that protects the active lytic peptide group of the delivery agent. This masking protection reduces or prevents interaction of the lytic peptide group with biological components (such as serum) before the lytic peptide group is activated within organelles in the endolysosomal pathway. The delivery agents of the present disclosure further comprise additional components that aid in their in vivo use and efficacy. By way of example only, and not intended to be limiting, a cleavable linker group can be positioned between the lytic peptide group and the masking peptide group to facilitate detachment of the lytic peptide group from the masking peptide group after entry into the endosome, such as by enzymatic cleavage with an enzyme (e.g., cathepsin B). By way of example only, the use of a cleavable linker, which may be cleavable by cathepsin B, can reduce the time the delivery agent and therapeutic agent are exposed to degradative enzymes. In yet some additional embodiments, an anchor group is used as a component of the delivery agent, thereby enhancing cell surface binding of the delivery agent and thus increasing cellular uptake of the delivery agent (and in some embodiments, one or more therapeutic agents), for example, through endocytosis as described above. In yet some additional embodiments, the anchor group can be substituted or supplemented with a targeting group that facilitates directing the delivery agent to a specific location.

[0096] A schematic illustrating the process by which a delivery agent according to the present disclosure acts to facilitate delivery of a therapeutic agent into the cytosol of a cell is shown in FIG. 1. As can be seen in FIG. 1, the anchor group 100 of the delivery agent 102 serves to guide and anchor the delivery agent to the extracellular membrane. The masking peptide group 104 serves to hide or protect the lytic peptide group 106 from the surrounding serum. There is also a therapeutic agent 108 located near or coupled (covalently or non-covalently bound) to the delivery agent 102. After endocytosis into a cell 110, the delivery agent 102 and therapeutic agent 108 are encapsulated within an endosome 112. When the pH within the endosome decreases, the cleavable linker 114 is cleaved, releasing the lytic peptide group 106 from the masking peptide group 104. This allows the lytic peptide group 106 to enter the cell membrane, and subsequently, the therapeutic agent 108 to pass through the endosomal membrane and enter the cytosol of the cell.

[0097] III. PEPTIDE-BASED DELIVERY AGENT EMBODIMENTS The present disclosure provides peptide-based delivery agents (also referred to herein as "delivery agents") that deliver therapeutic molecules to the cytosol of cells such that the therapeutic molecules delivered to the cytosol retain their therapeutic activity. Such therapeutic molecules can be covalently or non-covalently (e.g., electrostatically) bound to the delivery agent, or they can be unbound, such as simply associated with the delivery agent by being positioned in close proximity to each other. The disclosed delivery agents are water-soluble, amphiphilic, membrane-destabilizing structures. Without wishing to be bound by theory, after internalization into a cell, the disclosed delivery agents can destabilize (e.g., dissolve) the membrane of a membrane-bound compartment, such as an endosomal, endolysosomal, or lysosomal membrane within the cell, and / or form one or more pores in or otherwise disrupt the integrity of the cell's membrane (e.g., the membrane of an endosome, endolysosomal, or lysosomal) by transitioning from a polar to a non-polar form as the pH of the membrane-bound compartment decreases relative to physiological pH. Thus, the disclosed delivery agents can internalize the delivery agent and therapeutic molecule into the same membrane-bound compartment of a cell, and then deliver the therapeutic molecule to the cytosol of the cell by dissolving the membrane of the membrane-bound compartment, by forming one or more pores in the compartment membrane, or by locally disrupting the integrity of the compartment membrane. In some embodiments of the present disclosure, a cleavable linker that is rapidly cleaved is used, and the therapeutic molecule is released from the membrane-bound compartment (such as an endosome, endolysosome, or lysosome) by dissolution, pore formation, or local disruption by the delivery agent before the therapeutic molecule is exposed to many degradative enzymes.

[0098] The delivery agents described herein may have a structure including an N-terminal group, a lytic peptide group, a cleavable linker, a mask peptide group, an anchor group (and / or targeting group), a C-terminal group, and an optional fluorophore (which may be attached to different regions of the delivery agent, such as the C-terminus or N-terminus). In some embodiments of the present disclosure, the lytic peptide group provides the N-terminal group of the delivery agent, and the N-terminal amino acid of the lytic peptide group may be functionalized to provide the N-terminal group. In yet some additional embodiments, the anchor group (and / or targeting group) provides the C-terminal group of the delivery agent, and the C-terminal amino acid of the anchor group (and / or targeting group) may be functionalized to provide the C-terminal group. To reduce interactions between the delivery agent and biological components such as serum, which may otherwise reduce or eliminate the activity of the delivery agent (e.g., in a subject or in cell culture), the lytic peptide group is protected by a specially designed mask peptide group. In certain embodiments, the mask peptide group is configured to be symmetrical about the lytic peptide group from the N-terminus to the C-terminus. When the delivery agent is internalized in a cell within an endosome, endolysosome, or lysosome, the masking peptide group can separate from the lytic peptide group via cleavage of the cleavable linker, exposing the lytic peptide group within the endosome, endolysosome, or lysosome. In some embodiments of the present disclosure, the anchoring group improves the association (e.g., binding) of the delivery agent with a cell membrane, such as the plasma membrane. In embodiments that include a targeting group in addition to (or instead of) the anchoring group, the targeting group can facilitate targeted delivery of the delivery agent to a specific location.

[0099] According to some embodiments of the present disclosure, the delivery agent is a compound represented by the formula (1): 1 Y 1 Y 1 X 1 ] m wherein m is an integer selected from 2 to 8, and each X 1 and each Y 1 (1) a lytic peptide group, which is an amino acid; (2) a cleavable linker; and (3) a compound of the formula [X 2 Y 2 Y 2 X 2 ] m’wherein m' is an integer selected from 2 to 8, and each X 2 and each Y 2 may have a structure including, independently for each occurrence, a lytic peptide group, which is an amino acid, and (4) an anchor group (and / or targeting group). In some embodiments of the present disclosure, the delivery agent may further comprise a fluorophore group. In yet additional embodiments, the delivery agent may further comprise an attached therapeutic agent. Typically, the amino acid of the lytic peptide group may be functionalized to provide an N-terminal group, and the amino acid of the anchor group (and / or targeting group) may be functionalized to provide a C-terminal group.

[0100] In certain embodiments, the delivery agent may have a peptide backbone structure satisfying the general formula IA or IB shown below. [N-terminal group]-[X 1 Y 1 Y 1 X 1 ] m -[cleavable linker]-[X 2 Y 2 Y 2 X 2 ] m’ -[anchor / targeting group]-[C-terminal group] (Formula 1A) [N-terminal group]-[X 2 Y 2 Y 2 X 2 ] m’ -[anchor / targeting group]-[cleavable linker]-[X 1 Y 1 Y 1 X 1 ] m -[C-terminal group] (Formula 1B)

[0101] As shown in Formulas IA and IB, the lytic peptide group is indirectly conjugated to the masking peptide group via a cleavable linker. In some embodiments of the present disclosure, the bolded X of the lytic peptide group 1The group may be functionalized to provide an N-terminal group that may comprise a capping group or a fluorophore, or may itself provide the N-terminus of the peptide backbone of the delivery agent. The anchor group (or any targeting group) is attached to the C-terminal group, which may further comprise or be attached to any fluorophore group. Additional components, compounds, and / or structural features may be attached to the peptide backbone of the delivery agent to provide conjugates that comprise, for example, covalently attached therapeutic agents, targeting groups, etc. Representative embodiments of lytic peptide groups, cleavable linker groups, mask peptide groups, anchor groups (and / or targeting groups), optionally fluorophore groups, and suitable N- and C-terminal groups are described below.

[0102] (AN terminal group) In some aspects of the present disclosure, the N-terminal group of the delivery agent comprises a capping group (e.g., an acetyl group) or a fluorophore. In one embodiment, the N-terminal group is an amino acid of the lytic peptide group (e.g., X of the lytic peptide group). 1 In some aspects of the disclosure, the acetyl group is MeC(O)R, where R is the remainder of the peptide backbone of the delivery agent. In another embodiment, the N-terminal group comprises a 2-(methylamino)benzamide moiety (or a derivative thereof) attached to an amino acid of the lytic peptide group.

[0103] JPEG2025528284000002.jpg31158

[0104] In some embodiments of the present disclosure, one or more fluorophores are attached to one or more amino acids at or near the N-terminus of the disclosed delivery agent. In exemplary embodiments, the fluorophore may be 3-hydroxyisonicotinaldehyde and / or azulene, and the amino acid to which the fluorophore is attached may be a natural or unnatural amino acid present in the peptide backbone of the delivery agent and / or any branched peptide sequence resulting therefrom. In some such examples, one or more fluorophores may be attached to an amine-containing amino acid, such as lysine or other amine-containing amino acid derivative, on the N-terminus of the disclosed delivery agent.

[0105] B. Lytic Peptide Group According to some embodiments of the present disclosure, the lytic peptide group of the disclosed delivery agent can be a group that can dissolve cell membranes. In some embodiments of the present disclosure, the lytic peptide group dissolves membranes by transitioning from a polar form to a non-polar form as the pH in a membrane-bound compartment of a cell (such as an endosome, endolysosome, or lysosome) decreases compared to physiological pH. Without wishing to be bound by any particular theory, insertion of a delivery agent comprising a lytic peptide group in a non-polar form into a membrane can induce membrane dissolution, thereby delivering a therapeutic agent into the cytosol of a cell.

[0106] The lytic peptide group of the present disclosure may be a group capable of undergoing a transition between a hydrophobic α-helical form and a hydrophilic form. As described in more detail below, some embodiments of the lytic peptide group disclosure contain one or more pairs, preferably two or more pairs, of carbonyl-containing groups (e.g., carboxyl groups and / or amides), with the two carbonyl-containing groups of a pair separated by zero, one, two, or three amino acids. The composition and arrangement of the amino acids in the lytic peptide group are such that, in the presence of both aqueous and lipid-like phases, the lytic peptide group typically undergoes a reversible transition between a high-pH form, typically between low and neutral or high pH (e.g., pH 4.0-7.0), in which the side chains or terminal carbonyl-containing groups are entirely or predominantly in a non-hydrogen-bonded, ionic state, and a low-pH form in which the lytic peptide group has a substantially non-ionic α-helical structure rendered lipophilic by hydrogen bonding between the paired side chains or terminal carbonyl-containing groups. The hydrophobic low pH form partitions into the lipid environment, while the hydrophilic high pH form partitions preferentially into aqueous solution. Thus, the "reversible transition" between the lipophilic and hydrophilic forms of a polypeptide is the transition between a non-ionic α-helical structure with side chain carbonyl-containing groups participating in intramolecular hydrogen bonding favorable at low pH and a form with side chain carbonyl-containing groups in an ionic non-hydrogen-bonded state favorable at neutral or high pH. Such transitions can encompass the entire lytic peptide group or can occur in localized regions of the lytic peptide group, especially when the region is near the aqueous / lipid interface, or is particularly lipophilic and / or has a composition favorable for the formation of α-helices. Such localized regions of the lytic peptide group are often effective in initiating the entry of the delivery agent into a lipid phase, such as a membrane, even if the region of the delivery agent further away from the lipid phase may be in a hydrophilic structure. In yet another embodiment, the lytic peptide group comprises one or more basic amino acids, such as arginine, lysine, histidine, or a combination thereof. Without wishing to be limited to a particular theory, such examples may not undergo the transitions described above, but may still be used with certain types of delivery agents of the present disclosure.

[0107] In some embodiments of the present disclosure, the lytic peptide group has the formula [X 1 Y 1 Y 1 X 1 ] m In such embodiments, m can be an integer selected from 2 to 8 or more, and each X 1 may, independently for each occurrence, be a basic amino acid (or an ionized form thereof), an acidic amino acid (or an ionized form thereof), a nonpolar amino acid, or a derivative thereof, and each Y 1 may be, independently for each occurrence, a non-polar amino acid or derivative thereof. In some embodiments of the present disclosure, m is 2, 3, 4, 5, 6, 7, 8, or 8 or more. In a particularly non-limiting embodiment, m is 3. In some embodiments of the present disclosure, each X 1 is independently for each occurrence an acidic amino acid selected from glutamic acid (and / or glutamate), or aspartic acid (and / or aspartate), glutamine, or a derivative thereof, a basic amino acid selected from arginine, lysine, or a derivative thereof, or alanine (provided that at least one X 1 is other than alanine). In certain non-limiting embodiments, each X 1 is glutamic acid (or glutamate), aspartic acid (or aspartate), or a derivative thereof. In another specific, non-limiting embodiment, each X 1 is arginine or lysine, or an ionized form thereof, or a derivative thereof. In another specific, non-limiting embodiment, at least one X 1 At least one X 1 is alanine or a derivative thereof, provided that is other than alanine, e.g., a basic amino acid or an acidic amino acid. In some embodiments of the disclosure, each Y 1 is, independently for each occurrence, leucine, α-methylleucine, methionine, alanine, or 2-aminobutyric acid, or a derivative thereof. In certain non-limiting embodiments, each Y 1is leucine, α-methylleucine, or a derivative thereof. In another specific non-limiting embodiment, each Y 1 is α-methylleucine. In another specific, non-limiting embodiment, each Y 1 is leucine.

[0108] In some embodiments of the present disclosure, the lytic peptide group has an amino acid sequence at least 85% identical to SEQ ID NO:1 (ELLEELLEELLE, where E is glutamic acid and L is leucine; see Figure 2), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1. In certain non-limiting embodiments, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:1.

[0109] In some embodiments of the present disclosure, the lytic peptide group has an amino acid sequence at least 85% identical to SEQ ID NO:2 (DLLDDLLDDLLE, where D is aspartic acid, E is glutamic acid, and L is leucine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2. In certain non-limiting embodiments, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:2.

[0110] In some embodiments of the present disclosure, the lytic peptide group has an amino acid sequence at least 85% identical to SEQ ID NO:38 (ELLEELLEELLEELLE, where E is glutamic acid and L is leucine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:38. In certain non-limiting embodiments, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:38.

[0111] In some embodiments of the present disclosure, the lytic peptide group has an amino acid sequence at least 85% identical to SEQ ID NO:30 (ELLEQLLQELLE, where E is glutamic acid, Q is glutamine, and L is leucine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:30. In certain non-limiting embodiments, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:30.

[0112] In some embodiments of the present disclosure, the lytic peptide group has an amino acid sequence at least 85% identical to SEQ ID NO: 153 (QLLEQLLQQLLE, where E is glutamic acid, Q is glutamine, and L is leucine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 153. In certain non-limiting embodiments, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO: 153.

[0113] In some embodiments of the present disclosure, the lytic peptide group has an amino acid sequence at least 85% identical to SEQ ID NO:92 (RLLRRLLRRLLR, where R is arginine and L is leucine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:92. In certain non-limiting embodiments, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:92.

[0114] C. Cleavable Linkers According to certain aspects of the present disclosure, the linker of the disclosed delivery agent may be a cleavable linker that allows the lytic peptide group to be separated from the remainder of the delivery agent (including the masking peptide group) within a membrane-bound compartment (such as an endosomal, endolysosomal, or lysosomal compartment) after internalization of the delivery agent into a cellular endosome. Cleavage of the cleavable linker may allow the lytic peptide group to dissociate from the masking peptide group, thus allowing the lytic activity of the lytic peptide group to act within the membrane-bound compartment.

[0115] In some embodiments of the present disclosure, the cleavable linker group is cleavable by an enzyme present in an endosome, endolysosome, or lysosome. In certain embodiments of the present disclosure, the cleavable linker group can be cleaved by cathepsin B.

[0116] In some aspects of the disclosure, the cleavable linker comprises an amino acid sequence comprising 2 to 10 (e.g., 4 to 10 or 6 to 10) amino acids in length, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In certain non-limiting embodiments, the cleavable linker is 7 amino acids in length. In another specific non-limiting embodiment, the cleavable linker has an amino acid sequence at least 70% identical to SEQ ID NO:3 (GFGFVGG, where G is glycine, F is phenylalanine, and V is valine), e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3. In certain non-limiting aspects of the disclosure, the cleavable linker has an amino acid sequence comprising or consisting of SEQ ID NO:3. In another specific non-limiting embodiment, the cleavable linker has an amino acid sequence at least 70% identical to SEQ ID NO:233 (GGGVXGG, where G is glycine, X is citrulline (or Cit), and V is valine), e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:233. In certain non-limiting aspects of the disclosure, the cleavable linker has an amino acid sequence comprising or consisting of SEQ ID NO:233.In another specific, non-limiting embodiment, the cleavable linker has an amino acid sequence at least 70% identical to SEQ ID NO:234 ​​(GGGVKGG, where G is glycine, K is lysine, and V is valine), e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:234. In certain non-limiting aspects of the present disclosure, the cleavable linker has an amino acid sequence comprising or consisting of SEQ ID NO:234. In another specific, non-limiting embodiment, the cleavable linker has an amino acid sequence at least 70% identical to SEQ ID NO:227 (XGGXV, where G is glycine, each X is independently aminohexanoic acid or homoarginine, and V is valine), e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:227.

[0117] (D. Masked Peptide Group) According to some embodiments of the present disclosure, the masking peptide group of the disclosed delivery agent may be a group that can reduce or prevent the lytic peptide group of the delivery agent from being exposed to biological components (such as a cell culture system or a subject) before the delivery agent is internalized into a cell. In certain embodiments of the present disclosure, the masking peptide group hides the amino acid components of the lytic peptide group from serum, thereby reducing or avoiding interactions between the amino acid components and serum. In some embodiments of the present disclosure, hydrophobic residues (e.g., leucine) in the lytic peptide group sequence may adversely interact with serum (e.g., serum albumin) and reduce the activity of the delivery agent. The masking peptide group may reduce and / or prevent these interactions by hiding the hydrophobic residues until the delivery agent reaches its target (e.g., a membrane-bound compartment or other biological target region).

[0118] In some cases, the mask peptide group may contain one or more pairs, preferably two or more pairs, of carbonyl-containing groups (e.g., amides), with the two carbonyl-containing groups of a pair separated by zero, one, two, or three amino acids. Without being limited to a single theory, it is currently believed that the composition and position of the amino acids in the mask peptide group are such that, in the presence of an aqueous phase, the mask peptide group forms a substantially alpha-helical structure rendered lipophilic by hydrogen bonding between the paired amide side chains. Furthermore, it is believed that the alpha-helical character of the mask peptide facilitates masking of the lytic peptide. As described below, some embodiments of the mask peptide group of the present disclosure contain, in addition to one or more pairs, preferably two or more pairs, of carbonyl-containing groups (e.g., amides), one or more amino acids (e.g., glutamic acid) that may be negatively charged under physiological conditions. In some cases, the mask may be more soluble in an aqueous environment while retaining sufficient alpha-helical character of the mask peptide to facilitate masking of the lytic peptide.

[0119] In some embodiments of the present disclosure, the masking peptide group of the disclosed delivery agent is symmetrical to the lytic peptide group of the delivery agent. However, in some independent embodiments of the present disclosure, the masking peptide group of the disclosed delivery agent may have one, two, or three fewer amino acids than the lytic peptide group of the delivery agent. In certain embodiments of the present disclosure, the masking peptide group has a structure represented by the formula [X 2 Y 2 Y 2 X 2 ] m’ In such aspects of the disclosure, m' can be an integer selected from 2 to 8 or more, and each X 2 may be, independently for each occurrence, an acidic amino acid (e.g., glutamine, glutamic acid (and / or glutamate), aspartic acid (and / or aspartate)), a nonpolar amino acid (e.g., alanine), or a derivative thereof, and each Y 2 may, independently for each occurrence, be a non-polar amino acid or derivative thereof. In certain embodiments of the present disclosure, at least one X 2 is an acidic amino acid. In some aspects of the disclosure, m' is 2, 3, 4, 5, 6, 7, 8, or greater than 8. In particular, in non-limiting aspects of the disclosure, m' is 3. In certain non-limiting embodiments, each X 2 At least one X 2 is independently for each occurrence glutamic acid (and / or glutamate), aspartic acid (and / or aspartate), glutamine, alanine, or derivatives thereof, with the proviso that is other than alanine. In certain non-limiting embodiments, each X 2 is, independently for each occurrence, glutamic acid or a derivative thereof. In another non-limiting embodiment, each X 2 is, independently for each occurrence, aspartic acid or a derivative thereof. In another non-limiting embodiment, each X 2 is, independently for each occurrence, glutamine, or a derivative thereof. In another non-limiting embodiment, at least one X 2 at least one additional X 2is alanine, with the proviso that each Y is other than alanine. 2 is, independently for each occurrence, leucine, α-methylleucine, methionine, alanine, or 2-aminobutyric acid, or a derivative thereof. In certain non-limiting embodiments, each Y 2 is leucine, or a derivative thereof. In another specific non-limiting embodiment, each Y 2 is α-methylleucine or a derivative thereof. In another specific non-limiting embodiment, each Y 2 is, independently for each occurrence, alanine or a derivative thereof.

