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

Abstract

Disclosed herein is a delivery agent that facilitates efficient delivery of drugs and other compounds across the lipid layer. The delivery agents disclosed herein provide lipid solubility under selected conditions, provide water solubility under other conditions, and can effectively deliver compounds into cell cytosol. The disclosed delivery agents also avoid detrimental interactions with serum, thereby providing effective in vivo delivery of therapeutic agents.

Description

Field of the Invention

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

[0002] Incorporation of Electronic Sequence Listing

[0003] The electronic sequence listing was created on August 11, 2023, and is submitted in the form of the XML file 10413-107991-02.xml (341,757 bytes), which is hereby incorporated by reference in its entirety.

[0004] Cross-Reference to Related Applications

[0005] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 397,601, filed on August 12, 2022, the entire content of which is incorporated herein by reference. Background of the Invention

[0007] Lipid layers, such as those of cell membranes (including plasma membranes and vesicle membranes), can constitute a barrier to effective drug delivery. For optimal delivery, drugs should be freely soluble in the aqueous compartments of the body and in the lipid layers that surround these compartments. While many low to medium polarity, low molecular weight compounds can cross lipid layers directly, more polar and / or larger molecular weight compounds (such as nucleic acids and proteins) generally enter eukaryotic cells only through endocytosis or related processes. During endocytosis, the compound is brought into the cell by the progressive invagination of a membrane region, ultimately forming a closed vesicle or endosome within the cell. The endosome can then fuse with a lysosome, resulting in entrapment within the vesicle and exposing the internalized compound to degradative enzymes.

[0008] Accordingly, there is a need for improved agents to facilitate more efficient delivery of drugs and other compounds across lipid layers, such as agents that provide lipid solubility under selected conditions and water solubility under other conditions, and that can effectively deliver compounds into the cell cytosol. Summary of the Invention

[0010] Aspects of the present disclosure relate to a peptide-based delivery agent comprising: a solubilizing peptide group having the structure of formula [X 1 Y 1 Y 1 X 1 m wherein each X 1 is independently, upon each occurrence, a basic amino acid, an acidic amino acid, a nonpolar amino acid, or a derivative thereof, provided that at least one X 1 is a basic amino acid or an acidic amino acid; each Y 1 ​independently a non-polar amino acid or its derivative each time it appears; and m is an integer selected from 2 to 8; a cleavable linker group; having the formula [X 2 Y 2 Y 2 X 2 m’ of a masked peptide group of the structure, wherein each X 2 is independently an acidic amino acid, a non-polar amino acid or its derivative each time it appears, provided that at least one X 2 is an acidic amino acid; each Y 2 is independently a non-polar amino acid or its derivative each time it appears; and m' is an integer selected from 2 to 8; and an anchoring 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 peptidomimetic, an aptamer, a sugar or a small targeting molecule; or a combination thereof.

[0011] In some aspects of the present disclosure, the delivery agent has a structure of Formula IA or IB:

[0012] [N-terminal group]-[X 1 Y 1 Y 1 X 1 m -[Cleavable linker]-[X 2 Y 2 Y 2 X 2 m’ -[Anchoring / targeting group]-[C-terminal group]

[0013] Formula IA

[0014] [N-terminal group]-[X 2 Y 2 Y 2 X 2 m -[Anchoring / targeting group]-[Cleavable linker]-[X 1 Y 1 Y 1 X 1 m’ -[C-terminal group]

[0015] Formula IB

[0016] wherein each X 1 is independently a basic amino acid, an acidic amino acid, a non-polar amino acid or its derivative each time it appears, provided that at least one X 1 is a basic amino acid or an acidic amino acid; each Y​​​​​1 is independently a nonpolar amino acid or its derivative each time it appears; each m and m’ is independently an integer selected from 2 to 8 each time it appears; each X 2 is independently an acidic amino acid, a nonpolar amino acid or its derivative each time it appears, provided that at least one X 2 is an acidic amino acid; each Y 2 is independently a nonpolar amino acid or its derivative each time it appears; the cleavable linker is an amino acid sequence having a length of 2 to 10 amino acids; the anchoring group (if present) is selected from a heteroaliphatic group, a dibenzocyclooctyne compound, an antibody or antibody fragment, a dibenzocyclooctyne compound, a biotin group, an avidin group, a streptavidin group, a dimeric or neutral avidin group; the targeting group (if present) is selected from a cell, an antibody or antibody fragment, a peptide, a peptidomimetic, an aptamer, a sugar or a small targeting molecule; or a combination thereof; the C-terminal group includes an amino-terminated glycine moiety; and the N-terminal group includes a capping group or a fluorophore.

[0017] Aspects of the present disclosure also relate to a composition comprising: a peptide-based delivery agent according to the present disclosure; and a therapeutic agent.

[0018] Aspects of the present disclosure also relate to a method comprising contacting a cell with a delivery agent and / or composition according to the present disclosure.

[0019] Aspects of the present disclosure also relate to a method comprising administering to a subject a therapeutically effective amount of a delivery agent and / or composition according to the present disclosure.

[0020] Aspects of the present disclosure also relate to a method of identifying a therapeutic compound, comprising: contacting a cell with a peptide-based delivery agent according to the present disclosure and one or more compounds; determining the effect of the one or more compounds on the contacted cell; 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.

[0021] Aspects of the present disclosure also relate to a kit comprising a container, wherein the container contains a peptide-based delivery agent or composition according to the present disclosure, and wherein the container is selected from a syringe, a vial, a tube, an ampoule, a capsule or a bottle.

[0022] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description with reference to the accompanying drawings. Brief Description of the Drawings

[0024] Figure 1 is a schematic diagram showing the endocytic uptake of a peptide-based delivery agent and a therapeutic agent disclosed herein into the cytosol of a eukaryotic cell.

[0025] Figure 2A and 2B show a helical wheel diagram of an exemplary EXXE-motif-based lytic peptide group ( Figure 2A ) and how the distribution of acidic residues leads to the facing of polar and non-polar regions; and Figure 2B is a schematic diagram showing the interaction between the backbone of an exemplary lytic peptide group and the backbone of an exemplary masking peptide group.

[0026] Figure 3A and 3B are graphs showing the average luciferase activity (LUC) / protein of the delivery agents 23-6 and 23-7 described herein in combination with a control morpholino compound in 1% serum ( Figure 3A ) and 80% serum ( Figure 3B ).

[0027] Figure 4 is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 23-10, 23-16, 23-20 and a positive control in 1% serum.

[0028] Figure 5A and 5B are graphs showing the average luciferase activity (LUC) / protein of the delivery agents 30-2, 30-4, 30-6, 30-10, 30-14, 30-16, 30-18 and 30-22 described herein in 1% serum ( Figure 5A ) and 80% serum ( Figure 5B ).

[0029] Figure 6A and 6B are graphs showing the average luciferase activity (LUC) / protein of the delivery agents 40-1 and 40-46 at a delivery agent concentration of 12 μM in 80% serum ( Figure 6A ), and graphs showing the average luciferase activity (LUC) / protein of the delivery agents 42-9 and 42-39 at a delivery agent concentration of 6 μM in 80% serum ( Figure 6B ).

[0030] Figure 7 is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 40-1, 40-46, 42-9 and 42-39 in 1% serum.

[0031] Figures 8A - 8C is a graph showing the helical wheel diagrams of the delivery agents 23-26 ( Figure 8A ) and 50-22 ( Figure 8B ) and the average luciferase activity (LUC) / protein in 80% serum (Figure 8C )。

[0032] Figure 9 Is a graph showing the mean luciferase activity (LUC) / 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, and only the morpholino control described herein.

[0033] Figure 10 Is a graph showing the mean luciferase activity (LUC) / 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, and only the morpholino control described herein.

[0034] Figure 11 Is a graph showing the mean luciferase activity (LUC) / protein in 1% serum at different concentrations using delivery agents 56-3, 56-5, 23-26, and control described herein.

[0035] Figure 12A and 12B Is a graph showing the mean luciferase activity (LUC) / protein in 80% serum for delivery agents 60-16, 60-17, 60-4, 62-9, and 23-26 described herein.

[0036] Figure 13 Is a graph showing the mean luciferase activity (LUC) / protein in 80% serum using control and delivery agents 23-26, 88-8, 88-9, 88-10, 88-11, and 88-12 described herein.

[0037] Figure 14 Is a graph showing the mean luciferase activity (LUC) / 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 described herein.

[0038] Figures 15A - 15D Shows the helical wheel diagrams of delivery agents 114-12 ( Figure 15A ), 114-6 ( Figure 15B ), and 114-9 ( Figure 15C ), and the mean luciferase activity (LUC) / protein of these delivery agents and compound 104-3+4 in 80% serum ( Figure 15D ).

[0039] Figure 16Graph of the % fraction of compound bound to human serum albumin as a function of the number of amino acids in the solubilizing peptide moiety.

[0040] Figure 17 Graph showing the mean luciferase activity (LUC) / 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 described herein.

[0041] Figure 18A and 18B Graph showing the mean luciferase activity (LUC) / protein of delivery agents 21-11 and 19-1 described herein in 1% serum ( Figure 18A ) and 80% serum ( Figure 18B ).

[0042] Figure 19A and 19B Graph showing the mean luciferase activity (LUC) / protein of delivery agents 36-47 and 36-48 described herein in 1% serum ( Figure 19A ) and 80% serum ( Figure 19B ).

[0043] Figure 20 Graph showing the mean luciferase activity (LUC) / protein of delivery agents 96-1, 96-4, 96-5, 96-6, and 96-7 described herein in 80% serum.

[0044] Figure 21 Graph showing the mean luciferase activity (LUC) / protein of delivery agent 26-17, 26-2, and negative control in 80% serum.

[0045] Figure 22A and 22B Graph showing the mean luciferase activity (LUC) / protein of delivery agents 19-1, 19-3, 19-5, and two controls in 1% serum ( Figure 22A ) and 80% serum ( Figure 22B ).

[0046] Figure 23A and 23B Graph showing the mean luciferase activity (LUC) / protein of delivery agents 23-12, 23-13, and 23-14 in 1% serum ( Figure 23A ) and 80% serum ( Figure 23B ).

[0047] Figure 24A and 24Bis a graph showing the average luciferase activity (LUC) / protein of delivery agents 21-12, 21-13, and 19-1 with different anchor group tail lengths described herein in 1% serum ( Figure 24A ) and 80% serum ( Figure 24B ).

[0048] Figure 25 is a graph showing the average luciferase activity (LUC) / protein of delivery agents 23-15 and 19-1 described herein in 1% serum.

[0049] Figures 26A - 26D is a graph showing the average luciferase activity (LUC) / protein of delivery agents 19-1 and 21-8 at 3 μM in 1% serum ( Figure 26A ), at 9 μM in 80% serum ( Figure 26B ), at 2 μM in 1% serum ( Figure 26C ), and at 9 μM in 80% serum ( Figure 26D ).

[0050] Figure 27 is a graph showing the average luciferase activity (LUC) / protein of delivery agents 40-31 to 40-46 described herein in 80% serum.

[0051] Figure 28 is a graph showing the average luciferase activity (LUC) / protein of delivery agents 23-26 and 53-6 to 53-15 and an untreated control in 80% serum.

[0052] Figures 29A - 29C is a graph showing the average luciferase activity (LUC) / protein of delivery agents 96-10, 23-26, and a control at different DMSO concentrations at 1 or 3 μM in 1% serum ( Figure 29A ); a graph showing the average luciferase activity (LUC) / protein of delivery agents 96-10, 23-26, and a control at 12 μM in 80% serum ( Figure 29B ); and a graph showing the average luciferase activity (LUC) / protein of delivery agent 26-13A with and without DMSO at 9 or 18 μM in 80% serum ( Figure 29C ).

[0053] Figure 30A and 30B is a graph showing the average luciferase activity (LUC) / protein of delivery agents 21-1, 21-4, and 21-6 and a control in 80% serum ( Figure 30A ) and 1% serum ( Figure 30B ).

[0054] Figure 31 A graph showing the average luciferase activity (LUC) / protein of delivery agents 19-1, 21-4, and 21-6 at different concentrations described herein and a control in 80% serum.

[0055] Figure 32A and 32B A graph showing the average luciferase activity (LUC) / protein of delivery agents 23-27 and 23-4 in 80% serum.

[0056] Figure 33 A graph showing the average luciferase activity (LUC) / protein of delivery agents 19-1, 23-4, and 23-5 in 80% serum.