[0120] In some embodiments of the present disclosure, the masked peptide group has an amino acid sequence at least 85% identical to SEQ ID NO:4 (QLLQQLLQQLLQ, where Q is glutamine and L is leucine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4. In certain non-limiting embodiments of the present disclosure, the masked peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:4.

[0121] In some embodiments of the present disclosure, the masked peptide group has an amino acid sequence at least 85% identical to SEQ ID NO:51 (QLLQQLLQQLLQQLLQ, where Q is glutamine and L is leucine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:51. In certain non-limiting embodiments of the present disclosure, the masked peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:51.

[0122] In some embodiments of the present disclosure, the masked peptide group has an amino acid sequence at least 85% identical to SEQ ID NO: 154 (QLLQELLEQLLQ, where Q is glutamine, L is leucine, and E is glutamic acid), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 154. In certain non-limiting embodiments of the present disclosure, the masked peptide group has an amino acid sequence comprising or consisting of SEQ ID NO: 154.

[0123] In some embodiments of the present disclosure, the masked peptide group has an amino acid sequence at least 85% identical to SEQ ID NO: 153 (QLLEQLLQQLLE, where Q is glutamine, L is leucine, and E is glutamic acid), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 153. In certain non-limiting embodiments of the present disclosure, the masked peptide group has an amino acid sequence comprising or consisting of SEQ ID NO: 153.

[0124] In some embodiments of the present disclosure, the masked peptide group has an amino acid sequence at least 85% identical to SEQ ID NO: 148 (QLLAQLLAQLLQ, where Q is glutamine, L is leucine, and A is alanine), e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 148. In certain non-limiting embodiments of the present disclosure, the masked peptide group has an amino acid sequence comprising or consisting of SEQ ID NO: 148.

[0125] E. Anchor and / or Targeting Groups In some embodiments of the present disclosure, endocytosis of the disclosed delivery agents can be promoted by including one or more anchor groups (e.g., 1, 2, 3, or 4 anchor groups) in the structure of the delivery agent. In some embodiments of the present disclosure, the anchor group comprises a heteroaliphatic group, an antibody, a biotin group, an avidin group, a streptavidin group, or a neutravidin group. In certain embodiments of the present disclosure, the anchor group comprises a lipid anchor group comprising a tail group that promotes localization to the cell surface.

[0126] The anchor group of the delivery agent disclosed herein typically comprises an amino acid moiety attached to an amino acid group of the peptide backbone of the delivery agent. In some embodiments of the present disclosure, the amino acid moiety of the anchor group is attached to an amino acid of the mask peptide group, such as the amino acid of the mask peptide group located toward the C-terminal group of the delivery agent. The anchor group further comprises a tail group that facilitates targeting and accumulation of the delivery agent to a desired location, such as a cell surface (e.g., at a cell membrane). In certain embodiments of the present disclosure, the amino acid moiety of the anchor group further provides a C-terminal group that can be functionalized with different moieties described herein, such as the C-terminal group shown in Formula IA or IB, which may comprise an amine-terminated glycine moiety, a fluorophore, or the like, as described herein. In certain embodiments of the present disclosure, the amino acid moiety of the anchor group is lysine (K), and the tail group comprises an aliphatic tail. In some embodiments of the present disclosure, the amino acid moiety of the anchor group may be attached to two separate tail groups, such as when the anchor group terminates the delivery agent peptide backbone or when it terminates a branched group arising from the delivery agent peptide backbone. In some aspects of the present disclosure, the tail group may further comprise a functional group that facilitates attachment of the aliphatic tail to an amino acid moiety.

[0127] In some aspects of the present disclosure, the functional group may be provided by:

[0128] [ka]

[0129] In some embodiments of the present disclosure, the aliphatic terminus comprises an alkyl chain having a length of 6 to 12 carbon atoms (e.g., 6, 7, 8, 9, 10, 11, or 12). In yet some additional embodiments of the present disclosure, the amino group of the 2-aminoethylhydrogencarbonate group and / or the 4-aminobutanoate group can be functionalized to provide a quaternary amine. In certain embodiments of the present disclosure, the amino group is functionalized with an aliphatic group, such as a lower alkyl group having 10 or fewer carbon atoms. In some exemplary embodiments of the present disclosure, the amino group is functionalized with at least one methyl group, and in some specific examples, comprises two methyl groups. In certain embodiments of the present disclosure, the tail portion can have a structure selected from any of the structures shown below (the wavy line indicates the point of attachment of the anchor group to the amino acid portion):

[0130] [ka]

[0131] In some embodiments of the present disclosure, the anchor group may have a structure according to any of Formulas IIA-IID below.

[0132] [ka]

[0133] In some embodiments of the present disclosure, one or more anchor groups may be included in the delivery agent. In some embodiments of the present disclosure, two anchor groups may be directly bonded to each other or indirectly bonded via one or more amino acids. In some embodiments of the present disclosure, the two anchor groups may be identical or different in chemical structure. In some embodiments of the present disclosure, two (or more) anchor groups may be directly bonded to each other, and the amino acid moiety of one anchor group may be bonded to the amino acid moiety of the other anchor group via a peptide bond. By way of example only, if the amino acid moiety of each of the two anchor groups is lysine, the amino group of one lysine may be bonded to the mask peptide group, and the corresponding carboxylic acid group may be bonded to the amino group of the second lysine. The carboxylic acid group of the second lysine may be bonded to the C-terminal group of the delivery agent. In some embodiments of the present disclosure, the delivery agent may include two or more different anchor groups, such as one or more groups and one or more ligands that may have a structure according to Formula IIA, IIB, IIC, or IID. In receptor-mediated endocytosis, an anchoring group attached or conjugated to a composition targeted for uptake may bind (e.g., specifically bind) to a receptor on the cell surface (e.g., a receptor specific for a ligand). Such an anchoring group can be used to enhance general endocytic uptake or to target specific cell types.

[0134] The delivery agents disclosed herein may further comprise a targeting group, either in addition to or instead of any anchor group contained in the delivery agent. The targeting group may be utilized to facilitate specific delivery of the delivery agent (and the therapeutic agent attached thereto) to a particular target (e.g., a biological region, cell, or biological structure). The targeting moiety may include a cell, an antibody (or fragment thereof), a peptide, a biomimetic peptide, an aptamer, a sugar, a small targeting molecule, or a combination thereof. Exemplary antibodies may include anti-CD33, anti-CD30, anti-HER2, anti-CD22, anti-nectin-4, anti-nectin-1, anti-AXL, anti-CD74, anti-ALK, anti-PTK7, anti-PSMA, anti-TM4SF1, anti-CD276, anti-CD20, anti-CD19, anti-CD3, anti-CD71, etc. Exemplary antibody / target combinations may include interferon-α / β cell surface receptor complexes targeting type I interferons (IFNα, IFNβ), death receptors 4 and 5 (DR4 / 5) targeting tumor necrosis factor-related apoptosis-inducing ligand (TNI), EGFRvIII targeting antibodies or nanobodies (ENb), and CD36 targeting thrombospondin. Exemplary peptides and / or biomimetic peptides include, but are not limited to, iRGD (which binds to integrin receptors in tumors), PEN-221, octreotide, rabies virus glycoprotein-29, miniAp-4, angiopep-1, RGD-4C (cyclic), BT1718, PL1, and the like. Exemplary sugars include, but are not limited to, glucose, mannose, and galactose. Exemplary cells that can be used as targeting groups include, but are not limited to, erythrocytes, macrophages, neutrophils, monocytes, T cells, and the like. Exemplary small targeting molecules may include, but are not limited to, folic acid (for targeting cancer), bisphosphonates (for targeting bone), dibenzocyclooctyne compounds (or "DBCO") (for targeting the cell surface or other cellular features), etc. In some embodiments, dibenzocyclooctyne compounds may also be used as anchor groups to facilitate attachment of delivery agents to cell surfaces.

[0135] The targeting group may be attached to the delivery agent through an attachment point that may include an amino acid (e.g., a lysine or cysteine ​​group) of the delivery agent peptide backbone (or any such group present on a branch emerging from the delivery agent peptide backbone). In some embodiments of the present disclosure, the targeting group may be conjugated to the delivery agent by functionalizing one or more of these amino acids with an available amine, carboxyl, or side chain group of the amino acid. The attachment point may be included in the targeting group of the peptide backbone, a branch emerging from the peptide backbone, and / or the C-terminal group of the peptide backbone. By way of example only, the targeting group may be attached to the delivery agent through the amine terminus of a lysine group or the amine of a side chain of a lysine.

[0136] In some embodiments of the present disclosure, the anchor group and / or targeting group can be directly attached to the delivery agent via any of the bonds described above, or indirectly attached via a linking group. In some embodiments of the present disclosure, the linking group can comprise a carbonyl group, an amide group, an alkylene oxide group, or a combination thereof. In some embodiments of the present disclosure, the linking group has a structure according to Formula A, Formula B, or Formula C shown below, where the circled wavy bond in Formula A, Formula B, and Formula C indicates the point of attachment of the anchor and / or targeting group (or multiple independent anchor and / or targeting groups, as in the case of Formulas A and C), and the other wavy lines in Formula A, Formula B, and Formula C indicate attachment to the remainder of the delivery agent.

[0137] [ka]

[0138] In certain embodiments of the disclosure, the delivery agent has an amino acid sequence at least 85% identical to SEQ ID NO:272, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:272, wherein SEQ ID NO:272 is QLLEQLLQQLLEGFGFVGGQLLEQLLQQLLEKGXG, K at position 32 provides one or more DBCO groups attached to a Lys side chain having a linking group according to any one of Formulas A, B, or C, X is naphthylalanine (Nal), the C-terminal G at position 35 is a modified glycine comprising a -C(O)-NH group, and in some embodiments, the N-terminal Q comprises an N-terminus modified with an acetyl group. In certain non-limiting embodiments, the delivery agent has an amino acid sequence comprising or consisting of SEQ ID NO: 272. In any of these embodiments, K at position 32 provides (i) three DBCO groups attached to the Lys side chain by linking groups according to Formula A, (ii) one DBCO group attached to the Lys side chain by linking groups according to Formula B, or (iii) two DBCO groups attached to the Lys side chain by linking groups according to Formula C.

[0139] In certain embodiments of the disclosure, the delivery agent has an amino acid sequence at least 85% identical to SEQ ID NO:273, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:273, wherein SEQ ID NO:273 is ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKGXG, K at position 32 provides one or more DBCO groups attached to a Lys side chain with a linker group according to any one of Formulas A, B, or C, X is naphthylalanine (Nal), the C-terminal G at position 35 is a modified glycine comprising a -C(O)-NH group, and in some embodiments, the N-terminal Q comprises an N-terminus modified with an acetyl group. In certain non-limiting embodiments, the delivery agent has an amino acid sequence comprising or consisting of SEQ ID NO: 273. In any of these embodiments, K at position 32 provides (i) three DBCO groups attached to the Lys side chain by linking groups according to Formula A, (ii) one DBCO group attached to the Lys side chain by linking groups according to Formula B, or (iii) two DBCO groups attached to the Lys side chain by linking groups according to Formula C.

[0140] In certain embodiments of the present disclosure, the delivery agent has an amino acid sequence at least 85% identical to SEQ ID NO:274, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:273, wherein SEQ ID NO:273 is ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKGXG, K at position 32 provides one or more DBCO groups attached to a Lys side chain having a linking group according to any one of Formulas A, B, or C, X is naphthylalanine (Nal), the C-terminal G at position 35 is a modified glycine comprising a -C(O)-NH group, and in some embodiments, the N-terminal Q comprises an N-terminus modified with an acetyl group. In certain non-limiting embodiments, the delivery agent has an amino acid sequence comprising or consisting of SEQ ID NO: 273. In any of these embodiments, K at position 32 provides (i) three DBCO groups attached to the Lys side chain by linking groups according to Formula A, (ii) one DBCO group attached to the Lys side chain by linking groups according to Formula B, or (iii) two DBCO groups attached to the Lys side chain by linking groups according to Formula C.

[0141] (FC terminal group) In some embodiments of the present disclosure, the C-terminal group of the delivery agent includes an amine-terminated glycine moiety, a fluorophore, or both. In certain embodiments of the present disclosure in which the C-terminal group includes a fluorophore, the fluorophore can be naphthylalanine or tryptophan, and the glycine moiety can be flanked by one or both sides of the fluorophore. In certain embodiments of the present disclosure, the amine-terminated glycine moiety has a structure selected from -Nal-K-G', -GK-G', -Nal-C-G', -G-Nal-C-G', -GC-G', -Nal-G', -G-Nal-G', -G-Nal-K-G' (SEQ ID NO:5), -W-G', -GW-G', or -GWK-G', where Nal is naphthylalanine, G is glycine, G' is a modified glycine with a -C(O)-amine group, K is lysine, C is cysteine, and W is tryptophan. In some aspects of the present disclosure, any lysine or cysteine ​​at the C-terminus may serve as an attachment point and thus may be used to attach a targeting group as described herein. In certain non-limiting embodiments, the amine-terminated glycine moiety is G' or -G-Nal-G', where G' is -C(O)-N(R a ) groups, each R a are independently hydrogen or aliphatic (e.g., methyl, ethyl, propyl, i-propyl, t-butyl, i-butyl, n-butyl, etc.). In a further aspect of the disclosure, the amine-terminated glycine moiety is -G-Nal-K-G', where G' is -C(O)-N(R a ) groups, each R a are independently hydrogen or aliphatic (e.g., methyl, ethyl, propyl, i-propyl, t-butyl, i-butyl, n-butyl, etc.).

[0142] In some embodiments of the present disclosure, one or more other types of fluorophores can be attached to one or more amino acids present at the C-terminus of the disclosed delivery agents, such as unnatural amino acids. In such examples, one or more fluorophores can be attached to cysteine ​​or amine-containing amino acids present at the C-terminus of the disclosed delivery agents, such as lysine or other amine-containing amino acid derivatives. In some embodiments of the present disclosure, the fluorophore is selected from the group consisting of 3-hydroxyisonicotinaldehyde, azulene, Fmoc-Lys(Mca)-OH, Fmoc-Asp(EDANS)-OH, Fmoc-Glu(EDANS)-OH, Fmoc-Lys(Dabcyl)-OH, Fmoc-Lys(Dnp)-OH, p-cyanophenylalanine, 5-cyanotryptophan, 4-cyanotryptophan, 2-cyanophenylalanine, 7-azatryptophan, 7-cyanotryptophan, β-(1-azulenyl)-L-alanine, acridon-2-ylalanine, L-leucine 7-amido-4-methylcoumarin, trans-4-hydroxy-L-proline 7-amido-4-methylcoumarin, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 633, Alexa 647, Atto 465, Atto 488, Atto 532, Atto 550, Atto 565, Atto 647N, Atto 655, BODIPY-TMR, Cy3, Sulfo-Cy3, Cy3B, Sulfo-Cy5, Dyomics 654, Oregon Green 488, Oregon Green 514, sulforhodamine B, Texas Red, tetramethylrhodamine. In exemplary embodiments of the present disclosure, the fluorophore is selected from naphthylalanine, tryptophan, 3-hydroxyisonicotinaldehyde, and / or azulene.

[0143] In some embodiments of the present disclosure, a therapeutic agent or targeting group may be covalently or non-covalently attached to a delivery agent via a C-terminal group. In some embodiments of the present disclosure, a therapeutic agent may be attached to a delivery agent via a disulfide that can be cleaved to separate the delivery agent from the therapeutic agent. In some embodiments of the present disclosure, a therapeutic agent may be attached to a delivery agent via a second cleavable linker having a similar or identical sequence, or a different sequence, compared to that described for other cleavable linkers present in the delivery agent peptide backbone. In such embodiments of the present disclosure, the cleavable linker is attached to the amine-terminated glycine moiety. For example, the cleavable linker may be covalently attached to the glycine of the amine-terminated glycine moiety, or, in embodiments of the present disclosure comprising an amine-terminated glycine moiety having the structure described above, may be attached to a different amino acid of the amine-terminated glycine moiety, such as lysine or cysteine. In some embodiments of the present disclosure in which a therapeutic agent is attached to a delivery agent, it may be indirectly attached to the cleavable linker group via a chemical linker group. In some embodiments of the present disclosure, the chemical linker group may have a structure of any of Formulae IIIA-IIID. In further embodiments of the present disclosure, the chemical linker group may be a pyrophosphate diester linker (e.g., a pyrophosphate diester linker that can be cleaved by lysosomal phosphatases and pyrophosphate, such as those described in Kern et al., J. Am. Chem. Soc. 2016, 138(4):1430-1445; and Zheng et al., Acta Pharmaceutica Sinica B. 2021, 11(12):3889-3907, the structures of such pyrophosphate diester linkers disclosed by these documents are incorporated herein by reference); a β-glucuronide linker (e.g., a β-glucuronide linker that can be cleaved by β-glucuronidase, an enzyme present in lysosomes and overexpressed in some tumors, such as those described in Lu et al., Int J Mol Sci.2016, 17(4):561, the structure of such a β-glucuronide linker being incorporated herein by reference); a β-galactose linker (e.g., a β-galactose linker that is cleaved by β-galactosidase, an enzyme present in lysosomes and overexpressed in some tumors, as described in Komatsu et al., J. Am. Chem. Soc. 2006, 128(50):15946-15947; and Zheng et al., Acta Pharmaceutica Sinica B. 2021, 11(12):3889-3907, the structure of such a β-galactose linker being incorporated herein by reference); an aryl sulfate linker (e.g., an aryl sulfate linker that can be cleaved by sulfatase, an enzyme present in lysosomes and overexpressed in some cancers) and / or a photoresponsive cleavable linker, both of which are described in Zheng et al., Acta Pharmaceutica Sinica B. 2021, 11(12):3889-3907 (the structures in Table 1 are incorporated herein by reference), or other linkers that are cleavable in a low pH environment, such as orthoesters (Srinivasachar et al., Biochemistry, 1989, 28(6):2501-2509, the orthoesters described therein are incorporated herein by reference), silyl ethers (Parrott et al., J. Am. Chem. Soc. 2010, 132(50):17928-17932, the silyl ethers described therein are incorporated herein by reference), acetal linkers (Liu et al., J. Am. Chem. Soc. 2010, 132(5):1500-1501, the acetal linkers described therein are incorporated herein by reference), β-thiopropionate linkers (Oishi et al., Am. Chem. Soc.2005, 127(6):1624-1625 (the β-thiopropionate linkers described therein are incorporated herein by reference), phosphoramidate linkages (Jeong et al., Bioconjugate Chemistry, 2003, 14(2):473-479 (the phosphoramidate linkages described therein are incorporated herein by reference), imine linkers (Zhao et al., J. Am. Chem. Soc. 2010, 132(37):13016-13025 (the imine linkages described therein are incorporated herein by reference), vinyl ethers (Shin et al., J. Controlled Release, 2003, 91:187-200 (the vinyl ether linkages described therein are incorporated herein by reference), or hydrazones (Kale et al., Bioconjugate Chem. 2007, 18(2):363-370 (the hydrazone bond described therein is incorporated herein by reference), but may be selected from, but not limited to, Formula IIIA-IIID. With reference to Formulas IIIA-IIID, each p is independently an integer selected from 0 to 20, e.g., 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 4, and q is an integer selected from 0 or 1, where when q is 0, the second cleavable linker is absent and the chemical linker group is directly attached to the delivery agent.

[0144] [ka]

[0145] G. Exemplary Delivery Agent Embodiments In certain non-limiting embodiments, the delivery agent comprises (1) an N-terminal group (e.g., an acetyl group) and (2) a group of the formula [X 1 Y 1 Y 1 X 1 ] m wherein m is 3 and each X 1 is glutamic acid, and each Y 1 is leucine; (3) a cleavable linker having the amino acid sequence of SEQ ID NO:3; and (4) a peptide having the formula [X 2Y 2 Y 2 X 2 ] m’ wherein m is 3 and each X 2 is glutamic acid, and each Y 2 is leucine; (5) an anchor group having any structure of formula IIA-IID; and (6) an amine-terminated glycine moiety which may have the structure, for example, -G-Nal-G', where G is glycine, NaI is naphthylalanine, and G' is -C(O)-N(R a ) groups, each R a is independently hydrogen or aliphatic, and a C-terminal group.

[0146] In another non-limiting embodiment, the delivery agent has (1) an N-terminal group (e.g., an acetyl group) and (2) a group of formula [X 1 Y 1 Y 1 X 1 ] m wherein m is 3 and each X 1 is glutamic acid, and each Y 1 is leucine; (3) a cleavable linker having the amino acid sequence of SEQ ID NO:3; and (4) a peptide having the formula [X 2 Y 2 Y 2 X 2 ] m’ wherein m is 3 and each X 2 is glutamic acid, and each Y 2 is leucine; (5) an anchor group having any structure of formula IIA-IID; and (6) an amine-terminated glycine moiety which may have the structure, for example, -G-Nal-G', where G is glycine, NaI is naphthylalanine, and G' is -C(O)-N(R a ) groups, each R a is independently hydrogen or aliphatic, and a C-terminal group.

[0147] In some embodiments of the present disclosure, the amino acid sequence of the lytic peptide group, the cleavable linker, and the mask peptide group comprises any one of SEQ ID NO:6 or 9. In some embodiments of the present disclosure, the amino acid sequence of the lytic peptide group, the cleavable linker, and the mask peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:6 or 9, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO:6 or 9. In certain non-limiting embodiments of the present disclosure, the amino acid sequence of the lytic peptide group, the cleavable linker, and the mask peptide group has an amino acid sequence that comprises or consists of SEQ ID NO:6 or 9.

[0148] In some embodiments of the present disclosure, the amino acid sequences of the lytic peptide group, the cleavable linker, the mask peptide group, and the amino acid portion of the anchor group comprise any one of SEQ ID NOs:7 or 10. In some embodiments of the present disclosure, the amino acid sequences of the lytic peptide group, the cleavable linker, the mask peptide group, and the amino acid portion of the anchor group have an amino acid sequence that is at least 85% identical to SEQ ID NO:7 or 10, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:7 or 10. In certain non-limiting embodiments of the present disclosure, the amino acid sequence of the lytic peptide group, the amino acid sequence of the cleavable linker, the mask peptide group, and the amino acid portion of the anchor group has an amino acid sequence comprising or consisting of SEQ ID NO:7 or 10.

[0149] In some embodiments of the present disclosure, the amino acid sequences of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portion of the anchor group, and the C-terminal portion comprise any one of SEQ ID NOs:8, 11, 13-15, 39, 149, or 171. In some embodiments of the present disclosure, the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portion of the anchor group, and the C-terminal portion have an amino acid sequence that is at least 85% identical to SEQ ID NO:8, 11, 13-15, 39, 149, or 171, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO:8, 11, 13-15, 39, 149, or 171. In certain non-limiting embodiments of the present disclosure, the amino acid sequence of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portion of the anchor group, and the C-terminal portion have an amino acid sequence comprising or consisting of SEQ ID NO:8, 11, 13-15, 39, 149, or 171.

[0150] SEQ ID NO:6:ELLEELLEELLEGFGFVGGQLLQQLLQQLLQ, in some aspects of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group.

[0151] SEQ ID NO:7:ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKK, where each K provides an anchor group, and in some aspects of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group.

[0152] SEQ ID NO:8:ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGXG, where each K provides an anchor group, X is naphthylalanine (Nal), the C-terminal G at position 36 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group.

[0153] SEQ ID NO:9:DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQ, in some aspects of the disclosure, the N-terminal D comprises an N-terminus modified with an acetyl group.

[0154] SEQ ID NO:10:DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQKK, where each K provides an anchor group, and in some aspects of the disclosure, the N-terminal D comprises an N-terminus modified with an acetyl group.

[0155] SEQ ID NO:11:DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQKKGXG, wherein each K provides an anchor group, X is NaI, the C-terminal G at position 36 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal D comprises an N-terminus modified with an acetyl group.

[0156] SEQ ID NO:13:ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKG, wherein each K provides an anchor group, X is NaI, the C-terminal G at position 34 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group or a fluorophore.

[0157] SEQ ID NO:14:ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGXG, wherein each K provides an anchor group, X is NaI, the C-terminal G at position 36 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group.

[0158] SEQ ID NO:15:ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGWG, where each K provides an anchor group, the C-terminal G at position 36 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group.

[0159] SEQ ID NO:39:ELLEELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKG, where each K provides an anchor group, the C-terminal G at position 42 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group.

[0160] SEQ ID NO:149:ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKG, where each K provides an anchor group, the C-terminal G at position 33 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal E comprises an N-terminus modified with an acetyl group.

[0161] SEQ ID NO:171:QLLEQLLQQLLEGFGFVGGQLLEQLLQQLLEKG, where each K provides an anchor group, the C-terminal G at position 33 is a modified glycine with a -C(O)-NH group, and in some embodiments of the disclosure, the N-terminal Q comprises an N-terminus modified with an acetyl group.

[0162] With reference to SEQ ID NOs: 6-11, 13-15, 39, 149, and 171, all of the N-terminal E, D, or Q amino acids of these sequences contain an N-terminal amine functionalized with an acetyl group.

[0163] Further illustrative, non-limiting examples of delivery agents are shown below, which further comprise a conjugated therapeutic agent attached via a chemical linker group and / or a second cleavable linker group.

[0164] [ka]

[0165] [ka]

[0166] With reference to delivery agents 1 and 2, SEQ ID NO:12 (DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQKKGXKG, where X is NaI, the Ks at positions 32 and 33 each provide an anchor group, the K at position 36 is functionalized with a second cleavable linker that is conjugated to a therapeutic agent via a chemical linker group, and the C-terminal G at position 37 is functionalized with an NH group) is the combined amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and C-terminal portion of delivery agents 1 and 2 shown above.

[0167] [ka]

[0168] Referring to delivery agent 3, SEQ ID NO:108 (ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGXKG, where X is NaI, the Ks at positions 32 and 33 each provide an anchor group, the K at position 36 is functionalized with a chemical linker group that is attached to a therapeutic agent, and the C-terminal G at position 37 is functionalized with an NH group) is the combined amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and the C-terminal portion of delivery agent 3 shown above.

[0169] [ka]

[0170] With reference to delivery agent 3, R 1 , R 2 , R 3 , and R 4are each independently H, CH, or cyclobutyl. SEQ ID NO:108 is the combined amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and the C-terminal portion of delivery agent 4 shown above.

[0171] [ka]

[0172] Referring to delivery agent 5, SEQ ID NO:235 (ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGKG, where the Ks at positions 32 and 33 each provide an anchor group, the K at position 35 is functionalized with a second cleavable linker group that is conjugated to a therapeutic agent via a chemical linker group, and the C-terminal G at position 36 is functionalized with an NH group) is the combined amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and the C-terminal portion of delivery agent 5 shown above.

[0173] [ka]

[0174] With reference to delivery agent 6, R 1 , R 2 , R 3 , and R 4 are each independently H, CH, or cyclobutyl. SEQ ID NO:270 (ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGKG, where the Ks at positions 32 and 33 each provide an anchor group, the K at position 35 is functionalized with a chemical linker group that is attached to a therapeutic agent, and the C-terminal G at position 36 is functionalized with an NH group) is the combined amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and the C-terminal portion of delivery agent 6 shown above.

[0175] [ka]

[0176] Referring to delivery agent 7, SEQ ID NO:236 (ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKKGKG, where the Ks at positions 32 and 33 each provide an anchor group, the K at position 35 is functionalized with a second cleavable linker group that is conjugated to a therapeutic agent via a chemical linker group, and the C-terminal G at position 36 is functionalized with an NH group) is the combined amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and the C-terminal portion of delivery agent 7 shown above.

[0177] [ka]

[0178] With reference to delivery agent 8, R 1 , R 2 , R 3 , and R 4 are each independently H, CH, or cyclobutyl, and SEQ ID NO:237 (ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKGKG, where the K at position 32 provides the anchor group, the K at position 35 is functionalized with a chemical linker group that is attached to a therapeutic agent, and the C-terminal G at position 36 is functionalized with an NH group) is the combined amino acid sequence of the lytic peptide group, cleavable linker, mask peptide group, amino acid portions of the two anchor groups, and the C-terminal portion of delivery agent 8 shown above.

[0179] In some embodiments of the present disclosure, the amino acid sequences of the lytic peptide group, the cleavable linker, the mask peptide group, the amino acid portion of the anchor group, and the C-terminal portion (together, all or part of the amino acid sequence of the disclosed delivery agent) are set forth in SEQ ID NOs: 12, 13, 14, 15, 18, 19, 21, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 42, 52, 53, 54, 55, 56, 57, 58, 59, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79, 82, 83, 87, 88, 89, 90, 91, 102, 103, 104, 105, 106, 107, 108, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 232, 233, 240, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 37 14, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 138, 139, 142, 143, 144, 146, 147, 149, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 2 4, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 228, 231, 232, 235 , 236, 237, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280. In certain embodiments of the present disclosure, all or part of the amino acid sequence of the disclosed delivery agents comprises any one of SEQ ID NOs.NO:12, 13, 14, 15, 18, 19, 21, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 42, 52, 53, 54, 55, 56, 57, 58, 59, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79, 82, 83, 87, 88, 89, 90, 91, 102, 103, 104, 105, 106, 107, 108, 113, 114, 1 18, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 138, 139, 142, 143, 144, 146, 147, 149, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 182, 183, 184, 185, 186 , 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 228, 231, 232, 235, 236, 237, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314 46, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280, e.g., SEQ ID NO:NO:12, 13, 14, 15, 18, 19, 21, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 42, 52, 53, 54, 55, 56, 57, 58, 59, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 79, 82, 83, 87, 88, 89, 90, 91, 102, 103, 104, 105, 106, 107, 108, 113, 114, 118, 119, 120, 121, 122, 123, 124, 1 25, 126, 127, 128, 129, 130, 131, 132, 133, 135, 138, 139, 142, 143, 144, 146, 147, 149, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 , 201, 202, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 228, 231, 232, 235, 236, 237, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280. In certain non-limiting embodiments of the present disclosure, all or part of the amino acid sequence of the disclosed delivery agent is identified as SEQ ID NO: 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280.NO:12, 13, 14, 15, 18, 19, 21, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 42, 52, 53, 54, 55, 56, 57, 58, 59, 69 , 70, 71, 72, 73, 74, 75, 76, 77, 79, 82, 83, 87, 88, 89, 90, 91, 102, 103, 104, 105, 106, 107, 108, 113, 114 , 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 138, 139, 142, 143, 144, 146, 147, 149, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 182, 183, 184, 185 , 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 228, 231, 232, 235, 236, 237 , 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280. In other specific non-limiting aspects of the present disclosure, all or part of the amino acid sequence of the disclosed delivery agent is selected from the group consisting of SEQ ID NOs.NO:12, 13, 14, 15, 18, 19, 21, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 42, 52, 53, 54, 55, 56, 57, 58, 59, 69 , 70, 71, 72, 73, 74, 75, 76, 77, 79, 82, 83, 87, 88, 89, 90, 91, 102, 103, 104, 105, 106, 107, 108, 113, 114 , 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 138, 139, 142, 143, 144, 146, 147, 149, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 182, 183, 184, 185 , 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 228, 231, 232, 235, 236, 237 , 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280. In certain embodiments of the disclosure, all or a portion of the amino acid sequence of the disclosed delivery agents has an amino acid sequence comprising or consisting of any one of SEQ ID NOs:8, 11, 39, 149, and 171.

[0180] IV. Compositions and Methods of Administration Provided herein are compositions comprising the delivery agents disclosed herein. In some examples, the compositions further comprise one or more additional therapeutic agents, adjuvants, carriers, buffers, detergents (such as deoxycholic acid), salts, lipids, stabilizers, emulsifiers, solubilizers, or any combination thereof. In some embodiments of the present disclosure, the compositions comprise a therapeutic agent that is covalently or non-covalently bound to a peptide-based delivery agent, or simply associated with the delivery agent. In yet further embodiments of the present disclosure, two separate compositions can be administered, with one composition comprising a therapeutic agent and the other composition comprising a delivery agent.

[0181] The method of administering the disclosed delivery agent is routine and can be determined by a clinician.The disclosed delivery agent can be administered in various dosage regimens, such as being combined with one or more therapeutic agents.The disclosed delivery agent is generally administered topically, intranasally, intravenously, orally, intracranially, intramuscularly, parenterally, or as an implant, but rectal or vaginal administration is also possible in principle.In some embodiments of the present disclosure, the disclosed delivery agent is administered intraperitoneally, intratumorally, intravitreally, intracerebrally, transcorneally or intraocularly, intracerebrally, intraepithelially, intradermally, subcutaneously, transdermally, intramuscularly, or via intraventricular injection.The disclosed delivery agent and one or more therapeutic agents can be administered to a subject using a combination of these techniques.

[0182] The therapeutically effective amount may be administered in a single or multiple doses. Administration may include daily or several days, or less than daily (such as weekly or monthly), for a period of several days to several weeks or months, or even years. Specific, non-limiting examples include monthly administration, once every three weeks, once every two weeks, weekly administration, twice weekly administration, or daily administration, or combinations thereof. The specific mode / method of administration and dosing regimen will be selected by a laboratory technician, attending physician, or veterinarian (in the case of administration to non-human animal subjects) taking into account the characteristics of the case (subject, disease, associated symptoms / severity, specific administration, whether the treatment is prophylactic, etc.).

[0183] Multiple routes of administration of the delivery agent may be used, such as intratumoral, intravenous, intraperitoneal, intramuscular, subcutaneous, oral, or topical (e.g., in combination with one or more therapeutic agents), and certain routes may provide a more rapid and effective response than others. The disclosed delivery agent and one or more therapeutic agents may be administered by the same route or different routes. In some embodiments of the present disclosure, the delivery agent and one or more therapeutic agents are administered using any suitable administration route, such as intravenous administration or intratumoral administration. In exemplary embodiments of the present disclosure, the delivery agent and one or more therapeutic agents may be administered by intratumoral injection. Alternatively or additionally, the delivery agent and one or more therapeutic agents may be administered intravenously.

[0184] In some embodiments of the present disclosure, a subject may be administered one or more doses of a delivery agent and one or more therapeutic agents at one or more different time intervals, at various concentrations. In some embodiments of the present disclosure, the disclosed delivery agent and one or more therapeutic agents are administered so that the effective period of the delivery agent overlaps with the administration of one or more therapeutic agents. In some embodiments of the present disclosure, the administration of the delivery agent and one or more therapeutic agents is carried out so that the delivery agent and one or more therapeutic agents are internalized into cells, such as target cells, in the same endosome of the cell.

[0185] Pharmaceutical compositions containing a therapeutically effective amount of the disclosed delivery agents and / or one or more therapeutic agents are useful in the disclosed methods. These pharmaceutical compositions may include any suitable carrier. For example, formulations suitable for intratumoral, intravenous, intramuscular, subcutaneous, intraperitoneal, or topical administration may comprise a sterile aqueous solution of the active components. Such formulations may be prepared by dissolving the therapeutic agent and / or additional active and / or inactive components in water containing physiologically compatible substances, such as sodium chloride (e.g., 0.1-2.0 M), glycine, and the like, and having a buffered pH compatible with physiological conditions, to produce an aqueous solution, and then sterilizing the solution. The appropriate carrier may be the same for some (e.g., two or more) of the delivery agent and all of the one or more therapeutic agents being administered, or it may be different for all of the delivery agent and one or more therapeutic agents.

[0186] Suitable solid or liquid pharmaceutical formulation forms include, for example, aerosols, (micro)capsules, creams, drops, infusions or infusions in the form of ampoules, emulsions, granules, powders, suppositories, suspensions, syrups, tablets, coated tablets, and sustained-release preparations of active compounds, which are typically prepared using excipients and additives and / or adjuvants such as binders, coating agents, disintegrants, flavorings, lubricants, solubilizers, sweeteners, or bulking agents, as described above. Pharmaceuticals are suitable for use in various drug delivery systems. For a brief review of various methods of drug delivery, see Langer, "New Methods of Drug Delivery," Science 249:1527-1533 (1990). This document is incorporated herein by reference to the extent that it does not contradict the present disclosure.

[0187] The delivery agents of the present disclosure may be formulated into therapeutically active pharmaceutical agents that can be administered to a subject parenterally or orally. Parenteral routes of administration include, but are not limited to, epidermal, intra-arterial, intramuscular (IM and depo-IM), intraperitoneal (IP), intravenous (IV), intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, gastric injection, subcutaneous injection (subcutaneous (SQ and depo-SQ), transdermal, topical, and ophthalmic.

[0188] The disclosed delivery agents can be mixed or combined with suitable pharmaceutically acceptable excipients, such as in combination with one or more therapeutic agents, to prepare pharmaceutical preparations. Pharmaceutically acceptable excipients include, but are not limited to, alumina, aluminum stearate, buffers (such as phosphates), glycine, ion exchangers (such as those that aid in controlled release of charged substances), lecithin, partial glyceride mixtures of saturated vegetable fatty acids, potassium sorbate, serum proteins (such as human serum albumin), sorbic acid, water, salts or electrolytes (such as cellulose-based substances), colloidal silica, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols (such as polyethylene glycol), polyethylene polyoxypropylene block polymers, polyvinylpyrrolidone, potassium hydrogen phosphate, protamine sulfate, Group 1 halide salts (such as sodium chloride), sodium carboxymethylcellulose, waxes, wool fat, zinc salts, and the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers.

[0189] Upon mixing or adding one or more of the disclosed delivery agents, the resulting mixture may be a solid, solution, suspension, emulsion, or the like. These may be prepared according to a suitable method. The form of the resulting mixture depends on several factors, including the intended method of administration and the solubility of the agent in the selected carrier. Pharmaceutical carriers suitable for administering the disclosed delivery agents include any such carriers known to be suitable for the particular method of administration. In addition, the disclosed delivery agents may be mixed with other inactive or active substances (such as one or more therapeutic agents) that do not impair the desired action, or with substances that supplement the desired action or have a different action.

[0190] Solubilization methods can be used when the delivery agent is not sufficiently soluble in the carrier. Such methods include, but are not limited to, dissolution in aqueous bicarbonate, using cosolvents such as dimethyl sulfoxide (DMSO), and using surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, Delaware).

[0191] The disclosed delivery agents may be prepared with carriers that protect them from rapid elimination from the body, such as coatings or sustained-release formulations. Such carriers include, but are not limited to, controlled-release formulations, such as microencapsulated delivery systems. The disclosed delivery agents are contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically beneficial effect, typically an amount that avoids undesirable side effects in the treated subject. The therapeutically effective concentration can be empirically determined by testing the compound in known in vitro and in vivo model systems for the condition or disease being treated. For example, mouse models of a condition or disease of interest, such as a genetic disease, rare disease, cancer, immune disorder, or infectious disease, can be used to determine an effective amount or concentration, which can then be applied to other subjects, such as humans.

[0192] Injection solutions or suspensions can be formulated using suitable non-toxic, parenterally acceptable diluents or solvents such as 1,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer's solution, normal saline, or water, or sterile, bland fixed oils including synthetic mono- or diglycerides and fatty acids such as oleic acid, natural vegetable oils such as coconut oil, cottonseed oil, peanut oil, and sesame oil, glycerin, polyethylene glycol, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol and methylparabens, antioxidants such as ascorbic acid and sodium bisulfite, buffers such as acetates, citrates, and phosphates, suitable dispersing or wetting agents and suspending agents such as chelating agents such as ethylenediaminetetraacetic acid (EDTA), tonicity adjusting agents such as sodium chloride, dextrose, and combinations thereof. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, etc. may be incorporated as needed. For intravenous administration, suitable carriers include saline, phosphate buffered saline (PBS), and solutions containing viscosity enhancers and solubilizers, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.

[0193] For topical application, the disclosed delivery agent can be combined with one or more therapeutic agents and made into cream, lotion, ointment, solution or suspension in a suitable aqueous or non-aqueous carrier.Topical application can also be achieved by a transdermal patch or bandage containing a therapeutic agent.Additives such as buffering agents such as sodium metabisulfite or disodium edetate, preservatives such as phenylmercuric acetate or nitrate, bactericides and fungicides including benzalkonium chloride or chlorhexidine, thickeners such as hypromellose, etc. can also be included.

[0194] When the disclosed delivery agents are orally administered as a suspension, for example in combination with one or more therapeutic agents, the pharmaceutical preparation can be prepared according to appropriate pharmaceutical formulation techniques and can contain suspending agents such as alginic acid or sodium alginate, bulking agents such as microcrystalline cellulose, viscosity enhancers such as methylcellulose, and sweeteners / flavorings. Oral liquid preparations can contain conventional additives, such as suspending agents such as gelatin, glucose syrup, hydrogenated edible fats, methylcellulose, sorbitol, syrup, emulsifiers such as acacia, lecithin, and sorbitan monooleate, non-aqueous carriers (including edible oils) such as almond oil and fractionated coconut oil, oily esters such as glycerin, propylene glycol, and ethyl alcohol, preservatives such as methyl or propyl p-hydroxybenzoates or sorbic acid, and, if desired, conventional flavors or coloring agents. When formulated as immediate-release tablets, these agents may contain dicalcium phosphate, lactose, magnesium stearate, microcrystalline cellulose, starch, and / or other binders, diluents, disintegrants, excipients, fillers, and lubricants.

[0195] When oral administration is desired, the disclosed delivery agent can be provided in a composition that protects it from the acidic environment of the stomach, for example, in combination with one or more therapeutic agents.For example, the disclosed delivery agent can be formulated with an enteric coating that maintains its integrity in the stomach and releases active compound in the intestine.The disclosed delivery agent can also be formulated in combination with antacids or other such ingredients.

[0196] Oral compositions generally contain inert diluents or edible carriers, can be compressed into tablets, and can be enclosed in gelatin capsules.For oral therapeutic administration, the disclosed delivery agent can be combined with excipients and used in the form of capsules, tablets, or lozenges.Pharmaceutically compatible auxiliary substances or binders can be included as part of the composition.