[0057] Figure 34A and 34B A graph showing the average luciferase activity (LUC) / protein of delivery agents 26-7a / a’ and 26-7b / b’ at 18 μM in 80% serum ( Figure 34A ); and a graph showing the average luciferase activity (LUC) / protein of delivery agents 26-14A and 26-14B with different anchor group tail lengths described herein at 9 μM in 80% serum ( Figure 34B ).

[0058] Figure 35 A graph showing the average luciferase activity (LUC) / protein of delivery agents 26-14A-D with different anchor group tail lengths at different concentrations described herein in 80% serum.

[0059] Figure 36 A graph showing the average luciferase activity (LUC) / protein of delivery agents 26-15A / B with different anchor group tail lengths and a control at different concentrations described herein in 80% serum.

[0060] Figure 37 A graph showing the average luciferase activity (LUC) / protein of delivery agents 26-2A and 26-11 (without an anchor group) and delivery agents 26-2B and 26-13 and a control in 80% serum.

[0061] Figure 38 A graph showing the average luciferase activity (LUC) / protein of delivery agents 33-24 and 23-26 and a control and untreated in 80% serum.

[0062] Figure 39A and 39BGraph showing the mean luciferase activity (LUC) / protein of delivery agents 40-23A, 40-46, and control in 80% serum ( Figure 39A ); and graph showing the mean luciferase activity (LUC) / protein of delivery agents 40-23A, 40-23B, and 40-46 in 80% serum ( Figure 39B ).

[0063] Figure 40 Graph showing the mean luciferase activity (LUC) / protein of delivery agents 21-4a, 21-4b, 21-4b’, 21-6a, 21-6b, and 21-7a (each having a different anchor group tail length) in 80% serum.

[0064] Figure 41 Graph showing the mean luciferase activity (LUC) / protein of delivery agents 60-1b, 60-1a, and 23-26, and control in 80% serum.

[0065] Figure 42 Graph showing the mean luciferase activity (LUC) / protein of delivery agents 104-3+4, 109-1, and 109-8, and control in 80% serum.

[0066] Figure 43 Graph showing the mean luciferase activity (LUC) / protein of delivery agents 23-26, 78-1, and 78-2, and control in 80% serum.

[0067] Figure 44A and 44B Graph showing the mean luciferase activity (LUC) / protein of delivery agents 23-26, 85-2C, and 85-2P, and control in 80% serum ( Figure 44A ); and graph showing the mean luciferase activity (LUC) / protein of delivery agents 23-26, 85-2C, 85-2P, 86-1C, 86-1P, 86-4C, and 86-4P, and control in 80% serum ( Figure 44B ).

[0068] Figure 45 Graph showing the mean luciferase activity (LUC) / protein of delivery agents 23-26, 85-AC, and 86-5AP, and control in 80% serum.

[0069] Figure 46It is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 23-26, 50-23, 50-24, 50-25, and 50-26 described herein in 80% serum.

[0070] Figure 47A and 47B It is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 23-26, 30-3, 30-5, 30-7, 30-9, 30-11, 30-15, 30-17, and 30-22 described herein in 1% serum ( Figure 47A ) and 80% serum ( Figure 47B ).

[0071] Figure 48A and 48B It is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 19-1, 19-17A, and 19-17C described herein and two controls in 1% serum ( Figure 48A ) and 80% serum ( Figure 48B ).

[0072] Figure 49A and 49B It is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 21-4 and 21-5 described herein and a control in 1% serum ( Figure 49A ) and 80% serum ( Figure 49B ).

[0073] Figure 50A and 50B It is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 19-1 and 22-11 described herein and a control in 1% serum ( Figure 50A ) and 80% serum ( Figure 50B ).

[0074] Figure 51A and 51B It is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 24-1, 23-27, 23-1, and 23-4 described herein in 80% serum.

[0075] Figure 52A and 52B It is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 23-1 to 23-5 described herein in 1% serum ( Figure 52A ) and 80% serum ( Figure 52B ).

[0076] Figure 53A and 53BIs a graph showing the average luciferase activity (LUC) / protein of the delivery agents 21-4, 22-1, and 22-12 described herein, as well as the control, in 1% serum ( Figure 53A ) and 80% serum ( Figure 53B ).

[0077] Figure 54 Is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 21-1, 33-26a, 33-26b, 33-26c, 34-32, 34-33, and 34-34 described herein in 80% serum.

[0078] Figure 55 Is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 104-3+4 and 109-2 described herein in 80% serum.

[0079] Figure 56A and 56B Is a graph showing the average luciferase activity (LUC) / protein of different concentrations of the delivery agents 23-26, MO+23-26, Vivo-MO+23-26, Vivo-MO, and untreated in 80% serum ( Figure 56A ), as well as the average luciferase activity (LUC) / protein of the delivery agents MO+23-26, Vivo-MO, and Vivo-MO+23-26 described herein in 80% serum ( Figure 56B ).

[0080] Figure 57 Is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 23-26+a and 23-26+b described herein and untreated in 80% serum.

[0081] Figure 58 Is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 23-26+a and 23-26+b described herein and 23-26 without morpholino in 80% serum.

[0082] Figure 59 Is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 102-3 and 98-1-MO conjugate discussed herein and untreated in 80% serum.

[0083] Figure 60 Is a graph showing the average luciferase activity (LUC) / protein of the compounds and control discussed in Example 59 in 80% serum.

[0084] Figure 61A graph showing the average luciferase activity (LUC) / protein of the delivery agents 104-3+4P+MO and 107-8-MO conjugates and controls in 80% serum.

[0085] Figure 62A and 62B is a graph showing the cell viability results of compound 104-3+4 ( Figure 62A ) and the saporin delivery results when saporin toxin is combined with compound 104-3+4 ( Figure 62B ).

[0086] Figure 63 is an image showing mRNA amplified by RT-PCR (isolated from muscle tissue) and then analyzed using gel electrophoresis.

[0087] Figure 64 is an image showing mRNA amplified by RT-PCR (isolated from muscle tissue) and then analyzed using gel electrophoresis.

[0088] Figure 65A and 65B is a graph showing the average luciferase activity (LUC) / protein of compound 16-6 and two controls in 1% serum ( Figure 65A ) and 80% serum ( Figure 65B ).

[0089] Figure 66A and 66B is a graph showing the average luciferase activity (LUC) / protein of compound 19-1 and two controls in 1% serum ( Figure 66A ) and 80% serum ( Figure 66B ).

[0090] Figure 67A and 67B is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 215-2DBCO, 216-A DBCO, and 216-B DBCO in 80% serum ( Figure 67A ) and 10% serum ( Figure 67B ).

[0091] Figure 68 is a graph showing the average luciferase activity (LUC) / protein of the delivery agents 215-2FA, 216-A FA, 216-B, FA, 215-2DBCO, 216-A DBCO, 216-B DBCO in 10% serum, where "FA" is free amine.

[0092] Figure 69It is a graph showing the average luciferase activity (LUC) / protein of delivery agents 218-1, 218-2, 219-1, and 219-2 in 10% serum.

[0093] Figures 70A - 70C It is a graph showing the results of evaluating different delivery agents disclosed herein, where Figure 70A shows GFP expression of the control and delivery agents 215-2Ab, 216-2Ab, 216-1Ab, 215-2PEG12-BCN, and 216-1PEG12-BCN in 10% serum; Figure 70B shows the average luciferase activity (LUC) of HeLa Luc / 705 cells transfected with transferrin receptor (TFRC) in 10% serum; Figure 70C shows the average luciferase activity (LUC) of untransfected HeLa Luc / 705 cells in 10% serum.

[0094] Figure 71 shows the average luciferase activity (LUC) of delivery agents 224-2, 225-2, and 216-2 in 80% serum.

[0095] Figure 72 shows the average luciferase activity (LUC) of delivery agents 223-2 and 89-5 in 80% serum.

[0096] Sequence Listing

[0097] The nucleic acid sequences and amino acid sequences listed in the accompanying sequence listing are shown using the standard letter abbreviations for nucleobases and amino acids as defined in 37 C.F.R. § 1.822. Only one strand of each nucleic acid sequence is shown, but it should be understood that the complementary strand is included by any reference to the shown strand.

[0098] SEQ ID NO:1 is the amino acid sequence of the solubilizing peptide moiety.

[0099] SEQ ID NO:2 is the amino acid sequence of the solubilizing peptide moiety.

[0100] SEQ ID NO:3 is the amino acid sequence of the cleavable linker.

[0101] SEQ ID NO:4 is the amino acid sequence of the masking peptide moiety.

[0102] SEQ ID NO:5 is the amino acid sequence of the C-terminal portion.

[0103] SEQ ID NO:6 is the amino acid sequence of the solubilizing peptide moiety, cleavable linker, and masking peptide moiety of the delivery agent.

[0104] SEQ ID NO:7 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, and the amino acid portions of two anchoring groups of the delivery agent.

[0105] SEQ ID NO:8 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, the amino acid portions of two anchoring groups, and the C-terminal portion of the delivery agent.

[0106] SEQ ID NO:9 is the amino acid sequence of the solubilizing peptide group, cleavable linker, and masking peptide group of the delivery agent.

[0107] SEQ ID NO:10 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, and the amino acid portions of two anchoring groups of the delivery agent.

[0108] SEQ ID NO:11 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, the amino acid portions of two anchoring groups, and the C-terminal portion of the delivery agent.

[0109] SEQ ID NO:12 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, the amino acid portions of two anchoring groups, and the C-terminal portion of the delivery agent.

[0110] SEQ ID NO:13 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, the amino acid portions of two anchoring groups, and the C-terminal portion of the delivery agent, such as those included in delivery agents 21-4, 40-46, 42-39, 23-4, and 23-26.

[0111] SEQ ID NO:14 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, the amino acid portions of two anchoring groups, and the C-terminal portion of the delivery agent.

[0112] SEQ ID NO:15 is the amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, the amino acid portions of two anchoring groups, and the C-terminal portion of the delivery agent.

[0113] SEQ ID NO:16 is the amino acid sequence of the solubilizing peptide group.

[0114] SEQ ID NO:17 is the amino acid sequence of the solubilizing peptide group.

[0115] SEQ ID NO:18 is the amino acid sequence of delivery agent 23-6.

[0116] SEQ ID NO:19 is the amino acid sequence of delivery agent 23-7.

[0117] SEQ ID NO:20 is the amino acid sequence of the solubilizing peptide group.

[0118] SEQ ID NO:21 is the amino acid sequence of delivery agent 23-10.

[0119] SEQ ID NO:22 is the amino acid sequence of delivery agent 23-16.

[0120] SEQ ID NO:23 is the amino acid sequence of delivery agent 23-20.

[0121] SEQ ID NO:24 is the amino acid sequence of the solubilizing peptide group.

[0122] SEQ ID NO:25 is the amino acid sequence of delivery agent 30-2.

[0123] SEQ ID NO:26 is the amino acid sequence of the solubilizing peptide group.

[0124] SEQ ID NO:27 is the amino acid sequence of delivery agent 30-4.

[0125] SEQ ID NO:28 is the amino acid sequence of the solubilizing peptide group.

[0126] SEQ ID NO:29 is the amino acid sequence of delivery agent 30-6.

[0127] SEQ ID NO:30 is the amino acid sequence of the solubilizing peptide group.

[0128] SEQ ID NO:31 is the amino acid sequence of delivery agent 30-10.

[0129] SEQ ID NO:32 is the amino acid sequence of the solubilizing peptide group.

[0130] SEQ ID NO:33 is the amino acid sequence of delivery agent 30-14.

[0131] SEQ ID NO:34 is the amino acid sequence of the solubilizing peptide group.

[0132] SEQ ID NO:35 is the amino acid sequence of delivery agent 30-16.

[0133] SEQ ID NO:36 is the amino acid sequence of the solubilizing peptide group.

[0134] SEQ ID NO:37 is the amino acid sequence of delivery agent 30-18.

[0135] SEQ ID NO:38 is the amino acid sequence of the solubilizing peptide group.

[0136] SEQ ID NO:39 is the amino acid sequence of delivery agent 40-1.

[0137] SEQ ID NO:40 is the amino acid sequence of the solubilizing peptide group.

[0138] SEQ ID NO:41 is the amino acid sequence of the masking peptide group.

[0139] SEQ ID NO:42 is the amino acid sequence of delivery agent 50-22.