[0197] Capsules, pills, tablets, troches and the like may contain any of the following ingredients or compounds of a similar nature: binders such as, but not limited to, acacia, corn starch, gelatin, tragacanth, polyvinylpyrrolidone, or sorbitol; fillers such as calcium phosphate, glycine, lactose, microcrystalline cellulose, or starch; disintegrants such as, but not limited to, alginic acid and corn starch; lubricants such as, but not limited to, magnesium stearate, polyethylene glycol, silica, or talc; flow agents such as, but not limited to, colloidal silicon dioxide; sweeteners such as sucrose or saccharin; disintegrants such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and flavorings such as peppermint, methyl salicylate, or fruit flavors.

[0198] When the dosage unit is a capsule, it can contain, in addition to the above-mentioned materials, liquid carriers such as fatty oils.In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, such as sugar coating and other enteric agents.The disclosed delivery agent can also be administered as a component of elixirs, suspensions, syrups, wafers, tea, chewing gum, etc.Syrups can contain, in addition to the active compound, sucrose or glycerin as a sweetener, and certain preservatives, dyes, colorings, and flavorings.

[0199] When orally administered, compound can be administered in the usual dosage form for oral administration.These dosage forms include the usual solid unit dosage forms such as tablets and capsules, as well as the liquid dosage forms such as solution, suspension and elixir.When using solid dosage form, it can be sustained-release type, so that compound can be administered less frequently.

[0200] In some examples, the disclosed delivery agents, for example, in combination with one or more therapeutic agents, are injected into the stomach of a subject and incorporated into the subject's entire body, such as various muscle groups. Examples of methods and compositions for administering therapeutic substances, including proteins, include those discussed in Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 2nd ed. (2005); Mahato, Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids (2004); McNally, Protein Formulation and Delivery, 2nd ed. (2007); and Kumar et al., "Novel Delivery Technologies for Protein and Peptide Therapeutics," Current Pharm. Biotech., 7:261-276 (2006). Each of these is incorporated herein by reference to the extent not inconsistent with the present disclosure.

[0201] In some embodiments of the present disclosure, delivery agents and / or therapeutic agents can be administered via nanoparticle vehicles. Nanoparticles (NPs) are synthetic particles ranging in size from one to several hundred nanometers, containing an inorganic core surrounded by an organic layer. Nanoparticles featuring inorganic cores such as gold, silica, and superparamagnetic iron oxide (SPIO) are available. For example, in cancerous tissue, NPs leach out of the leaky cancer vasculature at a higher rate than in healthy tissue and remain in the area due to the enhanced permeability and retention (EPR) effect. In some embodiments of the present disclosure, the delivery agent and / or therapeutic agent may be administered via lipid nanoparticles, peptide-based nanoparticles (Bioactive Materials Volume 11, May 2022, pp. 268-282), nanotubes (e.g., NanoPortal, NanoPrecision Medical, Emeryville, CA, USA), subcutaneously implanted reservoirs (e.g., NANOPOR®, Delpore, Brisbane, CA, USA), long-acting injectables (e.g., SABER® or CLOUD™, Durect, Cupertino, CA, USA), and / or transdermal microneedle systems (Zosano Pharma, Fremont, CA, USA). In certain embodiments of the present disclosure, the nanoparticles may be combined with the delivery agent and / or therapeutic agent as follows: (1) the delivery agent and / or therapeutic agent is encapsulated within the nanoparticle vehicle and / or (2) the delivery agent and / or therapeutic agent is covalently bound, coupled, linked, or associated with the exterior of the nanoparticle vehicle. To assess the delivery performance of embodiments using nanoparticles in combination with a delivery agent and / or a therapeutic agent, the HeLa Luc / 705 cell luciferase assay described herein may be used. Alternatively or additionally, biological cell assays may be used that measure (i) changes in cellular or biomolecular phenotype, or (ii) changes in the expression of molecular markers due to delivery of a therapeutic agent or delivery of an exogenous biological molecule, such as, for example, measuring fluorescence from green fluorescent protein (GFP) expression after delivery of GFP RNA into cells.By way of example only, when GFP RNA with and without a delivery agent is encapsulated within nanoparticle vehicles, and when biological cells are treated with the nanoparticle vehicles in growth medium for 24 hours or more, the average increase in fluorescence from cells treated with nanoparticles containing the delivery agent is significantly greater than cells treated with nanoparticles without the delivery agent.

[0202] The dosage of each delivery agent and / or each one or more therapeutic agents administered to a subject should be sufficient to elicit a beneficial therapeutic response in the subject over time, such as reducing the burden of a genetic disease, rare disease, cancer, immune disease, or infectious disease in the subject, increasing survival in the subject, reducing the incidence of recurrence of a genetic disease, rare disease, cancer, immune disease, or infectious disease in the subject, or a combination thereof. A beneficial therapeutic response may require one or more administrations of one or more disclosed delivery agents and / or one or more therapeutic agents, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 or more administrations, administered simultaneously or at different times. In some embodiments, an effective amount of the disclosed delivery agent, such as when combined with a therapeutic agent, can be administered as a single dose every three or four months, monthly, weekly, or daily, or can be divided into at least two unit doses for administration over a period of time. Treatment can be continued for as long as necessary to achieve the desired results. For example, treatment can continue for 3 or 4 weeks up to 12-24 months or longer, including continuous treatment. The compound can be administered in several doses intermittently, such as every few days (e.g., at least every 2, 3, 4, 5, or 10 days) or every few weeks (e.g., at least every 2, 3, 4, 5, or 10 weeks).

[0203] Dosages may vary or may be the same for each subject. Appropriate dosages may be determined, for example, using routine experimentation. Generally, administration of the disclosed delivery agent and / or one or more therapeutic agents results in a potent, synergistic therapeutic effect for treating a subject's genetic disease, rare disease, cancer, immune disease, or infectious disease, for example, compared to using the therapeutic agent without the delivery agent. Thus, a specific administration regimen may be tailored to a particular subject, the condition being treated, or the desired outcome. For example, when the disclosed method is used to treat a target disease or condition (such as a genetic disease (e.g., muscular dystrophy), a rare disease, cancer, an immune disease, or an infectious disease), an initial treatment regimen may be applied to suppress symptoms. Such an initial treatment regimen may include administering a higher dose of the disclosed delivery agent or administering such substance more frequently, for example, daily. After the desired therapeutic outcome is achieved, a second treatment regimen may be applied, such as administering a lower dose of the delivery agent or administering such substance less frequently, for example, monthly, bimonthly, quarterly, or semi-annually. In such cases, the second treatment regimen can act as a "booster" to restore or maintain muscle regeneration at a desired level. Similar treatment regimens can also be used in other subjects with reduced or impaired muscle regeneration capacity, such as elderly subjects.

[0204] The effective amount for various therapeutic treatments of the present disclosure may depend on the severity of the symptom or disease, the body weight and general condition of the subject, the absorption, inactivation, and excretion rates of the delivery agent (and any one or more therapeutic agents administered to the subject with the delivery agent), the administration schedule, and the dosage, as well as other factors recognizable in the art, particularly with the benefit of the present disclosure. It will also be apparent that the exact dosage and frequency of administration will vary depending on the specific delivery agent or other therapeutic agent administered, the specific symptom being treated, the severity of the symptom being treated, the age, body weight, and general physical condition of the specific subject, and other medications the subject may be taking. Typically, dosages used in vitro can provide useful guidance regarding amounts useful for in vivo administration of pharmaceutical compositions, and animal models can be used to determine effective dosages for treating specific diseases. For example, determining effective dosages using mouse models of a particular symptom or disease (such as genetic disorders, rare diseases, cancer, immune disorders, infectious diseases, etc.) can be translated to dosages for other subjects, such as humans. Various considerations in determining dosage are described, for example, in Gilman et al. (eds.), Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press (1990), and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa. (1990), each of which is incorporated herein by reference to the extent not inconsistent with the present disclosure.

[0205] In specific examples, the disclosed delivery agents are administered to a subject in an amount sufficient to provide a dosage of the delivery agent in the range of 10 nmol / g to 500 nmol / g, e.g., 2 nmol / g to 20 nmol / g, or 2 nmol / g to 10 nmol / g. In additional examples, the delivery agents are administered to a subject in an amount sufficient to provide a dosage of 0.01 μg / kg to 1000 mg / kg or 0.1 mg / kg to 1000 mg / kg, and in certain examples, this amount is provided per day or per week. In another example, the disclosed delivery agents are administered to a subject in an amount sufficient to provide a drug dosage of between 0.2 mg / kg and 2 mg / kg. In another example, the disclosed delivery agent is administered to a subject in an amount sufficient to provide a drug dosage of between 1.0 mg / kg and 30 mg / kg, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg / kg. In a further example, the delivery agent is administered to a subject in an amount sufficient to provide a concentration of the delivery agent in the administered substance of between 5 nM and 500 nM, e.g., between 50 nM and 200 nM, or 100 nM. In a further example, the delivery agent is administered to a subject in an amount sufficient to provide a concentration of the delivery agent in the administered substance of between 0.1 μM and 500 μM, e.g., between 5 μM and 30 μM, or 10 μM. In other examples, the delivery agent is administered to a subject at 25 mg / ml to 1 mg / ml, e.g., 5 mg / ml to 1 mg / ml, 1000 μg / ml to 500 μg / ml, 500 μg / ml to 50 μg / ml, 500 μg / ml, 400 μg / ml, 300 μg / ml, 250 μg / ml, 200 μg / ml, 150 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 2.5 μg / ml, and 1.25 μg / ml.

[0206] Exemplary therapeutic agents that can be administered in combination with the disclosed delivery agents are provided herein.In some embodiments of the present disclosure, any therapeutic agent that is active in the cytosol of cells can be used with the disclosed delivery agents and methods.In certain embodiments of the present disclosure, the therapeutic agent used herein is an agent that, when combined with the disclosed delivery agent, can be internalized into cells (such as target cells in a subject or cell culture) at a higher rate than without the delivery agent, and / or can have a greater therapeutic effect in a subject than without the delivery agent.In some embodiments of the present disclosure, one or more therapeutic agents are chemotherapeutic agents, therapeutic antibodies, immunotherapeutic agents, antibiotics, therapeutic agents for psychiatric disorders, therapeutic agents for substance abuse disorders, or combinations thereof. In certain non-limiting embodiments of the present disclosure, the one or more therapeutic agents are saporin, cisplatin, methotrexate, fluorouracil, doxorubicin, cyclophosphamide, chlorambucil, vinblastine, vincristine, docetaxel, or paclitaxel, chlorhexidine, triclosan, xylitol, or octadecen-1-amine hydrofluoride, 1-hexadecylamine hydrofluoride, gefitinib, lapatinib, olaparib, mitomycin C, sunitinib, geftinib, nintedanib, PD173074, erdaftinib, sorafenib, or a combination thereof.

[0207] In specific examples, a therapeutic agent is administered to a subject in an amount sufficient to provide a dosage of the therapeutic agent in the range of 10 nmol / g to 500 nmol / g, e.g., 2 nmol / g to 20 nmol / g, or 2 nmol / g to 10 nmol / g. In additional examples, a therapeutic agent is administered to a subject in an amount sufficient to provide a dosage of 0.01 μg / kg to 1000 mg / kg, or 0.1 mg / kg to 100 mg / kg, or 100 mg / kg to 1000 mg / kg, and in certain examples, this amount is provided per day or per week. In another example, a disclosed therapeutic agent is administered to a subject in an amount sufficient to provide a dosage of the agent of 0.2 mg / kg to 2 mg / kg. In further examples, a therapeutic agent is administered to a subject in an amount sufficient to provide a concentration of the therapeutic agent in the administered substance of 5 nM to 500 nM, e.g., 50 nM to 200 nM, or 100 nM, etc. In a further example, the therapeutic agent is administered to a subject in an amount sufficient to provide a concentration of the therapeutic agent in the administered substance of between 0.1 μM and 500 μM, e.g., between 0.1 μM and 50 μM, or 20 μM. In other examples, the therapeutic agent is administered to a subject at between 500 μg / ml and 1 μg / ml, e.g., between 300 μg / ml and 3 μg / ml, 200 μg / ml and 20 μg / ml, 500 μg / ml, 400 μg / ml, 300 μg / ml, 250 μg / ml, 200 μg / ml, 150 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 2.5 μg / ml, and 1.25 μg / ml.

[0208] Therapeutic antisense and ribozyme molecules can be used in combination with the delivery agents disclosed herein and in the methods disclosed herein. The use of the disclosed delivery agents in combination with antisense or ribozyme molecules can enhance the internalization and therapeutic activity of antisense or ribozyme molecules, thereby increasing the efficacy of antisense or ribozyme molecules, such as in subjects with targeted symptoms or diseases (such as genetic diseases, rare diseases, cancer, immune diseases, or infectious diseases). Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In cells, antisense nucleic acids hybridize with corresponding mRNA to form double-stranded molecules. Because cells do not translate double-stranded mRNA, antisense nucleic acids interfere with mRNA translation. In some embodiments of the present disclosure, the antisense molecules used herein are mRNA, miRNA, siRNA, or CRISPR-associated molecules. In one embodiment, a therapeutic antisense oligomer of 10-25 nucleotides is administered to a subject in combination with a delivery agent as disclosed. The use of antisense methods to inhibit gene translation is described, for example, in Marcus-Sakura, Anal. Biochem. 172:289, 1988.

[0209] Generally, the principle behind antisense technology is that antisense compounds hybridize to target nucleic acids and affect the regulation of gene expression activity or function, such as transcription, translation, or splicing. Regulation of gene expression can be achieved, for example, by degradation or occupancy-based inhibition of the target RNA. One example of modulation of target RNA function through degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound, such as an antisense oligonucleotide. Antisense oligonucleotides can also be used to regulate gene expression, such as splicing, through occupancy-based inhibition, such as blocking access to splice sites. Antisense compounds provide sequence-specific target gene regulation. This sequence specificity makes antisense compounds effective tools for selectively regulating target nucleic acids of interest. In some disclosed embodiments, expression of one or more genes of interest is inhibited by at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to a control.

[0210] The disclosed delivery agents and delivery methods contemplate the use of any type of antisense compound that specifically targets and controls the expression of one or more genes of interest. Such antisense compounds include single-stranded compounds, such as antisense oligonucleotides, and double-stranded compounds, including compounds with at least a partial double-stranded structure (e.g., siRNA, miRNA, shRNA, ribozymes, etc.). Antisense molecules may also contain flanking sequences (e.g., regulatory sequences). Ribozymes may have any common structure, including, but not limited to, hairpin, hammerhead, or axehead structures, as long as the molecule cleaves RNA. In some embodiments of the disclosure, a viral vector containing a therapeutically effective amount of the disclosed delivery agent and a nucleic acid inhibitor is administered to a subject or cells in cell culture.

[0211] The antisense oligonucleotides used in the disclosed delivery agents and methods can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. Antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions. For example, antisense nucleic acid molecules can be chemically synthesized using various modified nucleotides, such as phosphorothioate derivatives and acridine-substituted nucleotides, designed to increase the biological stability of naturally occurring nucleotides or molecules or to increase the physical stability of the duplex formed between the antisense nucleic acid and the sense nucleic acid. Examples of modified nucleotides that can be used to generate antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylketone, inosine, etc. Methods exist for screening antisense compounds for specificity (see, eg, US Patent Application Publication No. 2003 / 0228689).

[0212] Ribozymes, which are RNA molecules that have the ability to specifically cleave other single-stranded RNA in a manner similar to DNA restriction endonucleases, are also useful.By modifying the nucleotide sequence encoding these RNAs, it is possible to design molecules that recognize and cleave specific nucleotide sequences within RNA molecules (Cech, J.Amer.Med.Assn.260:3030,1988).The advantage of this approach is that it is sequence-specific, so only mRNAs with specific sequences are inactivated.

[0213] There are two basic types of ribozymes: Tetrahymena-type (Hasselhoff, Nature 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences four bases long, while "hammerhead"-type ribozymes recognize sequences 11–18 bases long. The longer the recognition sequence, the greater the likelihood that the sequence will appear exclusively in the target mRNA species. As a result, hammerhead ribozymes are more suitable than Tetrahymena-type ribozymes for inactivating specific mRNA species, and 18-base recognition sequences are more suitable than shorter recognition sequences.

[0214] A variety of delivery systems are available that can be used to administer siRNA and other inhibitory nucleic acid molecules as therapeutic agents, including, for example, liposomes, microparticles, microcapsules, nanoparticles, encapsulation in recombinant cells capable of expressing the therapeutic molecule (see, e.g., Wu et al., J. Biol. Chem. 262, 4429, 1987), construction of the therapeutic nucleic acid as part of a retrovirus or other vector, and the like.

[0215] In other non-limiting aspects of the present disclosure, the therapeutic agent is a peptide nucleic acid (PNA) (e.g., as described in Nielsen and Egholm, Current Issues Molec. Biol. (1999) 1(2):89-104, the related PNAs of which are incorporated herein by reference). Like DNA, synthetic PNAs bind to complementary nucleic acid strands and can specifically target and regulate the expression of one or more genes of interest.

[0216] In certain non-limiting embodiments, the therapeutic agent is a morpholino. Representative morpholinos are described in Moulton et al., J Drug Discov Develop and Deliv, 3(2):1023, 2016, and related morpholinos are incorporated herein by reference. Morpholinos, such as phosphorodiamidate morpholino oligomers, are oligomeric molecules useful for blocking sites on nucleic acids, such as target RNA molecules, and have a wide range of potential therapeutic applications, including targeting pathogens and genetic diseases. Morpholinos are specific, soluble, non-toxic, stable, and effective antisense therapeutic agents. They can target a wide range of RNAs, for example, blocking the translation of RNA molecules (such as mRNAs), modifying pre-mRNA splicing, or inhibiting miRNA maturation and activity. Morpholinos can be delivered to a range of cultured cells, embryos, and multicellular subjects (e.g., mammals, including humans and non-human animals), and administering the disclosed delivery agents in combination with one or more therapeutic morpholinos can enhance the internalization and therapeutic effect of the one or more morpholinos. In another embodiment, the therapeutic agent is a peptide-modified morpholino oligomer (PPMO). In certain aspects of the present disclosure, morpholinos having any of the sequences GATCCATGGACATTTGACTGGTATTTCC (SEQ ID NO: 240), CAGCGAGACAACCAG (SEQ ID NO: 241), TCCACTGGAAGAAGTTGATTATTTC (SEQ ID NO: 242), AGAGACAGCGAGACA (SEQ ID NO: 243), and GCTTCCTGAATGCCCAAAGAAACAC (SEQ ID NO: 244) may be used.

[0217] (V. Method) (A. Cell Contact Method) Provided herein are methods of contacting cells with a disclosed composition comprising a delivery agent described herein (e.g., contacting cells with an effective amount of the disclosed delivery agent). In embodiments of the disclosure, the delivery agent of the composition delivers one or more therapeutic agents to the cytosol of the cell. The disclosed delivery agent can be contacted with the cell in the same composition as the one or more therapeutic agents or in a different composition, simultaneously with or at a different time from the one or more therapeutic agents. In some embodiments of the disclosure, the cell is contacted with the disclosed delivery agent (e.g., in combination with one or more therapeutic agents) for at least 1-60 minutes, e.g., at least 1-10 minutes, 10-20 minutes, 20-30 minutes, 30-40 minutes, 40-50 minutes, or 50-60 minutes, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 7 For example, the contacting may be for 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes. In some embodiments of the present disclosure, cells are contacted with a disclosed delivery agent (e.g., in combination with one or more therapeutic agents) for at least 1-40 days, e.g., at least 1 day, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, at least 35 days, 10-30 days, 10-20 days, 20-30 days, 15-25 days, e.g., at least 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, or 40 days. In some embodiments of the present disclosure, exosomes are added once to the cell culture medium. In other aspects of the present disclosure, exosomes are added to the culture medium of cells multiple times, such as 2-5 times, such as 2, 3, 4, or 5 times, over the course of the culture.

[0218] In specific examples, cells are contacted with the disclosed delivery agents at a dosage of 1 nM to 500 nM, e.g., 1 nM to 50 nM, 50 nM to 100 nM, 100 nM to 150 nM, 150 nM to 200 nM, 200 nM to 250 nM, 250 nM to 300 nM, 300 nM to 350 nM, 350 nM to 400 nM, 400 nM to 450 nM, or 450 nM to 500 nM of delivery agent. In additional examples, cells are contacted with a disclosed delivery agent at a delivery agent dosage of 0.5 μM to 100 μM, e.g., 0.50 μM to 1 μM, 1 μM to 5 μM, 5 μM to 10 μM, 10 μM to 25 μM, 25 μM to 50 μM, 50 μM to 75 μM, 75 μM to 100 μM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 μM. In another example, cells are contacted with a disclosed delivery agent at a delivery agent dosage of between 500 μg / ml and 1 μg / ml, e.g., between 300 μg / ml and 3 μg / ml, between 200 μg / ml and 20 μg / ml, 500 μg / ml, 400 μg / ml, 300 μg / ml, 250 μg / ml, 200 μg / ml, 150 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 2.5 μg / ml, and 1.25 μg / ml. In another example, cells are contacted with a delivery agent at a dosage of between 0.5 ng / kg and 500 ng / kg, e.g., between 0.5 μg / kg and 500 μg / kg, e.g., between 0.5 mg / kg and 500 mg / kg.

[0219] In specific examples, cells are contacted with one or more therapeutic agents at a dosage of 1 nM to 500 nM, e.g., 1 nM to 50 nM, 50 nM to 100 nM, 100 nM to 150 nM, 150 nM to 200 nM, 200 nM to 250 nM, 250 nM to 300 nM, 300 nM to 350 nM, 350 nM to 400 nM, 400 nM to 450 nM, or 450 nM to 500 nM of the one or more therapeutic agents. In additional examples, cells are contacted with one or more therapeutic agents at a dosage of one or more therapeutic agents between 0.5 μM and 100 μM, e.g., between 0.50 μM and 1 μM, between 1 μM and 5 μM, between 5 μM and 10 μM, between 10 μM and 25 μM, between 25 μM and 50 μM, between 50 μM and 75 μM, between 75 μM and 100 μM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 μM. In other examples, the cells are contacted with one or more therapeutic agents at one or more therapeutic agent dosages between 500 μg / ml and 1 μg / ml, e.g., between 300 μg / ml and 3 μg / ml, between 200 μg / ml and 20 μg / ml, 500 μg / ml, 400 μg / ml, 300 μg / ml, 250 μg / ml, 200 μg / ml, 150 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 2.5 μg / ml, and 1.25 μg / ml. In another example, the cells are contacted with one or more therapeutic agents at a dosage of 0.5 ng / kg to 500 ng / kg, for example, 0.5 μg / kg to 500 μg / kg, for example, 0.5 mg / kg to 500 mg / kg.

[0220] In some examples, contacting a cell with a disclosed delivery agent and one or more therapeutic agents increases the activity of the one or more therapeutic agents (e.g., as measured using methods such as IC50, Emax, and / or area under the dose-response curve (AUC)) by 2-fold to at least 2,000-fold, e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 90 ...0-fold, at least 1500-fold, at least 2000-fold, at least 250-fold, at least 300- 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, at least 800-fold, at least 850-fold, at least 900-fold, at least 950-fold, at least 1,000-fold, at least 1,100-fold, at least 1,150-fold, at least 1,200-fold, at least 1,250-fold, at least 1,300-fold, at least 1,350-fold, at least 1,400-fold, at least 1,450-fold, at least 1,500-fold, at least 1,550-fold, at least 1,600-fold, at least 1,650-fold, at least 1,700-fold, at least 1,750-fold, at least 1,800-fold, at least 1,850-fold, at least 1,900-fold, at least 1,950-fold, or at least 2,000-fold (e.g., when cells are contacted with one or more therapeutic agents and compared to the absence of the delivery agent).

[0221] In some examples, contacting cells with a disclosed delivery agent and one or more therapeutic agents increases cell viability (e.g., as measured using methods such as IC50, Emax, and / or area under the dose-response curve (AUC)) by 2-fold to at least 2,000-fold, e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, or at least 500-fold. , at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, at least 800-fold, at least 850-fold, at least 900-fold, at least 950-fold, at least 1,000-fold, at least 1,100-fold, at least 1,150-fold, at least 1,200-fold, at least 1,250-fold, at least 1,300-fold, at least 1,350-fold, at least 1,400-fold, at least 1,450-fold, at least 1,500-fold, at least 1,550-fold, at least 1,600-fold, at least 1,650-fold, at least 1,700-fold, at least 1,750-fold, at least 1,800-fold, at least 1,850-fold, at least 1,900-fold, at least 1,950-fold, or at least 2,000-fold (e.g., compared to contacting the cells with one or more therapeutic agents and no delivery agent).

[0222] In certain embodiments of the present disclosure, contacting a cell with the composition induces endosomal membrane dissolution after the therapeutic agent and delivery agent are internalized within the cell, thereby delivering the therapeutic agent into the cytosol of the cell. In other specific embodiments of the present disclosure, contacting a cell with the composition causes pores to form in the endosomal membrane after one or more therapeutic agents and the disclosed delivery agents are internalized within the cell, thereby delivering one or more therapeutic agents into the cytosol of the cell. In other embodiments of the present disclosure, contacting a cell with the composition causes local membrane destabilization and / or permeabilization after the therapeutic agent and delivery agent are internalized within the cell, thereby delivering the therapeutic agent into the cytosol of the cell (Pei and Buyanova, Bioconjug Chem. 30(2):273-283, 2019).

[0223] In embodiments of the present disclosure, the cell may be contacted in vitro or in vivo. In some embodiments of the present disclosure, the cell is a non-human mammalian cell (e.g., a cell from a laboratory animal, including but not limited to, zebrafish or Xenopus laevis, or a cell from a veterinary animal, including but not limited to, dogs and cats, as well as mice, rats, rabbits, sheep, horses, cows, and non-human primates) or a human cell.

[0224] Also disclosed herein are methods for identifying therapeutic agents using cells, such as cells in a cell culture system. In some embodiments of the disclosure, the methods include contacting cells with a disclosed delivery agent and one or more test compounds (such as one or more potential therapeutic agents) and determining the effect of the one or more compounds on the contacted cells relative to a control. In some embodiments of the disclosure, a decrease in cell viability indicates that one or more compounds (or combination of compounds) are therapeutic agents (or combination of therapeutic agents) that can be used to treat a particular condition or disease of interest. In some examples, the methods include determining the IC of one or more test compounds. 50 This includes determining the value.

[0225] In some examples, this method is a high-throughput screening method. By using automation, data processing / control software, liquid handling equipment, and highly sensitive detectors, high-throughput screening can rapidly test large numbers (e.g., thousands or millions) of molecules (e.g., test compounds) to determine the effectiveness of each molecule for a desired purpose, such as the effectiveness of each molecule for treating a symptom or disease of interest (e.g., the effectiveness of each molecule for treating a genetic disease, rare disease, cancer, immune disease, or infectious disease). In some examples, cell-based therapeutic drug identification is performed in a microplate, such as a 24-well, 48-well, 96-well, or 384-well microtiter plate. In some embodiments of the present disclosure, result indicators, such as fluorescent or bioluminescent signals, such as indicators of cell viability or indicators of a particular cell phenotype (e.g., increased or decreased expression of a molecule of interest, such as a protein of interest), are detected using a microplate reader. The microplate reader detects biological, chemical, or physical events within the microtiter plate. A high-intensity lamp shines light onto the microtiter wells, and a detector quantifies the light emitted by the reaction within the wells. Detection modes for microplate assays include absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. In some embodiments of the present disclosure, fluorescence intensity is measured using a microplate reader such as the SPECTRAMAX® M5 (Molecular Devices), the ELX800™ absorbance microplate reader (BioTek), SpectraFluor (Tecan), or the VICTOR3™ (Perkin Elmer). Wavelengths appropriate for excitation and corresponding emission wavelengths can be used. Such wavelengths can range from 425 nm to 800 nm, e.g., 450 nm to 500 nm, or 450 nm to 495 nm, or 485 nm to 505 nm, e.g., 495 nm to 500 nm, or 620 nm to 800 nm, e.g., 620 nm to 750 nm. In a specific example, fluorescence intensity is measured using a Tecan SpectraFluor microplate reader, measuring excitation at 485 nm and emission at 535 nm.

[0226] In some embodiments of the present disclosure, methods include contacting cells with a disclosed delivery agent and one or more test compounds, e.g., adding the delivery agent and one or more test compounds to intact cells that represent a particular condition or disease of interest (such as a genetic disease, a rare disease, cancer, an immune disorder, or an infectious disease). The delivery agent and one or more test compounds are incubated with the intact cells for a period of time to allow the delivery agent and compound(s) to enter the cells. The delivery agent and one or more test compounds may be incubated with the cells for 1 to 120 minutes, e.g., 10 to 100 minutes, 20 to 90 minutes, 30 to 80 minutes, 40 to 70 minutes, 50 to 60 minutes, e.g., at least 5 minutes, e.g., 5, 10, 15, 20, 30, 60, 90, or 120 minutes. In some embodiments of the present disclosure, the delivery agent and one or more test compounds may be incubated with cells for 2 to 24 hours, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. The incubation is performed at a temperature that allows the delivery agent to internalize the one or more test compounds into the cells and release them from the endolysosome into the cytosol of the cells. For example, the incubation of the delivery agent and one or more test compounds with intact cells may be at about room temperature, such as about 20°C to about 25°C. In further embodiments of the present disclosure, the cells are incubated at a temperature of about 4°C to about 56°C, such as about 15°C to about 50°C, about 22°C to about 45°C, about 25°C to about 40°C, or about 30°C to about 37°C. In a particular example, the delivery agent and one or more test compounds are incubated with the intact cells for 60 minutes at 37° C. In another particular example, the delivery agent and one or more test compounds are incubated with the intact cells for 18 hours at 37° C.

[0227] In some embodiments of the present disclosure, cells are washed after incubation with a delivery agent and one or more test compounds to remove any delivery agent or test compound not internalized within the cells. Washing can be performed by standard methods, such as centrifuging the cells, removing the resulting supernatant, and resuspending the cells in a solution. The cells can be resuspended in a physiological buffer, such as phosphate-buffered saline, Hank's balanced salt solution, lactated Ringer's solution, or cell culture medium (e.g., RPMI-1640 or DMEM). The buffer can contain a small amount of solvent (e.g., 0.5% to 2% ethanol or methanol) or a carrier molecule (e.g., 1% to 4% glucose or fructose). The washing step can be repeated one to six times, such as once, twice, three times, four times, five times, or six times. In a specific example, the cells are washed three times by centrifuging at 400 x g for 2 to 10 minutes, removing the resulting supernatant, and resuspending the cells in phosphate-buffered saline.

[0228] In some examples, the screening method further includes selecting test compounds identified as having therapeutic activity (e.g., those that decrease expression of a protein of interest, induce a phenotype of interest in a cell, and / or decrease or enhance cell survival). In some examples, the screening method further includes administering such selected compounds, individually or in combination with a disclosed delivery agent, to a research animal, such as a research mammal, such as a rabbit, non-human primate, cat, dog, mouse, or rat, and further analyzing the effectiveness of the delivery agent and one or more test compounds for treating or inhibiting a symptom or disease of interest (such as a genetic disease, a rare disease, cancer, an immune disease, or an infectious disease).

[0229] (B. Treatment Methods for Subjects) Also provided herein is a method for administering a therapeutically effective amount of the disclosed composition to a subject. In certain embodiments of the present disclosure, the therapeutically effective amount of the composition treats, improves, or prevents the onset of a symptom or disease in a subject, such as a genetic disease, a rare disease, cancer, an immune disease, an infectious disease, a psychiatric disorder, or any combination thereof. Thus, disclosed is a method for treating a subject with a symptom or disease by administering a delivery agent disclosed herein, for example, a therapeutically effective amount of the disclosed delivery agent. In some embodiments of the disclosure, the delivery agent is administered in combination with a therapeutically effective amount of one or more therapeutic agents. In some embodiments of the present disclosure, the symptom or disease treated using the disclosed method is a genetic disease, a rare disease, cancer, an immune disease, an infectious disease, or a psychiatric disorder.In some embodiments of the present disclosure, the condition or disease is cancer (e.g., solid cancer (sarcoma (e.g., rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, alveolar soft tissue sarcoma)), carcinoma (e.g., colon cancer), and lymphoma such as Hodgkin's lymphoma or non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell ... lymphoma, AIDS-related or central nervous system lymphoma, etc.), neuroblastoma, gynecological cancer (such as uterine or ovarian cancer), breast cancer, liver cancer, lung cancer, prostate cancer, skin cancer, bone cancer, pancreatic cancer, brain cancer (neuroblastoma or glioma), head and neck cancer, kidney cancer (such as Wilms' tumor), retinoblastoma, adrenocortical tumor, desmoid tumor, desmoplastic small round cell tumor, endocrine tumors, and / or blood cancer (myeloma such as multiple myeloma, Hodgkin's lymphoma or non-Hodgkin's lymphoma) lymphomas, such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma, or leukemias, such as acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML)); immune disorders, such as autoimmune diseases or transplant rejection; or infectious diseases, such as cytomegalovirus, adenovirus, respiratory syncytial virus, Epstein-Barr virus, HIV infection, hepatitis virus infection (such as hepatitis C virus or hepatitis B virus), coronaviruses (such as COVID-19), Ebola hemorrhagic fever, rabies, West Nile virus, yellow fever, herpesvirus types I and II, hantavirus pulmonary syndrome, Marburg virus infection, Lassa virus infection, and dengue fever.

[0230] In some examples, the method includes treating or inhibiting a genetic disease by administering a delivery agent disclosed herein in combination with one or more therapeutic agents to a subject using the methods disclosed herein. In some examples, the genetic disease is acid maltase deficiency / Pompe disease, ADCY5-induced dyskinesia; adenosine aminohydrolase deficiency (ADA); X-linked agamaglobulinemia, type 1; Alagille syndrome; global hypertrophic dilated cardiomyopathy; alopecia congenita (ALUNC); Alpers syndrome; alpha-mannosidosis; alpha-1-antitrypsin deficiency, antitrypsin deficiency; alpha-thalassemia - Southeast Asia; amyotrophic lateral sclerosis - Gehrig's disease; androgen insensitivity; anilisia; ankylosing spine disease. Spondylitis; APC-associated polyposis; Argininosuccinate lyase deficiency; Arrhythmogenic right ventricular dysplasia / cardiomyopathy; Aspartylglucosaminuria; Ataxia with oculomotor apraxia type 2; Vitamin E deficiency ataxia; Ataxia-telangiectasia, telangiectasia; Autoimmune polyendocrine syndrome, β-hydroxyisobutyrate CoA deacylation deficiency (HIBCH deficiency); β-mannosidosis; Biotin dehydrogenase deficiency; Ptosis-ptosis-entropion; Bloom's syndrome; Brachydactyly; Brachydactyly Hypertensive syndrome;Brachydactyly type B1;Segmental nail-renal syndrome;BRCA1;Campomery skeletal dysplasia;Canavan;Cerebral xanthomatosis;Ceroid lipofuscinosis-Baton;Charcot-Marie-Tooth disease type 2B;Charcot-Marie-Tooth neuropathy type 1B;Charcot-Marie-Tooth neuropathy type 2A2;Charge syndrome;Cherubism;Choroidal atrophy;Citrin deficiency;Citrullinemia type 1;Coffin-Lowry syndrome;Cohen syndrome;Collagen 4A5;Congenital adrenal hyperplasia;Congenital Cataracts, facial dysmorphism, and neuropathy; congenital glycosylation dysplasia type 1a; congenital myasthenic syndrome; Cornelia-de Lange syndrome; Crohn's disease; cystic fibrosis; cystinosis; Darier's disease; desmin storage myopathy; DFNA2 nonsyndromic hearing loss; Diamond-Blackfan anemia; DNMT1 complex disorder; double cortex syndrome; Duane syndrome; Duchenne / Becker muscular dystrophy; Ecker muscular dystrophy; dysferlinopathy; dyskeratosis congenita; early-onset familial Alzheimer's disease;Early-onset primary dystonia (DYT1); Ehlers-Danlos syndrome; Ehlers-Danlos syndrome, classic type; Ehlers-Danlos syndrome, hypermobility type; Ehlers-Danlos syndrome, Emery-Dreifuss muscular dystrophy X-linked; epidermolysis bullosa simplex; Fabry disease; facioscapulohumeral muscular dystrophy; familial dysautonomia nervosa (HSANIII); familial hyperinsulinemia (FHI); familial hypertrophic cardiomyopathy; familial transaminase Iretin amyloidosis; Fanconi anemia; Fatal familial insomnia; fibrodysplasia ossificans progressiva; Fragile X syndrome; Friedreich ataxia; FRMD7-associated infantile nystagmus; Flins syndrome; Fucosemia; Galactosemia; Galactosylcylic acidosis; Gaucher disease; Gerstmann-Sträussler-Scheiker syndrome; Glucose transporter 1 deficiency; Glycine encephalopathy; Glycogen storage disease type VI; Hemophagocytic lymphohistiocytosis; Hemophilia Hepatic veno-occlusive disease with immunodeficiency; hereditary hemorrhagic telangiectasia; hereditary neuropathy with a tendency to pressure palsies; hereditary nonpolyposis colorectal cancer; hexosaminidase A deficiency; HFE-associated hereditary hemochromatosis; Holt-Oram syndrome; Huntington's disease; hydroxymethylbilane synthase (HMBS) deficiency; hypophosphatemia; inclusion body myositis type 2; Bloch-Salzberger syndrome; juvenile polyposis syndrome; Kallmann syndrome; Krabbe disease; Leber congenital amaurosis; Leber congenital amaurosis 1 Type 0; Lactate-Resistant Leukoencephalopathy with Brainstem and Spinal Cord Involvement (LBSL); Li-Fraumeni Syndrome; Limb-Girdle Muscular Dystrophy; Calpainosis; LIS1-Related Lissencephaly; Long QT Syndrome; Lowe's Syndrome; Lysosomal Storage Disorders (e.g., Gaucher Disease); Malignant Hyperthermia Susceptibility; Maple Syrup Urine Disease; MAPT-Related Disorders; Mastocytosis; McCasick-Kaufman Syndrome; MECP2-Rett Syndrome; Menkes Syndrome; Metachromatic Leukodystrophy; Methylmalonic Acidemia; Mucolipidosis Type II; Multiple Endocrine Neoplasia Type 1; Muco Mucolipidosis type I; Mucolipidosis type II; Multiple endocrine neoplasia type 1; Congenital myotonia; Myotonic dystrophy type 1; Myotonic dystrophy type 2; Nail-patella syndrome; Nemaline myopathy; Neurofibromatosis type 1; Neurofibromatosis type 2; Nonant syndrome; X-linked oculocutaneous albinism; Oculocutaneous albinism type 1; Oculocutaneous albinism type 2; Oculopharyngeal muscular dystrophy;Optic atrophy type 1; ornithine transcarbamylase deficiency; osteogenesis imperfecta; Parkinson's disease; Pendred syndrome; peroxisome biogenesis disorders, Zellweger syndrome; polyneuropathy, deafness, ataxia, retinitis pigmentosa, and cataract (PHARC) disease, phenylketonuria; polycystic kidney disease; Pompe disease-GSD II; porphyria; primary ciliary dyskinesia; progeria / HGPS, retinitis pigmentosa; retinoblastoma; Säthle-Chötzen syndrome; Schindler disease; SCN9A-related hereditary erythromelalgia; SHOX-related haploinsufficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; Smith-MaGinnis syndrome; Satos syndrome; spastic paraplegia 3A; spastic paraplegia 7; spastic paraplegia 8; spastic paraplegia Selected from spinal muscular atrophy; spinocerebellar ataxia type 2; spinocerebellar ataxia type 3; spinocerebellar ataxia type 7; spinocerebellar ataxia type 1; Stickler syndrome; achondroplasia; thoracic aortic aneurysm and aortic dissection; Treacher-Collins syndrome; trimethylaminuria; tuberous sclerosis; Udd-type distal muscular dystrophy; Usher syndrome type 1; LBSL disease; very long-chain acyl-CoA dehydrogenase deficiency; von Hippel-Lindau disease; Waardenburg syndrome, Werner syndrome; Wilms tumor; Wilson's disease; Wiskott-Aldrich syndrome; X-linked adrenal hypoplasia; X-linked adrenoleukodystrophy; X-linked dystonia-parkinsonism; X-linked juvenile retinoschisis; X-linked myofibrillar myopathy; X-linked SCIDs; and Zellweger syndrome.