[0140] SEQ ID NO:43 is the amino acid sequence of the solubilizing peptide group.

[0141] SEQ ID NO:44 is the amino acid sequence of the solubilizing peptide group.

[0142] SEQ ID NO:45 is the amino acid sequence of the solubilizing peptide group.

[0143] SEQ ID NO:46 is the amino acid sequence of the solubilizing peptide group.

[0144] SEQ ID NO:47 is the amino acid sequence of the solubilizing peptide group.

[0145] SEQ ID NO:48 is the amino acid sequence of the solubilizing peptide group.

[0146] SEQ ID NO:49 is the amino acid sequence of the solubilizing peptide group.

[0147] SEQ ID NO:50 is the amino acid sequence of the solubilizing peptide group.

[0148] SEQ ID NO:51 is the amino acid sequence of the masking peptide group.

[0149] SEQ ID NO:52 is the amino acid sequence of delivery agent 51-11.

[0150] SEQ ID NO:53 is the amino acid sequence of delivery agent 51-12.

[0151] SEQ ID NO:54 is the amino acid sequence of delivery agent 51-13.

[0152] SEQ ID NO:55 is the amino acid sequence of delivery agent 51-14.

[0153] SEQ ID NO:56 is the amino acid sequence of delivery agent 51-15

[0154] SEQ ID NO:57 is the amino acid sequence of delivery agent 51-16.

[0155] SEQ ID NO:58 is the amino acid sequence of delivery agent 51-17.

[0156] SEQ ID NO:59 is the amino acid sequence of delivery agent 51-18.

[0157] SEQ ID NO:60 is the amino acid sequence of the solubilizing peptide group.

[0158] SEQ ID NO:61 is the amino acid sequence of the solubilizing peptide group.

[0159] SEQ ID NO:62 is the amino acid sequence of the solubilizing peptide group.

[0160] SEQ ID NO:63 is the amino acid sequence of the solubilizing peptide group.

[0161] SEQ ID NO:64 is the amino acid sequence of the solubilizing peptide group.

[0162] SEQ ID NO:65 is the amino acid sequence of the solubilizing peptide group.

[0163] SEQ ID NO:66 is the amino acid sequence of the solubilizing peptide group.

[0164] SEQ ID NO:67 is the amino acid sequence of the solubilizing peptide group.

[0165] SEQ ID NO:68 is the amino acid sequence of the solubilizing peptide group.

[0166] SEQ ID NO:69 is the amino acid sequence of delivery agent 51-24.

[0167] SEQ ID NO:70 is the amino acid sequence of delivery agent 51-25.

[0168] SEQ ID NO:71 is the amino acid sequence of delivery agent 51-26.

[0169] SEQ ID NO:72 is the amino acid sequence of delivery agent 51-27.

[0170] SEQ ID NO:73 is the amino acid sequence of delivery agent 51-28.

[0171] SEQ ID NO:74 is the amino acid sequence of delivery agent 51-29.

[0172] SEQ ID NO:75 is the amino acid sequence of delivery agent 51-30.

[0173] SEQ ID NO:76 is the amino acid sequence of delivery agent 51-31.

[0174] SEQ ID NO:77 is the amino acid sequence of delivery agent 50-23.

[0175] SEQ ID NO:78 is the amino acid sequence of the solubilizing peptide group.

[0176] SEQ ID NO:79 is the amino acid sequence of delivery agent 56-5.

[0177] SEQ ID NO:80 is the amino acid sequence of the solubilizing peptide group.

[0178] SEQ ID NO:81 is the amino acid sequence of the masking peptide group.

[0179] SEQ ID NO:82 is the amino acid sequence of delivery agent 60-16.

[0180] SEQ ID NO:83 is the amino acid sequence of delivery agent 60-17.

[0181] SEQ ID NO:84 is the amino acid sequence of the solubilizing peptide group.

[0182] SEQ ID NO:85 is the amino acid sequence of the solubilizing peptide group.

[0183] SEQ ID NO:86 is the amino acid sequence of the solubilizing peptide group.

[0184] SEQ ID NO:87 is the amino acid sequence of delivery agent 88-8.

[0185] SEQ ID NO:88 is the amino acid sequence of delivery agent 88-9.

[0186] SEQ ID NO:89 is the amino acid sequence of delivery agent 88-10.

[0187] SEQ ID NO:90 is the amino acid sequence of delivery agent 88-11

[0188] SEQ ID NO:91 is the amino acid sequence of delivery agent 88-12.

[0189] SEQ ID NO:92 is the amino acid sequence of the solubilizing peptide group.

[0190] SEQ ID NO:93 is the amino acid sequence of the solubilizing peptide group.

[0191] SEQ ID NO:94 is the amino acid sequence of the solubilizing peptide group.

[0192] SEQ ID NO:95 is the amino acid sequence of the solubilizing peptide group.

[0193] SEQ ID NO:96 is the amino acid sequence of the lysing peptide group.

[0194] SEQ ID NO:97 is the amino acid sequence of the lysing peptide group.

[0195] SEQ ID NO:98 is the amino acid sequence of the masking peptide group.

[0196] SEQ ID NO:99 is the amino acid sequence of the masking peptide group.

[0197] SEQ ID NO:100 is the amino acid sequence of the masking peptide group.

[0198] SEQ ID NO:101 is the amino acid sequence of the masking peptide group.

[0199] SEQ ID NO:102 is the amino acid sequence of the delivery agent 109-7.

[0200] SEQ ID NO:103 is the amino acid sequence of the delivery agent 114-1.

[0201] SEQ ID NO:104 is the amino acid sequence of the delivery agent 114-3.

[0202] SEQ ID NO:105 is the amino acid sequence of the delivery agent 114-12.

[0203] SEQ ID NO:106 is the amino acid sequence of the delivery agent 114-30.

[0204] SEQ ID NO:107 is the amino acid sequence of the delivery agent 114-33.

[0205] SEQ ID NO:108 is the amino acid sequence of the lysing peptide group, cleavable linker, masking peptide group, amino acid portions of two anchoring groups, and C-terminal portion of the delivery agent.

[0206] SEQ ID NO:109 is the amino acid sequence of the lysing peptide group.

[0207] SEQ ID NO:110 is the amino acid sequence of the lysing peptide group.

[0208] SEQ ID NO:111 is the amino acid sequence of the masking peptide group.

[0209] SEQ ID NO:112 is the amino acid sequence of the masking peptide group.

[0210] SEQ ID NO:113 is the amino acid sequence of the delivery agent 114-6.

[0211] SEQ ID NO:114 is the amino acid sequence of delivery agent 114-9.

[0212] SEQ ID NO:115 is the amino acid sequence of the solubilizing peptide group.

[0213] SEQ ID NO:116 is the amino acid sequence of the solubilizing peptide group.

[0214] SEQ ID NO:117 is the amino acid sequence of the solubilizing peptide group.

[0215] SEQ ID NO:118 is the amino acid sequence of delivery agent 97-1.

[0216] SEQ ID NO:119 is the amino acid sequence of delivery agent 97-2.

[0217] SEQ ID NO:120 is the amino acid sequence of delivery agent 97-3

[0218] SEQ ID NO:121 is the amino acid sequence of delivery agent 97-5.

[0219] SEQ ID NO:122 is the amino acid sequence of delivery agent 97-7.

[0220] SEQ ID NO:123 is the amino acid sequence of delivery agent 91-6.

[0221] SEQ ID NO:124 is the amino acid sequence of delivery agent 114-18.

[0222] SEQ ID NO:125 is the amino acid sequence of delivery agent 114-21.

[0223] SEQ ID NO:126 is the amino acid sequence of delivery agent 114-24.

[0224] SEQ ID NO:127 is the amino acid sequence of delivery agent 114-27.

[0225] SEQ ID NO:128 is the amino acid sequence of delivery agent 21-11.

[0226] 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.

[0227] SEQ ID NO:130 is the amino acid sequence of delivery agent 36-48.

[0228] SEQ ID NO:131 is the amino acid sequence of delivery agent 96-1.

[0229] SEQ ID NO:132 is the amino acid sequence of delivery agent 96-4.

[0230] SEQ ID NO:133 is the amino acid sequence of delivery agent 96-5.

[0231] SEQ ID NO:134 is the amino acid sequence of the masking peptide group.

[0232] SEQ ID NO:135 is the amino acid sequence of delivery agent 26-17.

[0233] SEQ ID NO:136 is the amino acid sequence of the masking peptide group.

[0234] SEQ ID NO:137 is the amino acid sequence of the masking peptide group.

[0235] SEQ ID NO:138 is the amino acid sequence of delivery agent 19-3.

[0236] SEQ ID NO:139 is the amino acid sequence of delivery agent 19-5.

[0237] SEQ ID NO:140 is the amino acid sequence of the masking peptide group.

[0238] SEQ ID NO:141 is the amino acid sequence of the masking peptide group.

[0239] SEQ ID NO:142 is the amino acid sequence of delivery agent 23-12.

[0240] SEQ ID NO:143 is the amino acid sequence of delivery agent 23-13.

[0241] SEQ ID NO:144 is the amino acid sequence of delivery agent 23-14.

[0242] SEQ ID NO:145 is the amino acid sequence of the masking peptide group.

[0243] SEQ ID NO:146 is the amino acid sequence of delivery agent 21-13.

[0244] SEQ ID NO:147 is the amino acid sequence of delivery agents 23-15 and 21-12.

[0245] SEQ ID NO:148 is the amino acid sequence of the masking peptide group.

[0246] SEQ ID NO:149 is the amino acid sequence of delivery agent 21-8.

[0247] SEQ ID NO:150 is the amino acid sequence of the lysis peptide group.

[0248] SEQ ID NO:151 is the amino acid sequence of the lysis peptide group.

[0249] SEQ ID NO:152 is the amino acid sequence of the lysis peptide group.

[0250] SEQ ID NO:153 is the amino acid sequence of the lysis peptide group and / or the masking peptide group.

[0251] SEQ ID NO:154 is the amino acid sequence of the masking peptide group.

[0252] SEQ ID NO:155 is the amino acid sequence of the masking peptide group.

[0253] SEQ ID NO:156 is the amino acid sequence of the masking peptide group.

[0254] SEQ ID NO:157 is the amino acid sequence of the masking peptide group.

[0255] SEQ ID NO:158 is the amino acid sequence of the masking peptide group.

[0256] SEQ ID NO:159 is the amino acid sequence of the delivery agent 40-31.

[0257] SEQ ID NO:160 is the amino acid sequence of the delivery agent 40-32.

[0258] SEQ ID NO:161 is the amino acid sequence of the delivery agent 40-33.

[0259] SEQ ID NO:162 is the amino acid sequence of the delivery agent 40-34.

[0260] SEQ ID NO:163 is the amino acid sequence of the delivery agent 40-35.

[0261] SEQ ID NO:164 is the amino acid sequence of the delivery agent 40-36.

[0262] SEQ ID NO:165 is the amino acid sequence of the delivery agent 40-37.

[0263] SEQ ID NO:166 is the amino acid sequence of the delivery agent 40-38.

[0264] SEQ ID NO:167 is the amino acid sequence of the delivery agent 40-39.

[0265] SEQ ID NO:168 is the amino acid sequence of the delivery agent 40-40.

[0266] SEQ ID NO:169 is the amino acid sequence of delivery agent 40-41.

[0267] SEQ ID NO:170 is the amino acid sequence of delivery agent 40-42.

[0268] SEQ ID NO:171 is the amino acid sequence of delivery agent 40-43.

[0269] SEQ ID NO:172 is the amino acid sequence of the masking peptide group.

[0270] SEQ ID NO:173 is the amino acid sequence of the masking peptide group.

[0271] SEQ ID NO:174 is the amino acid sequence of the masking peptide group.

[0272] SEQ ID NO:175 is the amino acid sequence of the masking peptide group.

[0273] SEQ ID NO:176 is the amino acid sequence of the masking peptide group.

[0274] SEQ ID NO:177 is the amino acid sequence of the masking peptide group.

[0275] SEQ ID NO:178 is the amino acid sequence of the masking peptide group.

[0276] SEQ ID NO:179 is the amino acid sequence of the masking peptide group.

[0277] SEQ ID NO:180 is the amino acid sequence of the masking peptide group.

[0278] SEQ ID NO:181 is the amino acid sequence of the masking peptide group.