[0231] In some examples, a subject with a genetic disease is administered a delivery agent disclosed herein and one or more therapeutic agents targeting the genetic disease. Exemplary therapeutic agents for treating or inhibiting genetic diseases are described, for example, in Bick et al., Am J Med Genet C Semin Med Genet. 2021, 187(1):48-54 and Gahl et al., Orphanet J Rare Dis. 2021, 16:308. Specific examples of therapeutic agents that can be used include ivacaftor, tezacaftor, lumacaftor, inhaled antibiotics, allopurinol, statins, granulocyte colony-stimulating factor, clonazepam, EDTA chelation therapy, ezetimibe, cholestyramine, sodium benzoate, Ravikty, metronidazole, cysteamine, chlorophyllin, growth hormone, insulin, testosterone, etidronate, antihypertensives, metreleptin, lumasiran, N -Carbamyl glutamate, nitisinone, bezafibrate, phenylbutyrate, sirolimus, diazoxide, somatostatin analogs, nifedipine, glucagon, insulin-like growth factor-1, mTOR inhibitors, GLP-1 receptor antagonists, oral antidiabetic drugs, glibenclamide, leflunomide, rapamycin, emapalumab, TNF inhibitors, corticosteroids, platelet transfusions, red blood cell transfusions, antibiotic prophylaxis, antifungal prophylaxis , interferon gamma, fludrocortisone, mineralocorticoid receptor antagonists, amiloride, triamterene, eplerenone, calcium channel blockers, clonidine, spironolactone, beta-blockers, antiepileptic drugs, phenytoin, lacosamide, eculizumab, meningococcal vaccine, Haemophilus influenzae type b vaccine, pneumococcal vaccine, purified C1 inactivator concentrate, ecallantide, icatibant, lanaderma bu, ezogabine, memantine, dextromethorphan, carbamazepine, stilipentol, perampanel, acetylcholinesterase inhibitors, albuterol, salbutamol, fluoxetine, quinidine, 3,4-diaminopyridine, ephedrine, acetazolamide, mexiletine, lamotrigine, quinine, dantrolene, vigabatrin, decarboxylase inhibitors, levodopa, dopamine agonists, MAO B inhibitors, pramipexole, L-threo-3,4-dihydroxyphenylserine,Givosiran, hemin, afamelanotide, methixanthine, valproic acid, cyclic pyranopterin monophosphate, epalrestat, burosumab, isotretinoin, patisiran, yegusedi, tafamidis, miglustat, ambroxol, factor VIII, factor IX, fibrinogen concentrate, factor XIII A subunit, fresh frozen plasma, cryopreside, factor XIII concentrate, eteplirsen, byondois 53, deferoxamine, mitapivat, growth hormone receptor antagonist, estrogen, thiazide diuretics, plerixafor, ruxoli Tinib, abatacept, leniolisib, rituximab, adalimumab, tacrolimus, nonsteroidal anti-inflammatory drugs, ofatumumab, angiotensin-converting enzyme inhibitors, febuxostat, voranesorsen, indomethacin, alfacalcidol, oxcarbazepine, DDAVP, anakinra, rilonacept, canakinumab, colchicine, infliximab, tocilizumab, etanercept, ustekinumab, secukinumab, mesalazine, cyclosporine, methotrexate, azathioprine, hydroxychloroquine, selumetinib, imatinib, sucralose Nitinib, tolvaptan, levothyroxine, tetrabenazine, omeprazole, ranirestat, chloroquine, inclisiran, evolocumab, lomitapide, desferrioxamine, baricitinib, deoxycytidine, deoxythymidine, benzoic and phenylacetic acids, sodium phenylbutyrate, carglumic acid, betaine, pegvalaiase, eliglustat, velaglucerase-alfa, imiglucerase, taliglucerase, agalsidase beta, agalsidase alfa, migalast, sebelipase alfa, alglucosidase alfa, bel Mannase alfa, laronidase, idursulfase, erosulfase alfa, galsulfase, bestronidase alfa, cerliponase alfa, cysteine ​​(enteric coated), cysteine ​​hydrochloride eye drops, rosuvastatin calcium, lomitapide, cholic acid, chenodeoxycholic acid, asfotase alfa, burosumab (twza), calcium acetate, alendronate, ascorbic acid, thiamine, trisodium citrate, levocarnitine, triheptanoin, riboflavin, uridine triacetate, potassium citrate, tiopronin,Penicillamine, trientine hydrochloride, zinc acetate, hydroxocobalamin, inotesen, patisiran sodium, teriflunomide, fingolimod hydrochloride, siponimod, rasagiline, selegiline, carbidopa / levodopa, pitriasan, oxybutate sodium, duotetrabenazine, baclofen, everolimus, folic acid, biotin, rufinamide, cannabidiol, midazolam, levetiracetam, clobazam, topiramate, gabapentin, riluzole, radicava, pyridostigmine bromide, amifampridine, mexiletine hydrochloride, nusinersen sodium, octocog alfa, rurioctocog alfa pegol, lonoctocog alfa, emicizumab, damoctocog alfa pegol, turoctocog alfa, simoctocog alfa, moroctocog Alfa, desmopressin acetate, recombinant factor VIII, efloctocog alfa, factor VIII / von Willebrand factor, bonicog alfa, eftrenonacog alfa, albutrenonacog alfa, nonacog alfa, human blood coagulation factor IX, nonacog beta pegol, nonacog gamma, recombinant factor IX preparation, activated eptacog alfa, recombinant factor VIIa preparation, human coagulation factor X preparation, catalidecog, human protein C preparation, hydroxyurea, epoetin alfa, eltrobopag, deferasirox, methylene blue injection, siltuximab, anagrelide hydrochloride, ravulizumab, macapegfilgrastim, busulfan, thiotepa, deferiprone, caplacizumab, romiprost, lopeginterferon Alpha-2b, immune globulin infusion, methylprednisolone, golimumab, mesalamine, 5-aminosalicylic acid, obeticholic acid, tocopherol, C1 inhibitor (human), icatibant acetate, danazol, tranexamic acid, C1-esterase inhibitor, human, Conestat alfa, IL-1 receptor antagonist anakinra, senegelmine, cyclosporine, dexamethasone, somatropin for injection, octreotide, lanreotide, pegvisomant, pasireotide, osilodrostat, ketoconazole, hydrocortisone, human chorionic gonadotropin, gonadotropin-releasing hormone, mecasermin, calcitonin for injection, human, parathyroid hormone, tasimelteon,Macitentan, tadalafil, ambrisentan, nitric oxide, sildenafil, bosentan hydrate, selexipag, iloprost, parenteral treprostinil, riociguat, mannitol, tezacaft or ivacaft, tobramycin, aztreonam, colistimethate sodium, lumacaft or ivacaft, levofloxacin, pirfenidone, nintedanib, caffeine citrate, bovine pegademase, ADA cDNA-transfected CD34+ cells, interferon gamma 1-b, cromolyn sodium, amiodarone, autologous human corneal stem cells, boretigene parvovec, teduglutide, defibrotide, ibuprofen, or dexrazoxane.

[0232] In some specific embodiments of the disclosure, the genetic disease is a muscular dystrophy. In specific examples, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, or Emery-Dreyfus muscular dystrophy. In some embodiments of the present disclosure, the muscular dystrophy is Duchenne muscular dystrophy. In some embodiments of the present disclosure, the disease is a demyelinating disease. In some further embodiments of the present disclosure, the demyelinating disease is multiple sclerosis, Charcot-Marie-Tooth disease, Pelizaeus-Merzbach disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, and / or Guillain-Barré syndrome. In some examples, the methods are utilized to treat a subject having one or more signs or symptoms of a muscle disease, such as, but not limited to, muscular dystrophy (DMD).

[0233] In certain examples, an effective amount of the disclosed delivery agent is administered to a subject in combination with one or more therapeutic agents that alleviate or inhibit one or more signs or symptoms associated with muscular dystrophy. Exemplary therapeutic agents may include α7β1 modulating agents or laminin-111 protein therapy, which stabilize sarcolemma and reduce muscle degeneration. The therapeutic agents used herein may further include components of the extracellular matrix, such as integrins, dystrophin, dystroglycan, utrophin, or growth factors. In some examples, the additional therapeutic agent reduces or promotes the expression of substances that promote the formation or maintenance of the extracellular matrix. In some examples, the additional substance includes aggrecan, angiostatin, cadherin, collagen (including collagen I, collagen III, or collagen IV), decorin, elastin, enactin, endostatin, fibrin, fibronectin, osteopontin, tenascin, thrombospondin, vitronectin, and combinations thereof. Biglycan, glycosaminoglycans (such as heparin), glycoproteins (such as dystroglycan), proteoglycans (such as heparan sulfate), and combinations thereof may also be administered. In a specific, non-limiting example, a morpholino is administered to a subject suffering from DMD in combination with the disclosed delivery agent. Exemplary morpholinos used to treat DMD include ETEPLIRSEN® (Sarepta Therapeutics).

[0234] In some examples, growth stimulants such as cytokines, polypeptides, and growth factors such as brain-derived neurotrophic factor (BDNF), CNF (ciliary neurotrophic factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), glial growth factor (GGF), glial maturation factor (GMF), glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), insulin, insulin-like growth factor, keratinocyte growth factor (KGF), nerve growth factor (NGF), neurotropin-3 and -4, PDGF (platelet-derived growth factor), vascular endothelial growth factor (VEGF), and combinations thereof may be administered in conjunction with a delivery agent using the disclosed methods.

[0235] In some examples, the method includes treating or inhibiting cancer, such as a hematological malignancy or solid tumor. In certain disclosed embodiments, administering a therapeutically effective amount of the disclosed combination therapy to a subject reduces the subject's cancer burden, improves the subject's survival rate, reduces the incidence of cancer recurrence in the subject, induces dendritic cell maturation in the subject, or a combination thereof. The method can include selecting a subject suffering from cancer. Examples of hematopoietic malignancies include acute leukemia (such as 11q23-positive acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), T-cell large granular lymphocyte leukemia, polycythemia vera, lymphoma, Hodgkin's lymphoma, and the like. lymphoma, non-Hodgkin's lymphoma (low-grade and high-grade, including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic, small lymphocytic, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0236] Examples of solid cancers, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, lymphomas (including low-grade and high-grade lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, central nervous system lymphoma, etc.), pancreatic pancreatic cancer, breast cancer (including basal breast cancer, ductal carcinoma, and lobular breast cancer), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, melanoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and central nervous system tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0237] In particular examples, hematological malignancies that may be inhibited or treated by the methods disclosed herein include, but are not limited to, multiple myeloma, chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, prolymphocytic / myelocytic leukemia, plasma cell leukemia, NK cell leukemia, Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma (low- and high-grade forms, including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma). In further specific examples, solid tumors that can be treated or inhibited by the methods disclosed herein include lung cancer, prostate cancer, pancreatic cancer (e.g., insulinoma), breast cancer, colon adenocarcinoma or squamous cell carcinoma, neuroblastoma, testicular cancer (e.g., seminoma), and ovarian cancer. In specific, non-limiting examples, the subject has chronic myeloid leukemia, acute monocytic leukemia, or non-Hodgkin's lymphoma (low- and high-grade forms, including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma).

[0238] In some examples, a subject (such as a cancer patient) is administered a delivery agent disclosed herein and one or more chemotherapeutic agents and / or radiation therapy. Chemotherapeutic agents useful in the disclosed methods include chemical or biological agents that have therapeutic utility in treating diseases characterized by abnormal cell proliferation. Specific examples of chemotherapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In one embodiment, the chemotherapeutic agent is a radioactive compound. Other chemotherapeutic agents that can be used are described in Sausville and Longo, "Principles of Cancer Treatment," Chapter 69 of Harrison's Principles of Internal Medicine (20th ed.), McGraw-Hill, 2018; Niederhuber et al., "Cancer Pharmacology," Chapter 25 of Abeloff's Clinical Oncology (6th ed.), Elsevier, 2019; Gullatte et al., "A Clinical Guide to Antineoplastic Therapy: Chemotherapy Handbook (4th ed.)," Oncology Nursing Society, 2020; Chabner and Longo, "Cancer Chemotherapy, Immunotherapy, and Biotherapy: Principles and Practice (6th ed.)," Lippincott Williams & Wilkins, 2018; and Skeel, Handbook of Cancer Chemotherapy (9th ed.), Lippincott Williams & Wilkins, 2016. The disclosed methods can include the administration of two or more chemotherapeutic agents, such as in combination with a delivery agent disclosed herein.Such chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, chlorambucil), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, dacarbazine), antimetabolites such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU, cytarabine), purine analogs (e.g., mercaptopurine, thioguanine), or natural products such as vinca alkaloids (e.g., vinblastine, vincristine, vindesine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitocycin C), and enzymes (e.g., L-asparaginase). Other agents include platinum complexes (such as cis-diamine-dichloroplatinum type II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), adrenocortical suppressants (such as mitotane, aminoglutethimide), and hormones and antagonists such as corticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magnestrol acetate), estrogens (such as diethylstilbestrol, ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone).Examples of the most commonly used chemotherapy drugs include adriamycin, melphalan (Alkeran®), AraC (cytarabine), carmustine, busulfan, lomustine, carboplatin, cisplatin, cyclophosphamide (Cytoxan®), daunorubicin, dacarbazine, 5-fluorouracil, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, and paclitaxel. These include cetaxel (or other taxanes such as docetaxel), vinblastine, vincristine, VP-16, etc., while newer drugs include gemcitabine (Gemzar®), trastuzumab (Herceptin®), irinotecan (CPT-11), leustatin, navelbine, rituximab (Rituxan®), imatinib (STI-571), topotecan (Hycamtin®), capecitabine, ibritumomab (Zevalin®), calcitriol, etc.

[0239] Treatment of the conditions and diseases described herein generally begins after the onset of the symptoms described herein or after the onset of a precursor condition (such as the onset of dysplasia or benign tumors). Treatment can be initiated at an early stage of cancer. Treatment can also begin before the subject exhibits symptoms of the condition, for example, at the time of diagnosis of stage I or dysplasia. However, treatment can also begin at any stage of the disease, including, but not limited to, stage I, stage II, stage III, and stage IV cancer. Treatment before the onset of symptoms, such as treatment upon detection of dysplasia or early (benign) precursor symptoms, is referred to herein as treatment of a subject "at risk" of developing the condition. In some embodiments of the disclosure, administration of the combination therapy can be performed during or after the onset of the symptoms described herein.

[0240] Therefore, subjects suffering from or at risk of developing cancers such as those disclosed herein, for example, colon cancer, renal cancer, or melanoma, can be selected for treatment. Typical subjects intended for administration of the combination therapy disclosed herein include humans, as well as non-human primates and other animals. To identify relevant subjects, approved screening methods are employed to identify and / or diagnose the risk factors associated with the target cancer or suspected cancer (such as colon cancer, renal cancer, or melanoma) of the subject, or to identify the subject's existing cancer state. These screening methods include, but are not limited to, conventional thorough examinations to determine the environment, family, occupation, and other such risk factors that may be associated with the target cancer or suspected cancer, as well as diagnostic methods such as various histopathological, morphological, and / or cytological analyses to identify or diagnose the target cancer. These methods and other conventional methods allow clinicians to select patients who require treatment. In accordance with these methods and principles, the combination therapies disclosed herein may be carried out as stand-alone preventative or treatment programs, or as follow-up, adjunctive, or coordinated therapeutic regimens to other treatments, in accordance with the teachings herein or other conventional methods.

[0241] In some examples, the method includes treating or inhibiting an immune disease or disorder by administering a delivery agent disclosed herein in combination with one or more therapeutic agents to a subject using the methods disclosed herein. The immune disease or disorder can be any type of immune system condition, such as cytokine storm, an immune system disorder (e.g., an inflammatory disease or an autoimmune disease), or an immune system condition associated with another condition and / or disease (e.g., HIV infection or exposure to microgravity). In some non-limiting examples, the immune system disease or disorder is an inflammatory disease. In certain embodiments of the present disclosure, the inflammatory disease can be rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel disease, or systemic lupus erythematosus. In other examples, the immune system disease or disorder is an autoimmune disease. In certain embodiments of the present disclosure, the autoimmune disease is type 1 diabetes, multiple sclerosis, lupus erythematosus, myasthenia gravis, ankylosing spondylitis, celiac disease, Crohn's disease, Graves' disease, Hashimoto's thyroiditis, transplant rejection, or autoimmune uveitis.

[0242] In some examples, a subject (e.g., a subject having an immune disease or disorder, such as an autoimmune disease, transplant rejection, or inflammatory disease) is administered one or more immunomodulatory therapies (e.g., muromonab, ipilimumab, abatacept, belatacept, tremelimumab, BMS-936558, CT-011, MK-3475, AMP224, BMS-936559, MPDL3280A, MEDI4736, MGA271, IMP321, BMS-663513, PF-05082566, CDX-1127, anti-OX40, huMAb, OX40L, and TRX518, both of which are incorporated by reference in their entireties, Yao et al., Nat Rev Drug Discov, 12(2)130-146,2013, and Kamphorst et al., Vaccine, 33(02)B21-B28,2015), immunomodulatory biologics, regulatory cytokines such as IL-7, mTOR modulators such as rapamycin, vaccinations, antifungals, and / or antibacterial therapies such as antibiotics), anti-inflammatory drugs (NSAIDs, anti-leukotrines, immunoselective anti-inflammatory derivatives, ImSAIDs, bioactive compounds with anti-inflammatory properties such as plumbagin and plumelysin, and / or steroids), disease-modifying antirheumatic drugs (methotrexate, sulfasalazine, leflunomide, hydroxychloroquine, tofacitinib, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, anakinra, abatacept, and / or or rituximab), antimalarials (such as chloroquine and hydroxychloroquine), medical procedures (including surgery and stem cell transplants), immunosuppressants (e.g., for preventing rejection of transplanted organs or tissues, for treating autoimmune and / or inflammatory diseases, e.g., glucocorticoids such as prednisone, dexamethasone, and hydrocortisone, cytostatics such as alkylating agents and antimetabolites, antibodies such as atgam, thymoglobulin, and antibodies targeting T-cell receptors and IL-2 receptors, immunophilin-targeting drugs such as cyclosporine, tacrolimus, sirolimus, and everolimus, interferons (IFNs) such as IFNλ, IFNβ, opioids, TNF-binding proteins such as infliximab, etanercept, and adalimumab, mycophenolate,Small biologics such as fingolimod, myriocin, etc.), immune tolerance therapy (e.g., for the treatment of subjects at risk for tissue or organ transplant rejection, subjects with allergies, and / or subjects with autoimmune diseases), T cell or B cell targeted drugs or T cell or B cell suppressive drugs (e.g., CAMPATH-1H, calcineurin inhibitors, rituximab, epratuzumab, belimumab, atacicept, etc.), anti-CD3 antibodies, abatacept, induction of hematopoietic chimerism, e.g., mixed hematopoietic chimerism in which the bone marrow of an organ or tissue transplant patient is replaced with donor bone marrow or a mixture of donor and recipient bone marrow to alleviate organ or tissue transplant rejection, antigen desensitization (see Nepom et al., Immunol Rev;241(1):49-62,2011, incorporated herein by reference), antihistamines, helminth therapy (e.g., deliberately infecting a subject with helminths or helminth eggs to treat an immune disorder) may also be administered.

[0243] In some examples, the methods include treating or inhibiting an infectious disease by administering a delivery agent disclosed herein in combination with one or more therapeutic agents to a subject using the methods disclosed herein. In some examples, the infectious disease is an arbovirus infection, botulism, brucellosis, candidiasis, campylobacteriosis, chickenpox, chlamydia, cholera, coronavirus infection, staphylococcal infection, coxsackievirus infection, Creutzfeldt-Jakob disease, cryptosporidiosis, cyclospora infection, cytomegalovirus infection, Epstein-Barr virus infection, dengue fever, diphtheria, ear infection, encephalitis, influenza virus infection, parainfluenza virus infection, giardiasis, gonorrhea, Haemophilus influenzae infection, hantavirus infection, viral hepatitis, herpes simplex virus infection, HIV / AIDS, Helicobacter infection, human papillomavirus (HPV) infection, infectious mononucleosis, legionellosis, leprosy, leptospirosis, listeriosis, rabies ... The infectious disease is selected from Immune disease, lymphocytic choriomeningitis, malaria, measles, Marburg hemorrhagic fever, meningitis, monkeypox, mumps, mycobacterial infections, mycoplasma infections, Norwalk virus infections, whooping cough, pinworm infections, pneumococcal infections, Streptococcus pneumoniae infections, Mycoplasma pneumoniae, Moraxella catarrhalis infections, Pseudomonas aeruginosa infections, rotavirus infections, psittacosis, rabies, respiratory syncytial virus infection (RSV), ringworm, Rocky Mountain spotted fever, rubella, salmonellosis, SARS, scabies, sexually transmitted diseases, shigellosis, shingles, sporotrichosis, streptococcal infections, syphilis, tetanus, trichinosis, tuberculosis, tularemia, typhoid fever, viral meningitis, bacterial meningitis, West Nile virus infection, yellow fever, adenovirus-borne infections and diseases, retrovirus-borne infections, and yersiniosis zoonosis. For example, the infectious disease may be influenza, parainfluenza, respiratory syncytial virus, or HIV. In particular examples, the infectious disease is human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), tuberculosis (TB), malaria, dental caries, or Helicobacter pylori infection.

[0244] In some examples, a subject (e.g., a subject suffering from an infectious disease such as HIV) is also administered one or more anti-infective agents (e.g., antibodies, antifungal agents, antiviral agents, and / or antiparasitic agents). In some examples, the anti-infective agent is an antibiotic such as penicillin, amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, amoxicillin, clavulanic acid, or levofloxacin. In some examples, the anti-infective agent is an antiviral agent such as tilorone, vicriviroc, arbidol, or nelfinavir. In particular examples, the infectious disease is HIV and the subject is also administered an antiretroviral agent such as a broad-spectrum inhibitor such as a nucleoside and nucleotide reverse transcriptase inhibitor (nRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor, an entry inhibitor (or fusion inhibitor), a maturation inhibitor, or a natural antiviral agent. Other exemplary agents include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine, and efavirenz.