[0279] SEQ ID NO:182 is the amino acid sequence of delivery agent 53-6.

[0280] SEQ ID NO:183 is the amino acid sequence of delivery agent 53-7.

[0281] SEQ ID NO:184 is the amino acid sequence of delivery agent 53-8.

[0282] SEQ ID NO:185 is the amino acid sequence of delivery agent 53-9.

[0283] SEQ ID NO:186 is the amino acid sequence of delivery agent 53-10.

[0284] SEQ ID NO:187 is the amino acid sequence of delivery agent 53-11.

[0285] SEQ ID NO:188 is the amino acid sequence of delivery agent 53-12.

[0286] SEQ ID NO:189 is the amino acid sequence of delivery agent 53-13.

[0287] SEQ ID NO:190 is the amino acid sequence of delivery agent 53-14.

[0288] SEQ ID NO:191 is the amino acid sequence of delivery agent 53-15.

[0289] SEQ ID NO:192 is the amino acid sequence of delivery agent 96-10.

[0290] SEQ ID NO:193 is the amino acid sequence of delivery agent 21-6 and / or 23-5.

[0291] SEQ ID NO:194 is the amino acid sequence of delivery agent 26-13.

[0292] SEQ ID NO:195 is the amino acid sequence of delivery agent 21-7b.

[0293] SEQ ID NO:196 is the amino acid sequence of delivery agent 109-8.

[0294] SEQ ID NO:197 is the amino acid sequence of delivery agent 78-2.

[0295] SEQ ID NO:198 is the amino acid sequence of delivery agent 86-5.

[0296] SEQ ID NO:199 is the amino acid sequence of delivery agent 50-23.

[0297] SEQ ID NO:200 is the amino acid sequence of delivery agent 50-24.

[0298] SEQ ID NO:201 is the amino acid sequence of delivery agent 50-25.

[0299] SEQ ID NO:202 is the amino acid sequence of delivery agent 50-26.

[0300] SEQ ID NO:203 is the amino acid sequence of the masking peptide group.

[0301] SEQ ID NO:204 is the amino acid sequence of the masking peptide group.

[0302] SEQ ID NO:205 is the amino acid sequence of the masking peptide group.

[0303] SEQ ID NO:206 is the amino acid sequence of the masking peptide group.

[0304] SEQ ID NO:207 is the amino acid sequence of the masking peptide group.

[0305] SEQ ID NO:208 is the amino acid sequence of the delivery agent 30-3.

[0306] SEQ ID NO:209 is the amino acid sequence of the delivery agent 30-5.

[0307] SEQ ID NO:210 is the amino acid sequence of the delivery agent 30-7.

[0308] SEQ ID NO:211 is the amino acid sequence of the delivery agent 30-9.

[0309] SEQ ID NO:212 is the amino acid sequence of the delivery agent 30-11.

[0310] SEQ ID NO:213 is the amino acid sequence of the delivery agent 30-15.

[0311] SEQ ID NO:214 is the amino acid sequence of the delivery agent 30-17.

[0312] SEQ ID NO:215 is the amino acid sequence of the delivery agent 19-17C.

[0313] SEQ ID NO:216 is the amino acid sequence of the delivery agent 21-5.

[0314] SEQ ID NO:217 is the amino acid sequence of the delivery agent 22-11.

[0315] SEQ ID NO:218 is the amino acid sequence of the delivery agent 23-1.

[0316] SEQ ID NO:219 is the amino acid sequence of the delivery agent 23-2.

[0317] SEQ ID NO:220 is the amino acid sequence of the delivery agent 23-3.

[0318] SEQ ID NO:221 is the amino acid sequence of the delivery agent 22-1.

[0319] SEQ ID NO:222 is the amino acid sequence of the delivery agent 22-12.

[0320] SEQ ID NO:223 is the amino acid sequence of delivery agent 34-32.

[0321] SEQ ID NO:224 is the amino acid sequence of delivery agent 34-33.

[0322] SEQ ID NO:225 is the amino acid sequence of delivery agent 34-34.

[0323] SEQ ID NO:226 is the amino acid sequence of delivery agent 109-2.

[0324] SEQ ID NO:227 is the amino acid sequence of the cleavable linker.

[0325] SEQ ID NO:228 is the amino acid sequence of delivery agent 33-3.

[0326] SEQ ID NO:229 is the nucleic acid sequence of morpholino 1.

[0327] SEQ ID NO:230 is the nucleic acid sequence of morpholino 2.

[0328] SEQ ID NO:231 is the amino acid sequence of delivery agent 96-6.

[0329] SEQ ID NO:232 is the amino acid sequence of delivery agent 16-6.

[0330] SEQ ID NO:233 is the amino acid sequence of the cleavable linker.

[0331] SEQ ID NO:234 is the amino acid sequence of the cleavable linker.

[0332] SEQ ID NO:235 is the amino acid sequence of delivery agent 5.

[0333] SEQ ID NO:236 is the amino acid sequence of delivery agent 7.

[0334] SEQ ID NO:237 is the amino acid sequence of delivery agent 8.

[0335] SEQ ID NO:238 is the nucleic acid sequence of the forward DNA primer spanning dystrophin exons 21 and 24.

[0336] SEQ ID NO:239 is the nucleic acid sequence of the reverse DNA primer spanning dystrophin exons 21 and 24.

[0337] SEQ ID NO:240 is the morpholino nucleic acid sequence.

[0338] SEQ ID NO:241 is a morpholino nucleic acid sequence.

[0339] SEQ ID NO:242 is a morpholino nucleic acid sequence.

[0340] SEQ ID NO:243 is a morpholino nucleic acid sequence.

[0341] SEQ ID NO:244 is a morpholino nucleic acid sequence.

[0342] SEQ ID NO:245 is the amino acid sequence of delivery agent 22-1 branch.

[0343] 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.

[0344] SEQ ID NO:247 is the amino acid sequence of delivery agent 62-9.

[0345] SEQ ID NO:248 is the amino acid sequence of delivery agent 60-4.

[0346] SEQ ID NO:249 is the amino acid sequence of delivery agents 96-7, 86-4 and 102-3.

[0347] SEQ ID NO:250 is the amino acid sequence of delivery agent 26-13A.

[0348] SEQ ID NO:251 is the amino acid sequence of delivery agent 26-15A.

[0349] SEQ ID NO:252 is the amino acid sequence of delivery agent 26-7a’.

[0350] SEQ ID NO:253 is the amino acid sequence of delivery agent 26-7b.

[0351] SEQ ID NO:254 is the amino acid sequence of delivery agent 26-7b’.

[0352] SEQ ID NO:255 is the amino acid sequence of delivery agent 26-14B.

[0353] SEQ ID NO:256 is the amino acid sequence of delivery agent 26-14D.

[0354] SEQ ID NO:257 is the amino acid sequence of delivery agent 26-2A.

[0355] SEQ ID NO:258 is the amino acid sequence of delivery agent 26-11.

[0356] SEQ ID NO:259 is the amino acid sequence of delivery agent 40-23B.

[0357] SEQ ID NO:260 is the amino acid sequence of delivery agent 21-4b.

[0358] SEQ ID NO:261 is the amino acid sequence of delivery agent 21-4b’.

[0359] SEQ ID NO:262 is the amino acid sequence of delivery agent 21-6b.

[0360] SEQ ID NO:263 is the amino acid sequence of delivery agent 60-1b.

[0361] SEQ ID NO:264 is the amino acid sequence of delivery agent 78-1.

[0362] SEQ ID NO:265 is the amino acid sequence of delivery agent 19-17A.

[0363] SEQ ID NO:266 is the amino acid sequence of delivery agent 22-12 branch.

[0364] SEQ ID NO:267 is the amino acid sequence of delivery agent 33-26a.

[0365] SEQ ID NO:268 is the amino acid sequence of delivery agent 33-26b.

[0366] SEQ ID NO:269 is the amino acid sequence of delivery agent 33-26c.

[0367] SEQ ID NO:270 is the amino acid sequence of delivery agent 6.

[0368] SEQ ID NO:271 is the amino acid sequence of delivery agent 40-23A.

[0369] SEQ ID NO:272 is the amino acid sequence of delivery agents 215-2DBCO, 215-2FA, 215-2PEG12-BCN, and 215-2Ab.

[0370] SEQ ID NO:273 is the amino acid sequence of delivery agents 216-A DBCO, 216-AFA, 216-1PEG12-BCN, and 216-1Ab.

[0371] SEQ ID NO:274 is the amino acid sequence of delivery agents 216-B DBCO, 216-B FA, and 217-2.

[0372] SEQ ID NO:275 is the amino acid sequence of delivery agent 218-2.

[0373] SEQ ID NO:276 is the amino acid sequence of delivery agent 219-1.

[0374] SEQ ID NO:277 is the amino acid sequence of delivery agent 219-2.

[0375] SEQ ID NO:278 is the amino acid sequence of delivery agent 224-2, which does not contain a masking peptide group.

[0376] SEQ ID NO:279 is the amino acid sequence of delivery agent 225-2, which does not contain a masking peptide group.

[0377] SEQ ID NO:280 is the amino acid sequence of delivery agent 223-2. Detailed Description of the Invention

[0379] I. Glossary of Terms and Overview

[0380] The following glossary of terms is intended to better describe the present disclosure and guide one of ordinary skill in the art in practicing the present disclosure. As used herein, "comprising" means "including," and the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. The term "or" refers to a single element of the recited alternative elements or a combination of two or more elements, unless the context clearly dictates otherwise.

[0381] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. Unless otherwise noted, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the present disclosure are apparent from the following detailed description and claims.

[0382] When a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated value or intermediate value within that range is encompassed within the present disclosure, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges, which may independently be included within the smaller ranges, are also encompassed within the present disclosure, except for any clearly excluded limit values within the range. When the range includes one or both of the limits, ranges excluding one or both of the included limits are also included within the present disclosure.

[0383] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, percentages, temperatures, times, and the like, used in the specification or claims, are to be understood as being modified by the term "about." Accordingly, unless otherwise indicated, whether implicit or explicit, the numerical parameters set forth are approximations that may depend upon the desired properties sought and / or the detection limits under standard test conditions / methods. When embodiments are directly and explicitly distinguished from any discussed prior art, the numbers of the embodiments are not approximations unless the term "about" is recited. In addition, not all alternatives recited herein are equivalent. Also, within any numerical range, the specific recited endpoints are considered part of the recited range.

[0384] The compounds disclosed herein may contain one or more asymmetric elements, such as stereocenters, chiral axes, etc., such as asymmetric carbon atoms, such that the chemical conjugates may exist in different stereoisomeric forms. These compounds may be, for example, racemates or optically active forms. For compounds having two or more asymmetric elements, these compounds may also be mixtures of diastereomers. For compounds having an asymmetric center, all optical isomers in pure form and their mixtures are encompassed. In these cases, a single enantiomer, i.e., an optically active form, may be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of a racemate. Resolution of a racemate may 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 these forms.

[0385] The stereochemical definitions and conventions used herein generally follow 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, i.e., they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule around its chiral center. The prefixes d and l or (+) and (-) are used to denote the sign of rotation of the plane-polarized light of the compound, where (-) or l indicates that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory.

[0386] All forms of the probe (e.g., solvates, optical isomers, enantiomeric forms, polymorphs, free compounds, and salts) may be used alone or in combination.

[0387] To facilitate review of various aspects of the present disclosure, explanations of specific terms are provided below. Some functional group terms contain 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 attached to the formula described herein. For example, a functional group having the formula "-OC(O)R b " is attached to an atom of a functionalized compound through the oxygen atom of the functional group next to the "-" symbol.

[0388] Administration: As used herein, administering a delivery agent and / or a therapeutic agent (e.g., the delivery agents and therapeutic agents described herein, such as morpholino or other therapeutic agents described herein) to a subject means applying, giving, or bringing into contact with the subject the agent by any effective route. Administration can be accomplished by a variety of routes, such as intravenous, intratumoral, topical, oral, subcutaneous, transdermal, intrathecal, intramuscular, intraperitoneal, intranasal, and similar routes, or combinations of these routes. Exemplary administration routes are described herein.

[0389] Aliphatic: A compound or group thereof that is substantially hydrocarbon-based (e.g., C 6 H 13 , representing a hexyl group), including alkanes, alkenes, alkynes, including their cyclic forms, and also including straight-chain and branched-chain arrangements, as well as all stereoisomers and positional isomers. Unless otherwise explicitly stated, aliphatic groups contain from 1 to 25 carbon atoms; for example, from 1 to 15, from 1 to 10, from 1 to 6, or from 1 to 4 carbon atoms. The term "lower aliphatic" refers to an aliphatic group containing from 1 to 10 carbon atoms. The aliphatic chain can be substituted or unsubstituted. Unless explicitly referred to as "unsubstituted aliphatic", aliphatic groups can be unsubstituted or substituted. Aliphatic groups can be substituted with one or more substituents (up to two substituents per methylene carbon in the aliphatic chain, or up to one substituent per carbon of a C═C double bond in the aliphatic chain, or up to one substituent on the carbon of a terminal methylene). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxy, oxo, sulfonamide, mercapto, thioalkoxy, or other functional groups. In some instances, substituted aliphatic groups include at least one sp 3 -hybridized carbon or two sp 2 -hybridized carbons bound to a double bond or at least two sp-hybridized carbons bound to a triple bond.

[0390] Alkyl: A hydrocarbon group having a saturated carbon chain. The chain can be cyclic (e.g., cycloalkyl), branched, or unbranched. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. The term lower alkyl indicates that the chain contains from 1 to 10 carbon atoms. The terms alkenyl and alkynyl refer to hydrocarbon groups having carbon chains that contain one or more double or triple bonds, respectively. In some examples, substituted alkyl includes at least one sp 3 hybridized carbon.

[0391] Aliphatic aryl: An aryl that is coupled or can be coupled to a compound disclosed herein, wherein the aryl is coupled or becomes coupled through an aliphatic group.

[0392] Aliphatic heteroaryl: A heteroaryl that is coupled or can be coupled to a compound disclosed herein, wherein the heteroaryl is coupled or becomes coupled through an aliphatic group.

[0393] Amine: -NR b R c wherein R b and R c are each independently selected from hydrogen, aliphatic, aryl, heteroaliphatic, aliphatic-aryl, heteroaryl, aliphatic-heteroaryl, heteroaliphatic-aryl, heteroaliphatic-heteroaryl, and any combination thereof.

[0394] Amino acid: An organic acid that contains both a basic amino group (e.g., -NH 2 ) and an acidic carboxyl group (e.g., -COOH). The amino acids that are components of proteins are α-amino acids, in which the -NH 2 group is attached to the carbon atom adjacent to the -COOH group.

[0395] Antibody: A polypeptide ligand (e.g., an immunoglobulin, an antigen-binding fragment, or a derivative thereof) that contains at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an epitope of an antigen. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), targeting antibodies, and antigen-binding fragments, provided that they exhibit the desired antigen-binding activity. Antibodies are characterized by reacting specifically with an antigen in a demonstrable manner. Therapeutic antibodies recognize and bind to antigen receptors to activate or inhibit a series of biological processes, such as for blocking cancer cell growth and / or triggering an immune system response.

[0396] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and antigen-binding fragments thereof that retain the binding affinity for the antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, dsFv, Fab’, Fab’-SH, F(ab’)2 ; bispecific antibodies; linear antibodies; single-chain antibody molecules (such as scFv and ds-scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments generated by modifying whole antibodies or synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dübel (Eds.), Antibody Engineering, Vols. 1-2, 2 nd ed., Springer-Verlag, 2010).

[0397] Antibodies also include genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). Antibodies also include the defucosylated forms of disclosed antibodies.

[0398] An antibody may have one or more binding sites. If there are more than one binding sites, the binding sites may be the same or different from each other. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or a Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

[0399] Mammalian immunoglobulin molecules are composed of heavy chains (H) and light chains (L), each having a variable region, which are respectively called variable heavy chain (V H ) region and variable light chain (V L ) region. The V H region and the V L region together are responsible for binding the antigen recognized by the antibody. Mammalian immunoglobulins have five major heavy chain classes (or isotypes), which 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 its function has some similarities with those of mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are present in amphibians.

[0400] The variable region of an antibody contains "framework" regions and hypervariable regions, called "complementary determining regions" or "CDRs". The CDRs are mainly responsible for binding to the epitope. The framework regions of the antibody are used 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 Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; Kabat numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273, 927-948, 1997; Chothia numbering scheme) and the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; IMGT numbering scheme). The Kabat and IMGT databases are maintained online.

[0401] A "monoclonal antibody" is an antibody produced by the cloning of a single lymphocyte or by a cell into which a single antibody-encoding sequence has been transfected. Monoclonal antibodies include humanized monoclonal antibodies.

[0402] Cancer: Cancer is characterized by abnormal or uncontrolled cell growth (malignant cells). Malignant tumors typically also have other characteristics, including metastasis, interference with the normal functioning of adjacent cells, release of cytokines or other secreted products at abnormal levels, inhibition or exacerbation of the inflammatory or immune response, invasion of surrounding or distant tissues or organs (such as lymph nodes), etc. "Metastatic disease" refers to the migration of cancer cells away from the primary cancer site, for example, through the blood or lymphatic system, to other parts of the body.

[0403] The "cancer burden" of a subject can be measured by the number, volume, and / or weight of one or more tumors. Tumors that have not metastasized are called "benign". Tumors that invade surrounding tissues and / or can metastasize are cancers (referred to as "malignant").

[0404] In some instances, the cancer is a hematological cancer such as leukemia (including acute leukemia (e.g., 11q23-positive acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) or chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myelocytic leukemia, and chronic lymphocytic leukemia)), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. In specific non-limiting instances, the lymphoid malignancy can be adult T-cell leukemia, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, Hodgkin lymphoma, or diffuse large B-cell lymphoma.

[0405] Examples of solid cancers such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, cholangiocarcinoma (especially intrahepatic cholangiocarcinoma), osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, uterine leiomyosarcoma, orbital rhabdomyosarcoma, colorectal cancer (e.g., colon cancer), lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast cancer, uveal melanoma, ductal carcinoma, and lobular breast cancer, triple-negative breast cancer), uterine / endometrial cancer, neuroendocrine cancer, lung cancer (including non-small cell lung cancer), ovarian cancer, prostate cancer, hepatocellular carcinoma, angiosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid cancer, papillary thyroid cancer, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, gallbladder cancer, esophageal cancer, kidney cancer (e.g., renal cell carcinoma), melanoma, liver cancer, bile duct cancer, choriocarcinoma, Wilms tumor, cervical cancer, brain cancer, pleural cancer, bronchogenic carcinoma, testicular cancer, seminoma, bladder cancer, and CNS cancers (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoma may be a solid cancer in some cases. Specifically, by non-limiting example, the cancer is colorectal cancer, kidney cancer, or melanoma.

[0406] Cathepsin B: A lysosomal cysteine protease that has endopeptidase activity (cleaving internal bonds) at neutral / alkaline pH and exopeptidase (carboxydipeptidase) activity at acidic pH and can play a role in protein turnover. Cathepsin B can hydrolyze proteins with broad specificity. However, its cleavage specificity preferably but 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, which are dimerically linked by disulfide bonds.

[0407] Chemical linker: A molecular or atomic group located between two moieties. For example, the delivery agents described herein may include a chemical linker between components of the delivery agents of the present disclosure (such as an anchoring group, a targeting group, a C-terminal group, etc.) and a therapeutic agent. Generally, a chemical linker is bifunctional, so the chemical linker contains functional groups at each end, where the functional groups are used to couple the linker to the delivery agent and the therapeutic agent. These two functional groups can be the same (referred to as a homobifunctional linker) or different (referred to as a heterobifunctional linker).

[0408] Chemotherapeutic agent: Any chemical or biological agent having a therapeutic effect for treating diseases characterized by abnormal cell growth. For example, chemotherapeutic agents can be used to treat solid cancers such as sarcoma, carcinoma, lymphoma, colorectal cancer, or skin cancer. Specific examples of chemotherapeutic agents that can be used include microtubule-binding agents, DNA intercalating agents 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 provided in the following literature: Sausville and Longo, Principles of Cancer Treatment, Chapter 69 in Harrison’s Principles of Internal Medicine (20 th ed.), McGraw-Hill, 2018; Niederhuber et al., Cancer Pharmacology, Ch. 25 in Abeloff’s Clinical Oncology (6 th ed.), Elsevier, 2019; Gullatte et al., Clinical Guide to Antineoplastic Therapy: A Chemotherapy Handbook (4 th(ed.), Oncology Nursing Society, 2020; Chabner and Longo, Cancer Chemotherapy, Immunotherapy and Biotherapy: Principles and Practice (6th ed.), Lippincott Williams & Wilkins, 2018; Skeel, Handbook of Cancer Chemotherapy (9th ed.), Lippincott Williams & Wilkins, 2016. Combination chemotherapy is the administration of more than one chemotherapeutic agent to treat cancer.

[0409] 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 located between a solubilizing peptide moiety and a masking peptide moiety. In some aspects of the present disclosure, the cleavable linker comprises a peptide sequence that can be cleaved to separate the solubilizing peptide moiety from the masking peptide moiety. In some aspects of the present disclosure, such cleavable linkers are cleaved by enzymes. Suitable cleavable linkers are described herein.

[0410] Combination, combination therapy: A mode of treatment in which two or more therapeutic components such as the delivery agents disclosed herein and one or more therapeutic agents are combined to treat a disorder or disease such as a genetic disease, rare disease, cancer, immune disease, or infectious disease. As used herein, the therapeutic components provided in the combination (e.g., the disclosed delivery agents, one or more therapeutic agents, or conjugates formed from these two therapeutic components) can be contacted with cells and / or administered to a subject at two or more different times, substantially simultaneously or sequentially, in any order, or a combination of these two modes. One or more of the therapeutic agents in the combination can target several biological pathways in a unique synergistic or additive manner to treat the disorder or disease. The combination therapies disclosed herein can be used to treat a class of disorders or diseases such as cancer (e.g., melanoma), or two or more different disorders or diseases such as two or more types of cancer (e.g., renal cancer and colorectal cancer).

[0411] Conservative variant: A "conservative" amino acid substitution refers to a substitution that does not significantly affect or reduce the affinity or activity of a protein such as an antibody or a peptide such as the solubilizing peptide moiety, masking peptide moiety, or cleavable linker disclosed herein. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parental amino acid, provided that the variant retains activity. Non-conservative substitutions are those that reduce protein activity.

[0412] There are conservative amino acid substitution tables that provide amino acids with similar functions. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0413] 1) Alanine (A), serine (S), threonine (T);

[0414] 2) Aspartic acid (D), glutamic acid (E);

[0415] 3) Asparagine (N), glutamine (Q);

[0416] 4) Arginine (R), lysine (K);

[0417] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0418] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0419] Contact: Placing in a manner of direct physical association, including in solid and liquid forms. In one instance, contact includes the association between a therapeutic component (e.g., a delivery agent and / or a therapeutic agent disclosed herein) in a liquid medium and one or more cells (e.g., cells of a subject or cultured cells). Contact can occur in vitro with isolated cells or tissues, or in vivo by administration to a subject.

[0420] Derivative: A compound derived from a similar compound, or a compound that can conceivably be produced from another compound, e.g., if one atom is replaced by another atom or group of atoms.

[0421] Direct or indirect coupling: As used herein, the phrase "direct coupling" means that the groups or compounds mentioned are chemically coupled to each other without any intermediate. The phrase "indirect coupling" as used herein means that the groups mentioned are chemically coupled to each other through another moiety (e.g., a functional group, a linker, or a combination thereof).

[0422] Effective amount, therapeutically effective amount: The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent (e.g., one or more delivery agents provided herein, administered alone, in combination with other therapeutic agents, or potentially in combination) sufficient to induce a desired biological outcome. The outcome can be introduction of a therapeutic agent into the cytosol of a cell, and / or amelioration or alleviation of the signs, symptoms, or causes of a disease (e.g., reduction of a cancer burden in a subject), or any other desired alteration of a biological system. The effective amount can vary depending on the disorder being treated, the stage of progression of the disorder, and the type and concentration of the formulation being applied. In some aspects of the present disclosure, the effective amount of a combination of therapeutic agents disclosed herein is an amount sufficient to produce a detectable therapeutic response when administered to a subject. Such a therapeutic response can include, for example, reduction of the burden of a genetic disorder, rare disease, cancer, immune disorder, or infectious disease. The appropriate amount in any given case will be apparent or can be determined by routine experimentation, such as administering a combination of therapeutic agents (e.g., combination therapy for treating a genetic disorder, rare disease, cancer, immune disorder, or infectious disease) and observing the response of the subject.