[0245] In some examples, the method includes administering a delivery agent disclosed herein in combination with one or more therapeutic agents to a subject using the methods disclosed herein to treat or inhibit a psychiatric disorder. The psychiatric disorder can be any type of psychiatric disorder, such as anxiety disorder, attention deficit hyperactivity disorder (ADHD), childhood behavioral and / or emotional disorder, bipolar affective disorder, depression, dissociative and dissociative disorder, eating disorder, obsessive-compulsive disorder, panic disorder, delusion, post-traumatic stress disorder, psychosis, and / or schizophrenia. In some non-limiting examples, the psychiatric disorder is depression.

[0246] In some examples, a subject (e.g., a subject with a psychiatric disorder, such as a subject suffering from depression) is also administered one or more antidepressants, such as, but not limited to, a selective serotonin uptake inhibitor (SSRI), a tricyclic antidepressant, and / or a monoamine oxidase inhibitor (MAOI). Tricyclic antidepressants include imipramine, amitriptyline, nortriptyline, and desipramine. MAOIs approved for the treatment of depression include phenelzine (NARDIL®), tranylcypromine (PARNATE®), isocarboxazid (MARPLAN®), and the like. SSRIs primarily affect the neurotransmitter serotonin and include escitalopram HBr (LEXAPRO®), fluoxetine (PROZAC®), sertraline (ZOLOFT®), fluvoxamine (LUVOX®), paroxetine (PAXIL®), and citalopram (CELEXA®). Additional medications affect both norepinephrine and serotonin, such as venlafaxine (EFFEXOR®) and nefazadone (SERZONE®), or phenelzine (NARDIL®), tranylcypromine (PARNATE®), mirtazepine (REMERON®), nefazodone (SERZONE®), triazolopyridine (TRAZODONE®), and bupropion (WELLBUTRIN®).

[0247] C. Methods of Making Delivery Agent Embodiments Also disclosed herein are methods for producing delivery agents according to the present disclosure. Delivery agents can be produced by conjugating components of the delivery agent as discussed herein, for example, by performing one or more chemical bonds. In some embodiments of the present disclosure, chemical conjugation can include appropriate peptide bond-forming reactions, conditions, and reagents. Chemical conjugation can be performed in any suitable order. For example, chemical conjugation can be performed sequentially, such that the C-terminal group is used as a building block for synthesizing the remaining peptide backbone of the delivery agent. In some embodiments of the present disclosure, a peptide bond is formed from the C-terminal group, thereby providing an anchor group attached to the C-terminal group, followed by a mask peptide sequence attached to the anchor group, followed by a cleavable linker group attached to the mask peptide sequence, followed by a lytic peptide group attached to the cleavable linker group. The lytic peptide can be functionalized to provide an N-terminal group as disclosed herein. Other components that can be conjugated to the delivery agent can be added during or after the synthesis of the delivery agent. For example, targeting groups and / or therapeutic agents can be attached to different attachment points within the delivery agent. By way of example only, lysine or cysteine ​​attachment points that are part of the delivery agent peptide backbone (or branches thereof) can be functionalized with targeting and / or therapeutic agents using chemical linker groups that can be covalently attached to the side chains or C- or N-termini of the lysine or cysteine. Such moieties can be attached using appropriate coupling conditions and reagents. In yet some additional embodiments of the present disclosure, click chemistry can be used to attach the therapeutic agent and / or targeting group to the chemical linker group. For example, click chemistry using azide-alkyne cycloaddition coupling conditions recognized by those skilled in the art can be used to link the chemical linker and therapeutic agent or targeting group. In further embodiments of the present disclosure, therapeutic agents can be covalently attached to the delivery agent using reaction conditions suitable for coupling an amine, cysteine, carboxylic acid group, or other reactive functional group of an amino acid to one or more functional groups of the therapeutic agent (e.g., activated carbonate, thiocarbonyl group, activated ester, α,β-unsaturated ketone, olefin, etc.), which is a particular advantage of the present disclosure.

[0248] In some aspects of the disclosure, suitable peptide bond forming conditions include 2-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-N,N,N',N'-hexafluorophosphate, 2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate, 1-hydroxybenzotriazole, dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-(3-dimethylamino)methylamino The reaction may involve the use of coupling reagents including, but not limited to, benzotriazol-1-yloxy-tris(dimethylaminopropyl)-N'-ethylcarbodiimide·HCl, benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate, benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate, bromotripyrrolidinophosphonium hexafluorophosphate, and the like, or combinations thereof, and a base, such as an amine base (e.g., diisopropylethylamine, isopropylamine, n-methylmorpholine, and the like).

[0249] (VI. Kit) Provided herein are kits useful for various aspects of the disclosure described herein. The kits may contain various materials and reagents (e.g., for carrying out the methods described herein). For example, the kits may include reagents, including but not limited to, a disclosed delivery agent, one or more therapeutic agents, cells (e.g., cells representative of a particular condition or disease, cells suitable for use in the methods of screening one or more test compounds described herein, etc.), cell culture medium, serum, and other solutions or buffers useful for carrying out the assays and other methods provided herein. The kits may also include control samples, materials useful for the methods described herein, and containers, syringes, vials, tubes, ampoules, capsules, or bottles, microtiter plates, etc., in which assay reactions can be carried out. The kits are packaged in a container that may include compartments for receiving the contents of the kit and may include instructions for carrying out the methods described herein.

[0250] The kit may include a label or package insert affixed to the kit's container or attached to the kit's container. The label or package insert may also typically include instructions for using the delivery agent, one or more therapeutic agents, and / or cells provided with the kit, e.g., for use in the methods disclosed herein. The instructions may be in written, electronic, or visual format (e.g., a video file).

[0251] For example, the kit can include: (1) a peptide-based delivery agent described herein (e.g., a composition comprising a delivery agent described herein); (2) one or more therapeutic agents (either in the same composition and / or container as the delivery agent or in different compositions and / or containers); (3) instructions for administering or using the peptide-based delivery agent of (1) and / or one or more therapeutic agents of (2); (4) one or more administration devices; or (5) a delivery agent of (1) and any combination of (2)-(4).

[0252] (VII. Overview of Some Examples) Provided herein are peptide-based delivery agents having the formula [X 1 Y 1 Y 1 X 1 ] m a lytic peptide group having a structure according to 1 At least one X 1 is a basic amino acid, an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 1 is, independently for each occurrence, a non-polar amino acid or derivative thereof, and m is an integer selected from 2 to 8, 1 Y 1 Y 1 X 1 ] m a lytic peptide group having a structure according to the formula [X 2 Y 2 Y 2 X 2 ] m’ a mask peptide group having a structure according to 2 At least one X 2 is an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 2 is, independently for each occurrence, a non-polar amino acid or derivative thereof, and m is an integer selected from 2 to 8, 2 Y 2 Y 2 X 2 ] m’ and an anchor group selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group; a targeting group selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule, or a combination thereof.

[0253] In any or all of the above examples, the delivery agent has a structure according to Formula IA or Formula IB: [N-terminal group]-[X 1 Y 1 Y 1 X 1 ] m -[cleavable linker]-[X 2 Y 2 Y 2 X 2 ] m’ -[anchor / targeting group]-[C-terminal group] (Formula 1A) [N-terminal group]-[X 2 Y 2 Y 2 X 2 ] m’ -[anchor / targeting group]-[cleavable linker]-[X 1 Y 1 Y 1 X 1 ] m -[C-terminal group] (Formula 1B) each X 1 At least one X 1 is a basic amino acid, an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 1 is, independently for each occurrence, a nonpolar amino acid or derivative thereof, and each of m and m' is, independently for each occurrence, an integer selected from 2 to 8; 1 Y 1 Y 1 X 1 ] m and each X 2 At least one X 2 is an acidic amino acid, a nonpolar amino acid or a derivative thereof, provided that, for each occurrence, 2is, independently for each occurrence, a nonpolar amino acid or derivative thereof; the cleavable linker is an amino acid sequence that is 2 to 10 amino acids in length; the anchor group, if present, is selected from a heteroaliphatic group, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group; the targeting group, if present, is selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule, or a combination thereof; the C-terminal group comprises an amine-terminated glycine moiety; and the N-terminal group comprises a capping group or a fluorophore.

[0254] In any or all of the above examples, each X 1 is X 1 is alanine, at least one further X 1 is glutamine, glutamic acid, aspartic acid, arginine, or a derivative thereof, provided that, for each occurrence, is glutamine, glutamic acid, aspartic acid, arginine, or a derivative thereof.

[0255] In any or all of the above examples, each X 1 is, independently for each occurrence, glutamic acid, aspartic acid, or a derivative thereof.

[0256] In any or all of the above examples, each X 1 is, independently for each occurrence, (i) glutamic acid or a derivative thereof, or (ii) aspartic acid or a derivative thereof, or (iii) a combination of glutamic acid and aspartic acid to provide a lytic peptide sequence of DLLE or ELLD, or (iv) any combination of (i), (ii), and (iii).

[0257] In any or all of the above examples, each Y 1 is calculated independently for each occurrence and for each Y 2is, independently for each occurrence, leucine, α-methylleucine, alanine, or a derivative thereof.

[0258] In any or all of the above examples, each Y 1 is calculated independently for each occurrence and for each Y 2 is, independently for each occurrence, leucine, or a derivative thereof.

[0259] Each Y 1 is calculated independently for each occurrence and for each Y 2 is, independently for each occurrence, α-methylleucine, or a derivative thereof.

[0260] In any or all of the above examples, each X 2 is X 2 is alanine, at least one further X 2 is glutamine, glutamic acid, aspartic acid, alanine, or a derivative thereof, provided that, for each occurrence, is glutamine, glutamic acid, aspartic acid, or a derivative thereof.

[0261] In any or all of the above examples, m is 3 and m' is 3, or m is 4 and m' is 4.

[0262] In any or all of the above examples, each X 1 is glutamic acid, and each Y 1 and each Y 2 is leucine and X 2 If X is alanine, then at least one other X 2 is glutamine, 2 is glutamine or alanine, m is 3, and m' is 3.

[0263] In any or all of the above examples, the cleavable linker is cleavable by cathepsin B.

[0264] In any or all of the above examples, the cleavable linker comprises an amino acid sequence between 2 and 10 amino acids in length.

[0265] In any or all of the above examples, the cleavable linker is 7 amino acids in length.

[0266] In any or all of the above examples, the cleavable linker comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:3.

[0267] In any or all of the above examples, the cleavable linker comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:3.

[0268] In any or all of the above examples, the cleavable linker comprises the amino acid sequence of SEQ ID NO:3.

[0269] In any or all of the above examples, the cleavable linker consists of the amino acid sequence of SEQ ID NO:3.

[0270] In any or all of the above examples, the anchor group comprises a lysine moiety, the side chain of the lysine moiety having the formula -C(O)-X-[CH] p functionalized with a heteroaliphatic group having Z, where X is oxygen or CH2, and Z is -N(R)2 or -N + (R)3, where each R is independently hydrogen or aliphatic, and p is an integer ranging from 1 to 3.

[0271] In any or all of the above examples, X is oxygen or CH2, Z is -N(R)2, one R group is hydrogen and the other R group is C6-C 12 alkyl and p is 2.

[0272] In any or all of the above examples, X is oxygen or CH2 and Z is -N +(R)3, where two R groups are methyl and the other R group is C6-C 12 alkyl and p is 2.

[0273] In any or all of the above examples, the anchor group is selected from:

[0274] [ka]

[0275] In any or all of the above examples, multiple anchor groups are present.

[0276] In any or all of the above examples, one, two, or three anchor groups may be present.

[0277] In any or all of the above examples, the lytic peptide group provides the N-terminal group of the peptide-based delivery agent, and the anchor group provides the C-terminal group of the peptide-based delivery agent.

[0278] In any or all of the above examples, (i) the N-terminal group is attached to a carbonyl-containing group, and (ii) the C-terminal group is attached to an amine-terminated glycine moiety, a fluorophore, or a combination thereof.

[0279] In any or all of the above examples, the carbonyl-containing group is an acetyl group or a fluorophore.

[0280] In any or all of the above examples, the fluorophore comprises 2-(methylamino)benzamide.

[0281] In any or all of the above examples, the amine-terminated glycine moiety has a structure selected from -Nal-K-G', -G-Nal-K-G', -GK-G', -Nal-C-G', -G-Nal-C-G', -GC-G', -Nal-G', -G-Nal-G', -W-G', -GW-G', or -GWK-G', where Nai is naphthylalanine, G is glycine, G' is a modified glycine with a -C(O)-amine group, W is tryptophan, K is lysine, and C is cysteine.

[0282] In any or all of the above examples, the amine-terminated glycine moiety is -G-Nal-G', where G' is -C(O)-N(R a ) groups, each R a are independently hydrogen or aliphatic.

[0283] In any or all of the above examples, the lytic peptide group has the structure [ELLE]3, the cleavable linker has the amino acid sequence of SEQ ID NO:3, the mask peptide group has the structure [QLLQ]3, and the anchor group has a structure according to any or all of the above examples, wherein (i) the lytic peptide group provides an N-terminus attached to an acetyl group, and (ii) the anchor group is attached to an amine-terminated glycine moiety having the structure -G-Nal-G', where G' is -C(O)-N(R a ) groups, each R a are independently hydrogen or aliphatic.

[0284] In any or all of the above examples, the lytic peptide group has the structure [ELLE]4, the cleavable linker has the amino acid sequence of SEQ ID NO:3, the mask peptide group has the structure [QLLQ]4, there are two anchor groups present and directly bonded to each other, each anchor group having a structure according to any or all of the above examples, (i) the lytic peptide group provides an N-terminus attached to an acetyl group, and (ii) the anchor group is -C(O)-N(R a ) groups, each R a are independently hydrogen or aliphatic.

[0285] In any or all of the above examples, the lytic peptide group has the structure [QLLE]-[QLLQ]-[QLLE], the cleavable linker has the amino acid sequence of SEQ ID NO:3, the mask peptide group has the structure [QLLE]-[QLLQ]-[QLLE], and the anchor group has a structure according to any or all of the above examples, wherein (i) the lytic peptide group provides an N-terminus bound to an acetyl group, and (ii) the anchor group is bound to an amine-terminated glycine moiety having the structure -GK-G', where G is glycine, K is lysine, and G' is -C(O)-N(R a ) groups, each R a are independently hydrogen or aliphatic.

[0286] In any or all of the above examples, the lytic peptide group has the structure [ELLE]3, the cleavable linker has the amino acid sequence of SEQ ID NO:3, the mask peptide group has the structure [QLLA]-[QLLA]-[QLLQ], and the anchor group has a structure according to any or all of the above examples, wherein (i) the lytic peptide group provides an N-terminus bound to an acetyl group, and (ii) the anchor group is bound to an amine-terminated glycine moiety having the structure -GK-G', where G is glycine, K is lysine, and G' is -C(O)-N(R a ) groups, each R a are independently hydrogen or aliphatic.

[0287] In any or all of the above examples, the peptide-based delivery agent has an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:272, 273, or 274, and K at position 32 comprises a linking group selected from formula A, B, or C, and the linking group is attached to one or more DBCO groups.

[0288] Also disclosed herein are exemplary compositions comprising a peptide-based delivery agent according to any or all of the above examples and a therapeutic agent.

[0289] In any or all of the above examples, the therapeutic agent is covalently or non-covalently attached to the peptide-based delivery agent.

[0290] In any or all of the above examples, the therapeutic agent is a chemotherapeutic agent, a morpholino, a therapeutic antibody, an immunotherapeutic agent, an antibiotic, an antidepressant, or a combination thereof.

[0291] In any or all of the above examples, the therapeutic agent is saporin, cisplatin, methotrexate, fluorouracil, doxorubicin, cyclophosphamide, chlorambucil, vinblastine, vincristine, docetaxel, or paclitaxel, chlorhexidine, triclosan, xylitol, or octadecen-1-amine hydrofluoride, 1-hexadecylamine hydrofluoride, gefitinib, lapatinib, olaparib, mitomycin C, sunitinib, geftinib, nintedanib, PD173074, erdaftinib, sorafenib, or a combination thereof.

[0292] In any or all of the above examples, the therapeutic agent is a morpholino.

[0293] In any or all of the above examples, the morpholino comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:229, 240, 241, 242, 243, and 244.

[0294] In any or all of the above examples, (i) the peptide-based delivery agent comprises an antibody as an anchoring or targeting group, and (ii) the therapeutic agent is a morpholino, which is indirectly covalently attached to the peptide-based delivery agent via a linking group.

[0295] In any or all of the above examples, the composition further comprises an auxiliary agent, carrier, buffer, detergent, or combination thereof.

[0296] In any or all of the above examples, the composition is formulated for administration by injection, aerosol delivery, intranasal administration, oral administration, topical administration, or a combination thereof.

[0297] Also disclosed herein are exemplary methods that include contacting a cell with a delivery agent and / or composition according to any or all of the above examples.

[0298] In any or all of the above examples, the peptide-based delivery agent of the composition delivers the therapeutic agent to the cytosol of the cell.

[0299] In any or all of the above examples, the host cell is a non-human mammalian cell or a human cell.

[0300] In any or all of the above examples, the cells may be contacted in vitro or in vivo.

[0301] In any or all of the above examples, contacting the cell with the composition induces lysis of the endosomal membrane after the therapeutic agent and peptide-based delivery agent are internalized, or induces pore formation in the endosomal membrane after the therapeutic agent and peptide-based delivery agent are internalized, or induces localized disruption / destabilization of the endosomal membrane after the therapeutic agent and peptide-based delivery agent are internalized, thereby delivering the therapeutic agent into the cytosol of the cell.

[0302] In any or all of the above examples, contacting the cell with the composition includes contacting the cell with the therapeutic agent and the peptide-based delivery agent simultaneously or at different times.

[0303] Also disclosed herein are exemplary methods that include administering to a subject a therapeutically effective amount of a delivery agent and / or composition according to any or all of the above examples.

[0304] In any or all of the above examples, the subject is a human or non-human mammal.

[0305] In any or all of the above examples, the therapeutically effective amount of the composition treats, ameliorate, or prevent the onset of a symptom or disease selected from a genetic disease, a rare disease, a cancer, an immune disease, an infectious disease, a psychiatric disorder, a substance abuse disorder, or any combination thereof.

[0306] In any or all of the above examples, administration includes injection, aerosol delivery, intranasal administration, oral administration, topical administration, or a combination thereof.

[0307] In any or all of the above examples, administering includes providing the therapeutic agent and the peptide-based delivery agent to the subject at the same time or at different times.

[0308] In any or all of the above examples, administering includes providing the therapeutic agent and the peptide-based delivery agent to the subject using the same route of administration or different routes of administration.

[0309] Also disclosed herein is an exemplary method for identifying a therapeutic compound, comprising contacting a cell with a peptide-based delivery agent described in any one of claims 1 to 15 and one or more compounds, and comparing the effect of the one or more compounds on the contacted cell with a control, wherein a differential effect of the one or more compounds on the contacted cell relative to the control indicates that the one or more compounds are therapeutic compounds.

[0310] In any or all of the above examples, the method further comprises determining the IC of one or more compounds. 50 This includes determining the value.

[0311] In any or all of the above examples, the method is a quantitative high-throughput screening method.

[0312] In any or all of the above examples, the method further includes selecting one or more compounds that have a differential effect on the contacted cells compared to a control.

[0313] In any or all of the above examples, the effect of the one or more compounds on the contacted cells includes a decrease in viability of the contacted cells compared to a control, an increase in viability of the contacted cells compared to a control, induction of a phenotype of interest in the contacted cells compared to a control, an increase in expression of one or more genes in the contacted cells compared to a control, and / or a decrease in expression of one or more genes in the contacted cells compared to a control.

[0314] Provided herein are examples of kits comprising a container, the container comprising any or all of the above example peptide-based delivery agents or any or all of the above example compositions, wherein the container is selected from a syringe, a vial, a tube, an ampoule, a capsule, or a bottle.

[0315] In any or all of the above examples, the kit further comprises: (i) instructions for administering or using any or all of the above example peptide-based delivery agents or any or all of the above example compositions; (ii) a container comprising a therapeutic agent; (iii) one or more administration devices; or (iv) any combination of (i)-(iii). [Example]

[0316] VIII. EXAMPLES The following examples are presented to provide a complete disclosure and description of how to make and use the exemplary peptide-based delivery agents and methods described herein, and are not intended to limit the scope of what the present inventors regard as their invention, nor are they intended to represent that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be expected. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric.

[0317] JPEG2025528284000017.jpg162159

[0318] Unless otherwise specified, the morpholinos used (also referred to as standard morpholinos in the examples) are as follows:

[0319] [ka]

[0320] Also, in examples that include a control, unless otherwise specified, the control sample may consist of the morpholino (by itself) or may include a delivery agent without the morpholino.

[0321] For in vivo studies in mice, a morpholino sequence ("morpholino" in the structure above, "morpholino 1" with the sequence 5'-GGCCAAACCTCGGCTTACCTGAAAT-3', SEQ ID NO:229) is complementary to mouse dystrophin pre-mRNA for targeted deletion of exon 23. For in vitro studies in cell culture, a morpholino sequence ("morpholino" in the structure above, "morpholino 2" with the sequence 5'-GCAATATGAAACCTCTTACCTCAGT-3', SEQ ID NO:230) targets an aberrant splice site that interrupts a firefly luciferase gene stably transfected into HeLa cells (Kang et al., Biochemistry 1998, 37, 18, 6235-6239).