[0423] In one embodiment, the therapeutically effective amount is the amount required to eliminate a tumor, reduce the size of a tumor, or prevent tumor metastasis, e.g., to reduce 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 even 100% compared to its size / volume / number prior to treatment, and / or to reduce 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 even 100%.

[0424] Fluorescent dyes, fluorochromes or fluorophores: Molecular components that cause a molecule to fluoresce. The component can be a functional group of the molecule that absorbs energy at a specific wavelength and re-emits the energy at a different (but also specific) wavelength. In some aspects of the present disclosure, the fluorophore can fluoresce when the compound (or sample or composition containing the compound) is exposed to an excitation source or after cleavage from a compound embodiment. In some aspects of the present disclosure, the molecular component that causes a molecule to fluoresce is a dye. In some aspects, the amount and wavelength of the emitted energy depend on both the dye and the chemical environment of the dye. Many dyes can include, but are not limited to, 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, acridone-2-ylalanine, L-leucine 7-amide-4-methylcoumarin, trans-4-hydroxy-L-proline 7-amide-4-methylcoumarin, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 633, Alexa647, 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 Green514, sulfonyl rhodamine B, Texas Red, tetramethylrhodamine, fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), tandem PE-Texas Red, tandem PE-Cy5, propidium iodide, EGFP, EYGP, ECF, DsRed, allophycocyanin (APC), PerCp, SYTOX Green, coumarin, Alexa Fluors (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), LDS751, Indo-1, Fluo-3, DCFH, DHR, SNARF, Y66F, Y66H, EBFP, GFPuv, ECFP, GFP, AmCyan1, Y77W, S65A, S65C, S65L, S65T, ZsGreen1, ZsYellow1, DsRed2, DsRed monomer, AsRed2, mRFP1, HcRed1, monochlorobimane, calcein, DyLight Fluors, cyanine, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Lucifer Yellow, NBD, PE-Cy5 conjugate, PE-Cy7 conjugate, APC-Cy7 conjugate, Red 613, fluorescein, FluorX, BODIPY-FL, TRITC, X-rhodamine, Lissamine rhodamine B, TruRed, and derivatives thereof.

[0425] Halogenated aliphatic: An aliphatic group in which one or more hydrogen atoms, e.g., from 1 to 10 hydrogen atoms, are independently replaced by halogen atoms, e.g., fluorine, bromine, chlorine, or iodine.

[0426] Halogenated aliphatic aryl: An aryl group coupled or couplable to a compound disclosed herein, wherein the aryl group is coupled or coupled through a halogenated aliphatic group.

[0427] Halogenated aliphatic heteroaryl: A heteroaryl group coupled or couplable to a compound disclosed herein, wherein the heteroaryl group is coupled or coupled through a halogenated aliphatic group.

[0428] Halogenated heteroaliphatic: A heteroaliphatic group in which one or more hydrogen atoms, e.g., from 1 to 10 hydrogen atoms, are independently replaced by halogen atoms, e.g., fluorine, bromine, chlorine, or iodine.

[0429] Heteroaliphatic: An aliphatic group containing at least 1 to 20 heteroatoms (e.g., 1 to 15 heteroatoms or 1 to 5 heteroatoms) within the group, the heteroatoms being selected from, but not limited to, oxygen, nitrogen, sulfur, selenium, phosphorus, and their oxidized forms. Exemplary heteroaliphatic groups include, but are not limited to, aliphatic groups containing ethers, thioethers, esters, amines, carboxyls, carbonyls, or amides.

[0430] Heteroaliphatic aryl: An aryl group that is coupled or can be coupled to a compound disclosed herein, wherein the aryl group is coupled or coupled through a heteroaliphatic group.

[0431] Heteroaryl: An aryl group containing at least 1 to 6 heteroatoms (e.g., 1 to 4 heteroatoms) within the ring, and the heteroatoms can be selected from, but not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorus, and their oxidized forms. Such heteroaryl groups can have a single ring or multiple fused rings, where the fused rings can be or not be aromatic and / or contain heteroatoms, provided that the point of attachment is through an atom of the aromatic heteroaryl group.

[0432] Heteroatom: An atom other than carbon or hydrogen, such as (but not limited to) oxygen, nitrogen, sulfur, silicon, boron, selenium, or phosphorus. In a specifically disclosed aspect, for example, when valence constraints do not allow, the heteroatom does not include halogen atoms.

[0433] Immune disease or disorder: A disease or disorder, such as an autoimmune disorder or disease, in which the immune system mounts an immune response (such as a B-cell or T-cell response) against an endogenous antigen, thereby causing damage to tissues. The damage may be limited to some organs, such as thyroiditis, or may involve specific tissues in different locations, such as Goodpasture disease, or may be systemic, such as lupus erythematosus.

[0434] In some instances, autoimmune diseases include systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, type I diabetes, Wegener's granulomatosis, inflammatory bowel disease, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Achalasia, autoimmune encephalitis, Addison's disease, adrenitis, Graves' disease, eosinophilic granulomatosis with polyangiitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, atrophic gastritis, chronic hepatitis, lupoid hepatitis, atherosclerosis, Alzheimer's disease, demyelinating diseases, multiple sclerosis (including Balo disease), subacute cutaneous lupus erythematosus, 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 dysmotility, sclerodactyly, and telangiectasia), adult diabetes (type II diabetes), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, Hurst's disease, Crohn's disease, mixed connective tissue disease, polyarteritis, systemic necrotizing vasculitis, juvenile rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, post-cardiac syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird fancier's lung, allergic diseases, allergic encephalomyelitis, toxic epidermal necrolysis, hair loss, Alport's syndrome, alveolitis, allergic alveolitis, fibrotic alveolitis, inclusion body myositis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, ocular cicatricial pemphigoid, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki disease, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum diutinum, psoriasis, fetal erythrocytosis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochromic chronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, paroxysmal nocturnal hemoglobinuria, 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, Waldenstrom's macroglobulinemia, Berger's disease, rubella virus infection, and Evan's syndrome. Diseases that may cause or contribute to autoimmune diseases include Alzheimer's disease, parvovirus infection, rubella virus infection, dengue virus infection, Epstein-Barr virus infection, Hodgkin lymphoma and non-Hodgkin lymphoma, renal cell carcinoma, multiple myeloma, and malignant melanoma.,

[0435] Infectious diseases: Infectious diseases, also known as transmissible or communicable diseases, are diseases caused by infection. Infection is caused by infectious agents, including viruses, viroids, prions, bacteria; nematodes such as parasitic roundworms and pinworms; arthropods such as ticks, mites, fleas, and lice; fungi such as ringworm; and other large parasites such as tapeworms and other worms. The host uses the immune system to resist infection, such as the innate response (e.g., in mammals), including inflammation, followed by the adaptive response. Drugs used to treat infections include antibiotics, antivirals, antifungals, antiprotozoals, and anthelmintics. Specific non-limiting examples of infectious diseases include human immunodeficiency syndrome (HIV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HVC), tuberculosis (TB), and malaria.

[0436] Genetic disorders: Disorders caused by one or more germline mutations inherited from one or both parents. Such disorders may be only inherited and thus classified as Mendelian or monogenic disorders, or may be complexly inherited (e.g., multifactorial inheritance) and classified as non-Mendelian genetic disorders. Mutations associated with genetic disorders (also known as genetic diseases or disorders, or congenital disorders, diseases, or abnormalities) can include substitutions, insertions, inversions, point mutations, deletions, mismatches, copy number variations, and / or translocations. Exemplary genetic disorders that can be treated or inhibited using the disclosed delivery agents include those described herein and 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=2-369235).

[0437] Suppressing or treating a disorder or disease: For example, suppressing the overall development of a disease or disorder in a subject having or at risk of having a genetic disease, rare disease, cancer, immune disorder or infectious disorder. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms (e.g., reduces the cancer burden in a subject) after the onset of a disease or pathological condition (e.g., cancer). The term "improvement" with respect to a disease or pathological condition refers to any observable beneficial effect of the treatment. The beneficial effect can be demonstrated, for example, by a delay in the onset of the clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all of the clinical symptoms of the disease, a slowing of disease progression, an improvement in the overall health or well-being of the subject, or by other parameters specific to the particular disease, such as an increase in the survival rate of a subject having a genetic disease, rare disease, cancer, immune disorder or infectious disorder. Treatment can be evaluated by objective or subjective parameters; including but not limited to the results of physical examination, imaging or blood tests. "Preventive" treatment is administered to a subject who does not show signs of the disease or shows only early signs, with the aim of reducing the risk of developing pathological changes, such as preventing the occurrence or recurrence of cancer, immune disorders or infectious diseases.

[0438] Fragment: A fragment is a segment of a molecule, or a part of a conjugate.

[0439] Morpholino: An oligomeric molecule used to modify (e.g., reduce or prevent) gene expression. The molecular structure of a morpholino can include DNA bases attached to a backbone of methylene morpholine rings linked by phosphorodiamidate groups. A morpholino blocks access of other molecules to a small (usually about 25 base) specific sequence on the RNA base-pairing surface, e.g., by binding to a complementary sequence of RNA (or single-stranded DNA) by standard nucleic acid base pairing.

[0440] Muscular dystrophy: Refers to a group of genetic diseases that cause progressive muscle weakness. Muscular dystrophy can lead to skeletal muscle weakness and skeletal muscle protein defects, resulting in impaired various physiological functions. Existing treatments usually focus on reducing the impact of the disease and improving the quality of life of patients, e.g., by physical therapy or providing orthopedic devices.

[0441] The mutated genes associated with muscular dystrophy are responsible for encoding a variety of proteins related to the costameric protein network. These proteins include laminin-2, collagen, dystroglycan, integrin, caveolin-3, ankyrin, dystrophin, α-dystrobrevin, vinculin, netrin, BPAG1b, muscle LIM protein, desmin, actin-related LIM protein, α-actin, titin, telethonin, cypher, myotilin and the dystroglycan / sarcospan complex.

[0442] The most common muscular dystrophy is Duchenne's muscular dystrophy (DMD), which affects 1 in 3,500 live male births. DMD is an X-linked recessive genetic disorder characterized by mutations in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein with a size close to 430 kDa. The role of this protein is to connect the cytoskeleton of the cell to the extracellular matrix. The absence of dystrophin in DMD patients leads to the loss of attachment of muscle fibers to the extracellular matrix during contraction, ultimately resulting in progressive damage to the fibers, membrane leakage, and loss of muscle function. Most patients die of respiratory or heart failure before the age of 30.

[0443] Becker's muscular dystrophy (also known as benign pseudohypertrophic muscular dystrophy) is related to DMD, and both are caused by mutations in the dystrophin gene. However, DMD does not produce functional dystrophin, so DMD is much more severe than BMD. BMD is an X-linked recessive genetic disorder characterized by progressive weakness in the leg and pelvic muscles. BMD is a dystrophinopathy, including a series of muscle diseases in which insufficient dystrophin is produced in muscle cells, leading to instability of the muscle cell membrane structure. This is caused by mutations in the dystrophin gene encoding dystrophin. The symptom development pattern of BMD is similar to that of DMD, but the progression is later and much slower.

[0444] Congenital muscular dystrophy is caused by gene mutations. Fukuyama congenital muscular dystrophy (FCMD) and congenital muscular dystrophy type 1A (MDC1A) are examples of congenital muscular dystrophy. MDC1A is a congenital muscular dystrophy caused by gene mutations in the LAMA2 gene, resulting in the absence or complete loss of the laminin-α2 protein. The loss of this laminin-α2 leads to the absence of laminin-211 / 221. Laminin-211 / 221 is a major component of the extracellular matrix and plays a key role in muscle cell development. During muscle cell differentiation, laminin binds to the α7β1 integrin. Without laminin-α2, muscle fibers cannot adhere to the basement membrane, and myotubes will undergo apoptosis. Muscle regeneration also fails, resulting in the loss of muscle repair ability, increased muscle fibrosis, and inflammation. This chronic tissue damage is the main cause of morbidity and death in MDC1A.

[0445] Congenital muscular dystrophy (CMD) and limb-girdle muscular dystrophy (LGMD) are common forms of highly heterogeneous muscular dystrophies, which can be distinguished according to the age of onset. For CMD, symptoms appear at birth or within 6 months after birth; for LGMD, symptoms appear in late childhood, adolescence or even adulthood. The inheritance of LGMD can be autosomal dominant (LGMD type 1) or autosomal recessive (LGMD type 2), while CMD is recessive. CMD and LGMD may overlap clinically and genetically.

[0446] MDC1A is a progressive muscle atrophy disease that causes children to be confined to a wheelchair, require ventilator assistance for breathing, and even die prematurely. Symptoms are detected at birth, presenting as hypotonia and "floppy" infant syndrome. DMD, BMD, and LGMD are progressive muscle degenerative diseases, usually diagnosed at the age of 3 to 5 years, when children show developmental delays, including the ability to walk and climb stairs. This disease is progressive, and children usually become confined to a wheelchair and require ventilator assistance in their teens.

[0447] FCMD is a genetic disease that mainly affects muscles, the brain, and the eyes. Congenital muscular dystrophy is a group of genetic diseases that cause muscle weakness and wasting (atrophy) to begin very early in life. Fukuyama congenital muscular dystrophy affects skeletal muscles, that is, the muscles used by the body for movement. The initial symptoms of this disease appear in early infancy and include weak cry, poor feeding, and hypotonia (hypotonicity). Muscle weakness in the face usually results in a distinctive facial appearance, including drooping eyelids (ptosis) and an open mouth. In 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 impairs brain development. People with this disease develop a brain abnormality called cobblestone lissencephaly, that is, the brain surface has an uneven, irregular appearance (like cobblestones). These changes in brain structure lead to severe delays in the development of speech and motor skills and moderate to severe intellectual disability. Social skills are less impaired. Most children with Fukuyama congenital muscular dystrophy will never be able to stand or walk, although some can sit without support and can slide on the ground while sitting. More than half of the affected children also have seizures. Other signs and symptoms of Fukuyama congenital muscular dystrophy include impaired vision, other eye abnormalities, and slowly progressive heart problems after the age of 10. As the disease progresses, patients may develop difficulty swallowing, which can lead to a bacterial lung infection called aspiration pneumonia. Due to the serious medical problems associated with Fukuyama congenital muscular dystrophy, most people with this disease only live to late childhood or adolescence.

[0448] FCMD occurs almost exclusively in Japan and is the second most common form of muscular dystrophy in children (after Duchenne muscular dystrophy). It is estimated that 2 to 4 out of every 100,000 Japanese infants have Fukuyama congenital muscular dystrophy.

[0449] FCMD is caused by mutations in the FKTN gene, which encodes fukutin. The most common mutations in the FKTN gene reduce the amount of fukutin produced within cells. The lack of fukutin may prevent the normal modification of α-dystroglycan, thereby disrupting the normal function of this protein. Without functional α-dystroglycan to stabilize muscle cells, muscle fibers are damaged by repeated contraction and relaxation. The damaged fibers weaken and die over time, leading to progressive weakness and atrophy of skeletal muscle.

[0450] Defective α-dystroglycan also affects the migration of neurons during early brain development. Some neurons do not stop when they reach their intended destination but instead cross the surface of the brain and enter the fluid-filled space surrounding the brain. Since FCMD involves α-dystroglycan dysfunction, this disease is referred to as a dystroglycanopathy.

[0451] Facioscapulohumeral muscular dystrophy (FSHD) is a form of muscular dystrophy associated with progressive muscle weakness and loss of muscle tissue. Unlike DMD and BMD, which mainly affect the lower body, FSHD mainly affects the upper body muscles of the face, shoulders, and upper arms. However, it can affect the muscles around the pelvis, hips, and calves. The symptoms of FSHD usually do not appear until before the age of 10 to 26, but it is not uncommon for symptoms to appear later. In some cases, symptoms never appear. The symptoms are usually mild and deteriorate very slowly. Facial muscle weakness is common and may include drooping eyelids, inability to whistle, reduced facial expression, a sad or angry facial expression, difficulty speaking, shoulder muscle weakness (resulting in deformities such as winging of the scapulae (scapular winging) and shoulder tilting), lower limb weakness, hearing loss, and possible heart disease.

[0452] Peptide: Any chain of amino acids, regardless of its length or post-translational modifications (such as glycosylation or phosphorylation). "Peptide" can be used interchangeably with protein or polypeptide and refers to a polymer of amino acid residues in this article. "Peptide" applies to amino acid polymers, including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids, such as artificial chemical mimics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimic incorporated into a polypeptide through an amide bond or amide bond mimic. Peptides have an amino terminus (N-terminus) and a carboxyl terminus (C-terminus).

[0453] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers useful in the context of the present disclosure are conventional. Remington: The Science and Practice of Pharmacy, (23 rd nd ed.) by Adeboye Adejare, Academic Press (2020), describes compositions and formulations for drug delivery suitable for the therapeutic and delivery agents disclosed herein.

[0454] In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable liquids that include pharmaceutically and physiologically acceptable liquids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as vehicle. For solid compositions (such as in the form of powders, pills, tablets or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grade 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, etc., such as sodium acetate or sorbitan monolaurate.

[0455] Polarity: A polar compound is a compound in which the electrons are not equally or almost equally shared between atoms, so that regions of positive and negative charge are permanently separated. Polar compounds are usually soluble in water (hydrophilic). Examples of polar molecules are polar amino acids such as arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, threonine and tyrosine.

[0456] Mental disorder: A mental disorder, also known as a mental health disorder, is a variety of mental health conditions that may affect mood, thinking 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 lack of energy.

[0457] Non-polarity: A non-polar compound is a compound in which the electrons are equally or almost equally shared between atoms. Non-polar compounds are usually insoluble in water (hydrophobic). Examples of non-polar molecules are non-polar amino acids such as alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine.

[0458] Rare disease: A disease or disorder that affects fewer than 200,000 people in the United States.

[0459] Specific binding: A binding reaction in which, under specified conditions, an antibody preferentially binds to a specific target protein, peptide, or polysaccharide (such as a tumor-associated antigen) and does not bind significantly to other proteins or polysaccharides present in a sample or subject (such as on the cell surface). Specific binding also occurs between streptavidin and biotin, between avidin and biotin, or between neutravidin and biotin. Specific binding can be determined by suitable methods.

[0460] For an antibody-antigen complex, the specific binding of the antigen and the antibody can have a K -7 of less than 10 D moles, for example less than 10 -8 moles, 10 -9 or even less than 10 -10 moles. As an example, the affinity of streptavidin for biotin is described below.

[0461] K D refers to the dissociation constant of a given interaction, such as a polypeptide ligand interaction or an antibody-antigen interaction. For example, for a bimolecular interaction between an antibody or an antigen-binding fragment and an antigen, it is the concentration of each component in the bimolecular interaction divided by the concentration of the complex.

[0462] The antibodies used in the methods disclosed herein specifically bind to a determined target, such as a tumor-associated antigen, for example on the 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 expressing the tumor-associated antigen. Streptavidin, avidin, and neutravidin substantially bind to biotin. Of course, it is recognized that a certain degree of non-specific interaction can occur between an antibody or a conjugate comprising an antibody (such as an antibody that specifically binds to a target antigen or a conjugate comprising such an antibody) and a non-target (such as a cell that does not express the antigen). Generally, specific binding results in a much stronger binding between the antibody and a protein or cell bearing the antigen than between the antibody and a protein or cell lacking the antigen. Similarly, the specific binding of streptavidin, avidin, or neutravidin is the specific binding between streptavidin, avidin, or neutravidin and a biotinylated molecule (such as an antibody or an aptamer), rather than between a molecule labeled with a different label (such as an antibody or an aptamer). Specific binding generally results in an increase in the amount of antibody bound (per unit time) to a protein or cell or tissue expressing the target epitope that contains the epitope (or is labeled with biotin) by more than 2-fold, for example more than 5-fold, more than 10-fold, or more than 100-fold compared to a protein lacking such an epitope (or lacking biotin).

[0463] Subject: An organism, such as a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sport animals, and pets. 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 samples to be analyzed using the methods and devices of the present disclosure.

[0464] Sugar: A compound that can be used to target a specific tissue in the context of the present disclosure. For example, a sugar can be used in combination with a delivery agent of the present disclosure (e.g., by conjugating it to the delivery agent) to facilitate delivery of the delivery agent to a specific tissue. In some aspects of the present disclosure, a sugar can serve as a targeting group for a delivery agent. By way of example only, the sugar GalNAc can be used to target liver tissue, such that the delivery agent is used to treat liver diseases. Other sugars can include, but are not limited to, glucose (e.g., for targeting the GLUT1 receptor of the blood-brain barrier), mannose (e.g., for targeting the mannose-6-phosphate receptor in the lung, brain, and immune cells), and the like.

[0465] Therapeutic agent: Therapeutic agents include therapeutic formulations, prophylactic formulations, and replacement formulations. Thus, a therapeutic agent can be any substance or any combination of substances that can be used to achieve an end or result, such as improving a set of specific conditions in a subject suffering from a disease or disorder, e.g., substances or combinations of substances in a combination therapy for treating a genetic disease, rare disease, cancer, immune disorder, or infectious disease, e.g., substances or combinations of substances that can be used to inhibit cancer growth or metastasis in a subject. Therapeutic agents include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Exemplary therapeutic agents include nucleic acid-based therapeutic agents (e.g., oligonucleotides, such as nucleic acid analogs, e.g., morpholinos), protein-based therapeutic agents, and derivatives thereof. In examples, the therapeutic agents used herein can be chemotherapeutic agents, therapeutic antibodies, immunotherapeutic agents, antidepressants, antiviral agents, or antibiotics. Exemplary therapeutic agents further include saponin, cisplatin, methotrexate, fluorouracil, doxorubicin, cyclophosphamide, chlorambucil, vinblastine, vincristine, docetaxel, or paclitaxel, chlorhexidine, triclosan, xylitol, or octadec-1-ylamine hydrofluoride, hexadec-1-ylamine hydrofluoride, or combinations thereof.

[0466] II. Introduction

[0467] Multiple therapeutic molecules must achieve intracellular delivery and enter the cell cytosol to exert their activity. The functional delivery of larger and more polar therapeutic molecules into the cell cytosol remains a challenge as these molecules typically cannot directly cross the cell membrane and are instead internalized via endocytosis or similar mechanisms. Endocytosis is a process by which extracellular substances are brought into the cell through invagination of the cell membrane, which closes to form a vesicle within the cell called an endosome. Endocytosis can be receptor-mediated, where an extracellular compound binds to a specific receptor on the cell surface, or the extracellular compound may be nonspecifically internalized due to its presence near the cell membrane. The latter process is also known as fluid-phase endocytosis or pinocytosis. A related process, caveolin-mediated endocytosis, brings compounds into the cell via vesicles (called caveolae) near the cell surface. In these processes, the vesicles enclosing the extracellular compound become increasingly acidic after vesicle formation.

[0468] While peptide compositions for endosomal delivery exist in the art, they exhibit significant drawbacks that adversely affect in vivo use / activity. For example, such peptide compositions interact with biological components such as serum, leading to excretion from the body, e.g., via the kidneys. These interactions can reduce the free active concentration of the drug and decrease its suitability for clinical and other in vivo applications. However, the delivery agents of the present disclosure can avoid such problems of 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 due to the use of specially designed masking peptide groups to protect the active solubilizing peptide groups of the delivery agent. Such masking protection reduces or prevents the interaction of the solubilizing peptide groups with biological components (such as serum) before the solubilizing peptide groups are activated within the organelles of the endolysosomal pathway. The delivery agents of the present disclosure further comprise additional structural components that contribute to their use and efficacy in the in vivo environment. By way of example only, and not intended to be limiting, a cleavable linker group can be located between the solubilizing peptide group and the masking peptide group to facilitate separation of the solubilizing peptide group from the masking peptide group once inside the endosome, e.g., by enzymatic cleavage by an enzyme such as cathepsin B. By way of example only, a cleavable linker cleavable by cathepsin B can be used to reduce the exposure of the delivery agent and the therapeutic agent to degrading enzymes. In still some other aspects, an anchoring group is used as a structural component of the delivery agent, thereby enhancing the cell surface binding of the delivery agent and thus increasing the uptake of the delivery agent (and in some aspects, one or more therapeutic agents) into the cell, e.g., via endocytosis as described above. In still some other aspects, the anchoring group can be replaced or supplemented with a targeting group to facilitate directing the delivery agent to a specific location.