[0322] In some cases, endoporters (Gene Tools, Philomath, Oregon) were used as a positive control. Endoporters are peptides developed to facilitate the delivery of morpholinos to cultured cells. Endoporters are endosomal delivery agents that are uncharged at neutral pH but become cationic when protonated at the low pH of late endosomes. Endoporters release the contents of acidic endosomes into the cytosol. This is demonstrated with morpholinos and oxygen-responsive fluorescent probes. Endoporters are poorly soluble in water but soluble in DMSO. Cultured cells are immersed in fresh medium containing the cargo to be delivered (e.g., morpholinos), and an endoporter solution, usually formulated in DMSO, is added to the medium. When endoporters enter the aqueous medium, they aggregate into particles. Cultures must be immediately agitated to disperse the endoporators and limit the size of the aggregates. The endoporter particles slowly settle to the bottom of the culture vessel and come into contact with adherent cells. When using endoporators to grow suspension cultures, the cultures must be gently and continuously agitated (e.g., with a low-speed orbital shaker) to keep the particles in suspension (Moulton et al., J Drug Discov Develop and Deliv, 3(2):1023, 2016).

[0323] Unless otherwise noted, the activity of compounds administered to cell cultures in the following examples was measured using a luciferase reporter assay. Briefly, the HeLa LUC / 705 cell line, a positive test system for antisense compounds, was obtained from the University of North Carolina. HeLa cells were stably transfected with a plasmid containing a firefly luciferase gene interrupted by a mutated human β-globin intron. The mutation (IVS2-705) causes aberrant splicing of the luciferase pre-mRNA, preventing translation. Treatment with morpholino oligos targeted to the splice site corrects the splicing and restores luciferase activity.

[0324] For the assay, cells were cultured in DMEM containing glucose, glutamine, sodium pyruvate (e.g., Corning 10-013-CV), 10% fetal bovine serum (e.g., Corning 35-010-CV), and penicillin / streptomycin. In some cases, the medium was supplemented with glutamine (MP Biomedicals catalog number 1680149). Cells were trypsinized with 0.25% trypsin-EDTA (e.g., Corning 25-053-CI) for passaging. Morpholinos (sequences provided herein, specific examples are commercially available from Gene Tools LLC, Philomath, OR, USA) with or without a delivery agent were mixed with the medium and / or added at various stock concentrations to 60-90% confluent Luc / 705 HeLa cell plate wells containing medium with serum. The final solution concentrations of test / control compounds and the various percentages of serum used are listed in each example. After the incubation period (16–22 h), cells were washed with phosphate-buffered saline (PBS 1x, Invitrogen / ThermoFisher Scientific) and lysed with reporter lysis buffer (Promega, Madison, WI, USA). After centrifugation, luciferase activity was assessed from the lysate supernatant using a luciferase assay system (E1501, Promega, Madison, WI, USA) in a luminometer (Turner Biosystems). In some cases, luciferase activity was assessed using the Bright-Glo™ Luciferase Assay System (E2610, Promega, Madison, WI, USA) according to the manufacturer's protocol, and luminescence was measured using a Spectramax M3 microplate reader (Molecular Devices). In some cases, protein levels were quantified and normalized using a protein colorimetric assay reagent (e.g., Bio-Rad Laboratories Inc. kit #5000001, USA).

[0325] To assess cytotoxicity and saporin delivery, HeLa cells in 96-well clear-bottom microplates (e.g., Corning, Corning, NY, USA) were treated with the delivery agent, saporin protein (Millipore, Sigma, S9896) dissolved in water, or a combination of both diluted in DMEM medium alone, which was added to the plate wells at the volumes indicated in the Examples for 1 hour. The treatment was removed and replaced with DMEM-supplemented medium with 10% FBS (culture medium described above). After 24 hours, the medium was removed and the cells were washed with 1x PBS. Next, the cells were treated with 2 μM calcein AM (Biotium, San Francisco, CA, USA) for 30 minutes, and fluorescence was measured using a SpectraMax fluorescence microplate reader (Molecular Devices) according to the manufacturer's protocol.

[0326] Methods for assessing in vivo morpholino delivery have been previously published and adapted (Morcos et al., Biotechniques 2008 45:613-623). Successful morpholino delivery results in the deletion of dystrophin exon 23 sequences from mRNA. Studies were conducted at Gene Tools LLC using wild-type C57Bl mice obtained from the Jackson Laboratory. Morpholino 1 (sequence 5'-GGCCAAACCTCGGCTTACCTGAAAT-3', SEQ ID NO: 229, Gene Tools LLC) was dissolved in water and mixed with a delivery agent in water. The mixture was intravenously infused once daily for two or three days, after which the animals were euthanized at least one day after the final infusion. RNA from excised muscle tissue was isolated using the MELT Total Nucleic Acid Isolation System (Ambion, Inc., Austin, TX, USA). RT-PCR from total RNA was performed using the SuperScript One-Step RT-PCR System (Invitrogen). Forward (5'-TTCTGGATGCAGACTTTGTGGCCT-3'; SEQ ID NO: 238) and reverse (5'-AGGGCAGGCCATTCCTCTTTCA-3'; SEQ ID NO: 239) DNA primers (Integrated DNA Technologies, USA) spanned dystrophin exon 21 and exon 24. RT-PCR reaction conditions were 55°C for 30 minutes, 94°C for 2 minutes, 30 cycles of 94°C for 15 seconds, 57°C for 30 seconds, and 68°C for 1 minute, with a final extension at 68°C for 5 minutes. The predicted size of the amplified cDNA product is 517 bp if exon 23 is present and 304 bp if exon 23 is deleted. Additional details and explanations are provided in the Examples.

[0327] Exemplary methods for synthesizing exemplary delivery agent examples and / or their components (e.g., exemplary cleavable linkers, exemplary soluble and / or masking peptide groups, exemplary anchor groups, and / or exemplary morpholino groups) according to the present disclosure are described below. The methods below can be modified (with the benefit of this disclosure and / or general knowledge in the art) to create other delivery agent examples and / or their components contemplated by the present disclosure. Modification of appropriate reagents, reaction conditions, and / or delivery agent components will be recognizable to those of skill in the art, particularly in light of the present disclosure and other available information regarding chemical synthesis and / or peptide synthesis.

[0328] Synthesis of an Exemplary Delivery Agent (Peptide 88-8 of Example 11) Resin activation: The resin is activated with an amino acid (e.g., Gly) selected as the C-terminal amino acid of the delivery agent backbone. In this example, Rink Amide ChemMatrix resin (Gyros Protein Technologies, catalog number RCM-10-HL) was used, but other resins can also be used, such as Rink Amide MBHA resin (Millipore Sigma, catalog number 85003), NovaSyn TGR resin (Millipore Sigma, catalog number 855009), or HMBA resin (AAPPTec, catalog number ROZ005; the peptide is cleaved from the resin with a strong base).

[0329] [ka]

[0330] Synthesis of delivery agent components: The backbone of the delivery agent (including the lytic peptide group, cleavable linker, mask peptide group, and amine-terminated glycine moiety) is assembled on an Fmoc-protected glycine-functionalized resin from the C-terminus to the N-terminus, starting with the naphthylalanine group (Nal) and ending with glutamic acid.

[0331] [ka]

[0332] Deprotection and N-terminal addition: The N-terminal Fmoc is then removed with 20% piperidine / DMI and the N-terminal cap / group is added.

[0333] [ka]

[0334] Addition of anchor group: The protected lysine group (K' = Lys(Mtt)) is deprotected and a lipid anchor group is added to functionalize the side chain of the lysine moiety.

[0335] [ka]

[0336] A...

Claims

1. 1. A peptide-based delivery agent comprising: Formula [X １ Y １ Y １ X １ ] ｍ A lytic peptide group having a structure according to Each X １ is at least one X of the lytic peptide group １ is, independently for each occurrence, an amino acid or derivative thereof, provided that is a basic amino acid or an acidic amino acid; Each Y １ is, independently for each occurrence, a nonpolar amino acid or derivative thereof; m is an integer selected from 2 to 8; Formula [X １ Y １ Y １ X １ ] ｍ a lytic peptide group having a structure according to a cleavable linker group; and Formula [X ２ Y ２ Y ２ X ２ ] ｍ’ A mask peptide group having a structure according to each X ２ is at least one X of the mask peptide group ２ is an amino acid or derivative thereof, independently for each occurrence, provided that is glutamine; Each Y ２ is, independently for each occurrence, a nonpolar amino acid or derivative thereof; m' is an integer selected from 2 to 8; Formula [X ２ Y ２ Y ２ X ２ ] ｍ’ a mask peptide group having a structure according to an anchor group selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group, a targeting group selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule, or a combination thereof; A peptide-based delivery agent comprising:

2. and further comprising an N-terminal group and a C-terminal group, wherein the peptide-based delivery agent has a structure according to Formula IA or Formula IB: [N-terminal group]-[X １ Y １ Y １ X １ ] ｍ -[cleavable linker]-[X ２ Y ２ Y ２ X ２ ] ｍ’ -[anchor / targeting group]-[C-terminal group] (Formula 1A) [N-terminal group]-[X ２ Y ２ Y ２ X ２ ] ｍ’ -[anchor / targeting group]-[cleavable linker]-[X １ Y １ Y １ X １ ] ｍ -[C-terminal group] (Formula 1B) the cleavable linker is an amino acid sequence that is 2 to 10 amino acids in length; the anchor group, if present, is selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a biotin group, an avidin group, a streptavidin group, or a neutravidin group; said targeting group, if present, is selected from a cell, an antibody or antibody fragment, a peptide, a biomimetic peptide, an aptamer, a sugar, or a small targeting molecule, or a combination thereof; the C-terminal group comprises an amine-terminated glycine moiety; the N-terminal group comprises a capping group or a fluorophore; The peptide-based delivery agent of claim 1 .

3. Each X １ is a certain X １ is alanine, at least one further X １ is independently for each occurrence glutamic acid, glutamine, alanine, aspartic acid, or a derivative thereof, with the proviso that, optionally, each X １ is, independently for each occurrence, glutamic acid, aspartic acid, or a derivative thereof, and further optionally, each X １ is, independently for each occurrence, (i) glutamic acid or a derivative thereof, or (ii) aspartic acid or a derivative thereof, or (iii) a combination of glutamic acid and aspartic acid to provide a lytic peptide sequence of DLLE or ELLD, or (iv) any combination of (i), (ii), and (iii).

4. Each Y １ is independently for each occurrence and each Y ２ is, independently for each occurrence, leucine, α-methylleucine, alanine, or a derivative thereof, and optionally, (i) each Y １ is independently for each occurrence and each Y ２ is, independently for each occurrence, leucine or a derivative thereof; or (ii) each Y １ is independently for each occurrence and each Y ２ The peptide-based delivery agent of any one of claims 1 to 3, wherein, independently for each occurrence, is α-methylleucine or a derivative thereof.

5. At least one X of the masked peptide group is glutamine according to claim 1. ２ Each X other than ２ 5. The peptide-based delivery agent of claim 1, wherein, for each occurrence, is independently glutamine, glutamic acid, aspartic acid, alanine, or a derivative thereof.

6. 6. The peptide-based delivery agent of claim 1, wherein m is 3 and m' is 3, or m is 4 and m' is 4.

7. Each X １ is glutamic acid, and each Y １ and each Y ２ is leucine, and at least one X of the masked peptide group is glutamine according to claim 1 ２ Each X other than ２ is glutamine or alanine, m is 3 and m' is 3, the peptide-based delivery agent of claim 1 or 2.

8. 8. The peptide-based delivery agent of any one of claims 1 to 7, wherein the cleavable linker is cleavable by cathepsin B, and optionally the cleavable linker comprises an amino acid sequence 2 to 10 amino acids in length, and further optionally the cleavable linker is 7 amino acids in length.

9. 9. The peptide-based delivery agent of claim 1, wherein the cleavable linker comprises an amino acid sequence at least 70% identical or 85% identical to SEQ ID NO: 3, and optionally the cleavable linker consists of the amino acid sequence of SEQ ID NO:

3.

10. The anchor group is (i) an amino acid moiety that is a lysine moiety; (ii) tail groups, such as: (a) 2-aminoethyl hydrogen carbonate, 4-aminobutanoic acid, —CH(NH ２ )C(O)— group, or —CH(N ＋ Me ３ ) a functional group provided by a C(O)— group, wherein the 2-aminoethyl hydrogen carbonate group or the 4-aminobutanoic acid group is optionally functionalized to provide a quaternary amine, and (b) an aliphatic tail comprising 6 to 12 carbon atoms attached to the functional group.

10. A peptide-based delivery agent according to any one of claims 1 to 9.

11. The anchor group is 【Chemical 1】 The peptide-based delivery agent of claim 10, selected from:

12. 12. The peptide-based delivery agent of claim 1, wherein multiple anchor groups are present, optionally 1, 2, or 3 anchor groups.

13. The lytic peptide group provides an N-terminal group of the peptide-based delivery agent, and the anchor group provides a C-terminal group of the peptide-based delivery agent, and optionally (i) the N-terminal group is bonded to a carbonyl-containing group, and optionally the carbonyl-containing group is a fluorophore comprising an acetyl group or 2-(methylamino)benzamide, and (ii) the C-terminal group is bonded to an amine-terminated glycine moiety, a fluorophore, or a combination thereof, and optionally the amine-terminated glycine moiety is -Nal-K-G and wherein, in this document, NaI is naphthylalanine, G is glycine, G' is a modified glycine comprising a -C(O)-amine group, W is tryptophan, K is lysine, and C is cysteine; and further optionally, said amine-terminated glycine moiety is -G-Nal-G', and G' is -C(O)-N(R ａ ) ２ and each Ra is independently hydrogen or aliphatic.

14. (i) the lytic peptide group has the structure [ELLE] ３ wherein the cleavable linker has the amino acid sequence of SEQ ID NO: 3, and the mask peptide group has the structure [QLLQ] ３ and the anchor group has the structure of claim 11, wherein (a) the lytic peptide group provides an N-terminus bound to an acetyl group; and (b) the anchor group is bound to an amine-terminated glycine moiety having the structure -G-Nal-G', where G' is -C(O)-N(R ａ ) ２ is a modified glycine comprising a group, ａ are independently hydrogen or aliphatic, or (ii) the lytic peptide group has the structure [ELLE] ４ wherein the cleavable linker has the amino acid sequence of SEQ ID NO: 3, and the mask peptide group has the structure [QLLQ] ４ wherein there are two anchor groups directly bonded to each other, each anchor group having the structure of claim 11, wherein (a) the lytic peptide group presents an N-terminus bonded to an acetyl group, and (b) the anchor group has the structure -C(O)-N(R ａ ) ２ is a modified glycine comprising a group, ａ are independently hydrogen or aliphatic, or (iii) the lytic peptide group has the structure [QLLE]-[QLLQ]-[QLLE], the cleavable linker has the amino acid sequence of SEQ ID NO:3, the mask peptide group has the structure [QLLE]-[QLLQ]-[QLLE], and the anchor group has the structure of claim 11, wherein: (a) the lytic peptide group provides an N-terminus bound to an acetyl group; and (b) the anchor group is bound to an amine-terminal glycine moiety having the structure -G-K-G', where G is glycine, K is lysine, and G' is -C(O)-N(R ａ ) ２ is a modified glycine containing a group, ａ are independently hydrogen or aliphatic, or (iv) the lytic peptide group has the structure [ELLE] ３ wherein the cleavable linker has the amino acid sequence of SEQ ID NO: 3, the mask peptide group has the structure [QLLA]-[QLLA]-[QLLQ], and the anchor group has the structure of claim 11, wherein (a) the lytic peptide group provides an N-terminus bound to an acetyl group, and (b) the anchor group is bound to an amine-terminated glycine moiety having the structure -G-K-G', where G is glycine, K is lysine, and G' is -C(O)-N(R ａ ) ２ is a modified glycine containing a group, ａ are independently hydrogen or aliphatic; The peptide-based delivery agent of claim 1 .

15. 2. The peptide-based delivery agent of claim 1, having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 272, 273, or 274, wherein K at position 32 comprises a linking group selected from formula A, B, or C, and wherein the linking group is attached to one or more DBCO groups.

16. 1. A composition comprising: The peptide-based delivery agent of any one of claims 1 to 15; and and a therapeutic agent selected from a chemotherapeutic agent, a morpholino, a therapeutic antibody, an immunotherapeutic agent, an antibiotic, an antidepressant, or a combination thereof, wherein optionally (i) the therapeutic agent is covalently or non-covalently attached to the peptide-based delivery agent, and / or (ii) the therapeutic agent is selected from saporin, cisplatin, methotrexate, fluorouracil, doxorubicin, cyclophosphamide, chlorambucil, vinblastine, vincristine, docetaxel, or paclitaxel. ritaxel, chlorhexidine, triclosan, xylitol, or octadecen-1-amine hydrofluoride, 1-hexadecylamine hydrofluoride, gefitinib, lapatinib, olaparib, mitomycin C, sunitinib, geftinib, nintedanib, PD173074, erdaftinib, sorafenib, or a combination thereof, optionally wherein the composition further comprises an auxiliary agent, carrier, buffer, detergent, or a combination thereof.

17. 17. The composition of claim 16, wherein the therapeutic agent is a morpholino, and optionally the morpholino comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 229, 240, 241, 242, 243, and 244.

18. 18. The composition of claim 16 or 17, wherein (i) the peptide-based delivery agent comprises an antibody as the anchor group or the targeting group, and (ii) the therapeutic agent is a morpholino, and the morpholino is indirectly covalently attached to the peptide-based delivery agent via a linking group.

19. 19. The composition of any one of claims 16 to 18, formulated for administration by injection, aerosol delivery, intranasal administration, oral administration, topical administration, or a combination thereof.

20. 20. A method comprising contacting a cell in vitro or in vivo with a composition of any one of claims 16 to 19, optionally wherein the cell is a non-human mammalian cell or a human cell, and / or wherein the peptide-based delivery agent of the composition delivers a therapeutic agent to the cytosol of the cell.

21. Contacting the cell with the composition comprises: Inducing lysis of the endosomal membrane after the therapeutic agent and the peptide-based delivery agent have been internalized within the cell; or Inducing pore formation in the endosomal membrane after the therapeutic agent and the peptide-based delivery agent are internalized within the cell; or inducing local disruption / destabilization of endosomal membranes after the therapeutic agent and the peptide-based delivery agent have been internalized within the cell; The method of claim 20, whereby the therapeutic agent is delivered to the cytosol of the cell, and optionally, contacting the cell with the composition comprises contacting the cell with the therapeutic agent and the peptide-based delivery agent simultaneously or at different times.

22. 20. The composition of any one of claims 16 to 19 for use in treating, ameliorating, or preventing the onset of a symptom or disease in a subject selected from a genetic disease, a rare disease, cancer, an immune disease, an infectious disease, a psychiatric disorder, a substance abuse disorder, or any combination thereof.

23. 23. The compound of claim 22, wherein the subject is a human or non-human mammal, and the composition is administered using a technique selected from injection, aerosol delivery, intranasal administration, oral administration, topical administration, or a combination thereof, and optionally, administering the composition comprises providing the therapeutic agent and the peptide-based delivery agent to the subject at the same time or at different times.

24. 1. A method for identifying a therapeutic compound, comprising: contacting a cell with the peptide-based delivery agent of any one of claims 1 to 15 and one or more compounds; determining the effect of one or more compounds on said contacted cells; and comparing the effect of the one or more compounds on the contacted cells relative to a control, wherein a differential effect of the one or more compounds on the contacted cells relative to the control indicates that the one or more compounds are therapeutic compounds, and optionally the method further comprises: (i) determining the IC of the one or more compounds. ５０ and / or (ii) selecting one or more compounds that have a differential effect on the contacted cells relative to said control.

25. 25. The method of claim 24, wherein the method is a quantitative high-throughput screening method.

26. The effect of the one or more compounds on the contacted cells is a decrease in viability of the contacted cells relative to the control; an increase in viability of the contacted cells relative to the control; induction of a phenotype of interest in said contacted cells relative to said control; increased expression of one or more genes in the contacted cells relative to the control; and / or 26. The method of claim 24 or 25, comprising decreasing expression of one or more genes in the contacted cells compared to the control.

27. 20. A kit comprising a container, the container comprising the peptide-based delivery agent of any one of claims 1 to 15 or the composition of any one of claims 16 to 19, the container being selected from a syringe, a vial, a tube, an ampoule, a capsule, or a bottle, and optionally the kit further comprising: (i) instructions for administering or using the peptide-based delivery agent of any one of claims 1-15 or the composition of any one of claims 16-19; (ii) a container comprising a therapeutic agent; (iii) one or more administration devices; or (iv) any combination of (i) to (iii).

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