[0469] Figure 1 A schematic illustration shows the process by which the delivery agent according to the present disclosure facilitates the delivery of a therapeutic agent to the cell cytosol. From Figure 1It can be seen that the anchoring group 100 of the delivery agent 102 helps to direct and anchor the delivery agent to the extracellular membrane. The masking peptide group 104 helps to mask or protect the lysing peptide group 106 from the surrounding serum. There is also a therapeutic agent 108 located near or conjugated (covalently or non-covalently) to the delivery agent 102. After endocytosis into the cell 110, the delivery agent 102 and the therapeutic agent 108 are encapsulated within the endosome 112. As the pH within the endosome decreases, the cleavable linker 114 is cleaved, releasing the lysing peptide group 106 from the masking peptide group 104. This further enables the lysing peptide group 106 to enter the cell membrane, and subsequently the therapeutic agent 108 to cross the endosomal membrane into the cytosol of the cell.

[0470] III. Peptide-based Delivery Agent Embodiments

[0471] The present disclosure provides a peptide-based delivery agent (also referred to herein as a "delivery agent") that delivers a therapeutic molecule into the cytosol of a cell such that the therapeutic molecule retains its therapeutic activity when delivered into the cytosol. Such therapeutic molecules can be covalently or non-covalently (e.g., electrostatically) bound to the delivery agent, or they can be unbound, e.g., merely associated with the delivery agent because they are in close proximity to each other. The delivery agents of the present disclosure are water-soluble, amphiphilic, membrane-destabilizing constructs. Without being bound by a particular theory, after internalization into a cell, the delivery agents of the present disclosure can destabilize (e.g., lyse) the membrane of a membrane-bound compartment, such as an endosome, endolysosome, or lysosome membrane in a cell, and / or can form one or more pores or otherwise disrupt the integrity of the cell membrane (e.g., the membrane of an endosome, endolysosome, or lysosome) due to a transition from a polar form to a non-polar form when the pH in the membrane-bound compartment decreases relative to the physiological pH. Thus, the delivery agents of the present disclosure can deliver a therapeutic molecule into the cytosol of a cell, e.g., by lysing the membrane of a membrane-bound compartment, or forming one or more pores in the compartment membrane or by locally disrupting the integrity of the compartment membrane, and then internalizing the delivery agent and the therapeutic molecule into the same membrane-bound compartment of the cell. In some aspects of the present disclosure, where a cleavable linker that can be rapidly cleaved is used, the therapeutic molecule is released from the membrane-bound compartment (e.g., an endosome, endolysosome, or lysosome) by cleavage or pore formation or local disruption of the delivery agent before the therapeutic molecule is exposed to many degradative enzymes.

[0472] The delivery agents described herein can have a structure comprising an N-terminal group, a solubilizing peptide group, a cleavable linker, a masking peptide group, an anchoring group (and / or targeting group), a C-terminal group, and optionally a fluorophore (which can bind to different regions of the delivery agent, such as at the C-terminus or N-terminus). In some aspects of the present disclosure, the solubilizing peptide group provides the N-terminal group of the delivery agent, as the N-terminus of the amino acids of the solubilizing peptide group can be functionalized to provide the N-terminal group. In still other aspects, the anchoring group (and / or targeting group) provides the C-terminal group of the delivery agent, as the C-terminus of the amino acids of the anchoring group (and / or targeting group) can be functionalized to provide the C-terminal group. To reduce interactions between the delivery agent and biological components (such as serum), which could otherwise reduce or eliminate delivery agent activity (e.g., in a subject or in cell culture), the solubilizing peptide group is protected by a specifically designed masking peptide group. In certain aspects, the masking peptide group is configured to be symmetric with the solubilizing peptide group from the N-terminus to the C-terminus. When the delivery agent is internalized into a cell in an endosome, endolysosome, or lysosome, the masking peptide group can be separated from the solubilizing peptide group by cleavage of the cleavable linker, exposing the solubilizing peptide group within the endosome, endolysosome, or lysosome. In some aspects of the present disclosure, the anchoring group improves the association (e.g., binding) of the delivery agent with the cell membrane (such as the plasma membrane). In aspects that include a targeting group in addition to (or instead of) the anchoring group, the targeting group can facilitate the targeted delivery of the delivery agent to a specific location.

[0473] According to some aspects of the present disclosure, the delivery agent can have a structure comprising: (1) a solubilizing peptide group having the formula [X 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 is independently an amino acid each time it appears; (2) a cleavable linker; (3) a masking peptide group having 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 ​​independently an amino acid each time it appears; and (4) an anchoring group (and / or targeting group). In some aspects of the present disclosure, the delivery agent may further comprise a fluorophore group. In additional aspects, the delivery agent may further comprise a conjugated therapeutic agent. Generally, the amino acids of the solubilizing peptide group can be functionalized to provide an N-terminal group, and the amino acids of the anchoring group (and / or targeting group) can be functionalized to provide a C-terminal group.

[0474] In a specific aspect, the delivery agent may have a peptide backbone structure that satisfies General Formula IA or IB, as shown below:

[0475] [N-terminal group]-[X 1 Y 1 Y 1 X 1 m -[cleavable linker]-[X 2 Y 2 Y 2 X 2 m’ -[anchoring / targeting group]-[C-terminal group],

[0476] General Formula IA

[0477] [N-terminal group]-[X 2 Y 2 Y 2 X 2 m -[anchoring / targeting group]-[cleavable linker]-[X 1 Y 1 Y 1 X 1 m’ -[C-terminal group],

[0478] General Formula IB

[0479] As shown in General Formulas IA and IB, the solubilizing peptide group is indirectly coupled to the masking peptide group through a cleavable linker. In some aspects of the present disclosure, the bold X 1 groups of the solubilizing peptide group can be functionalized to provide an N-terminal group, which may comprise a capping group or a fluorophore, or which itself may provide the N-terminus of the peptide backbone of the delivery agent. The anchoring group (or any targeting group) is bound to the C-terminal group, which may also comprise or be conjugated to an optional fluorophore group. Other components, compounds, and / or structural features can be attached to the peptide backbone of the delivery agent to provide conjugates comprising, for example, covalently bound therapeutic agents, targeting groups, and the like. Representative aspects of the solubilizing peptide group, the cleavable linker group, the masking peptide group, the anchoring group (and / or targeting group), the optional fluorophore group, and suitable N-terminal and C-terminal groups are described below. ​​​​

[0480] A. N-terminal group

[0481] In some aspects of the present disclosure, the N-terminal group of the delivery agent comprises a capping group (such as an acetyl group) or a fluorophore. In one embodiment, the N-terminal group comprises an acetyl group that binds to the amino acid of the solubilizing peptide group (such as the X 1 group) of the solubilizing peptide group. In some aspects of the present 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) that binds to the amino acid of the solubilizing peptide group. In certain aspects, the N-terminus comprises where R represents the remainder of the peptide backbone of the delivery agent.

[0482] In some aspects of the present disclosure, one or more fluorophores are attached to one or more amino acids at or near the N-terminal group of the delivery agent of the present disclosure. In an exemplary aspect, the fluorophore can be 3-hydroxyisonicotinaldehyde and / or azulene, and the amino acid to which the fluorophore is attached can be a natural or unnatural amino acid present in the peptide backbone of the delivery agent and / or any resulting branched peptide sequence. In some such instances, one or more fluorophores can be attached to an amine-containing amino acid, such as lysine or other amine-containing amino acid derivatives, on the N-terminal group of the delivery agent of the present disclosure.

[0483] B. Solubilizing peptide group

[0484] In some aspects according to the present disclosure, the solubilizing peptide group of the disclosed delivery agent can be a group capable of solubilizing cell membranes. In some aspects of the present disclosure, as the pH in the membrane-bound compartments of the cell (such as endosomes, endolysosomes or lysosomes) decreases relative to physiological pH, the solubilizing peptide group solubilizes the membrane by transitioning from a polar form to a non-polar form. Without being bound by a particular theory, inserting a delivery agent comprising a non-polar form of the solubilizing peptide group into the membrane can induce membrane solubilization, thereby delivering the therapeutic agent into the cytosol of the cell.

[0485] The solubilizing peptide groups of the present disclosure can be groups capable of interconverting between a hydrophobic α-helical form and a hydrophilic form. As described below, the solubilizing peptide groups of some aspects of the present disclosure contain one or more pairs, and preferably two or more pairs, of carbonyl-containing groups (such as carboxyl and / or amide), where the two carbonyl-containing groups in a pair are separated by 0, 1, 2, or 3 amino acids. The composition and positioning of the amino acids of the solubilizing peptide group are such that in the presence of an aqueous phase and a lipid phase, the solubilizing peptide group typically undergoes a reversible transition between a high-pH form and a low-pH form between low pH and neutral or high pH (e.g., between pH 4.0 and 7.0). The high-pH form includes a multiply ionized hydrophilic structure in which the side-chain or terminal carbonyl-containing groups are completely or predominantly in a non-hydrogen-bonded ionic state, and the low-pH form includes a substantially non-ionic α-helical structure that exhibits lipophilicity through hydrogen bonding between paired side-chain or terminal carbonyl-containing groups. The hydrophobic, low-pH form partitions into the lipid environment, while the hydrophilic, high-pH form preferentially partitions into the aqueous solution. Thus, the "reversible transition" between the lipophilic and hydrophilic forms of the polypeptide is a transition between a non-ionic α-helical conformation in which side-chain carbonyl-containing groups that favor low pH values participate in intramolecular hydrogen bonding and a form in which side-chain carbonyl-containing groups that favor neutral or high pH values are in an ionic non-hydrogen-bonded state. This transition can encompass the entire solubilizing peptide group, or it can occur in a local region of the solubilizing peptide group, particularly when that region is near the water / lipid interface or has a particularly lipophilic and / or particularly α-helix-forming composition. Such a local region of the solubilizing peptide group typically effectively initiates the delivery agent into the lipid phase, such as a membrane, even though regions of the delivery agent that are remote from the lipid phase may be in a hydrophilic conformation. In still other aspects, the solubilizing peptide group includes one or more basic amino acids, such as arginine, lysine, histidine, or combinations thereof. Without being limited to a particular theory, these examples do not undergo the transitions described above but can still be used in some types of delivery agents of the present disclosure.

[0486] In some aspects of the present disclosure, the solubilizing peptide group has the structure of formula [X 1 Y 1 Y 1 X 1 m In these aspects, m can be an integer from 2 to 8 or greater; each X 1 independently can be a basic amino acid (or its ionized form), an acidic amino acid (or its ionized form), a nonpolar amino acid, or a derivative thereof each time it appears; and each Y 1 independently can be a nonpolar amino acid or a derivative thereof each time it appears. In some aspects of the present disclosure, m is 2, 3, 4, 5, 6, 7, 8, or greater than 8. Specifically, in a non-limiting aspect, m is 3. In some aspects of the present disclosure, each X 1 ​independently each occurrence is 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 not alanine). In certain non-limiting embodiments, each X 1 is glutamic acid (or glutamate), aspartic acid (or aspartate) or a derivative thereof. In another certain non-limiting embodiment, each X 1 is arginine or lysine, or an ionized form thereof, or a derivative thereof. In another certain non-limiting embodiment, at least one X 1 is alanine or a derivative thereof, provided that at least one X 1 is not alanine, such as a basic or acidic amino acid. In some aspects of the present disclosure, each Y 1 independently each occurrence is leucine, α-methyl leucine, methionine, alanine or 2-aminobutyric acid, or a derivative thereof. In certain non-limiting embodiments, each Y 1 is leucine, α-methyl leucine, or a derivative thereof. In another specific non-limiting embodiment, each Y 1 is α-methyl leucine. In another specific non-limiting embodiment, each Y 1 is leucine.

[0487] In some aspects of the present disclosure, the lysis peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:1 (ELLEELLEELLE, where E is glutamic acid and L is leucine, see Figure 2), such as 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 aspects, the lysis peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:1.

[0488] In some aspects of the present disclosure, the lytic peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:2 (DLLDDLLDDLLE, where D is aspartic acid, E is glutamic acid, and L is leucine), such as an amino acid sequence that is 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 aspects, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:2.

[0489] In some aspects of the present disclosure, the lytic peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:38 (ELLEELLEELLEELLE, where E is glutamic acid and L is leucine), such as an amino acid sequence that is 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 aspects, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:38.

[0490] In some aspects of the present disclosure, the lytic peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:30 (ELLEQLLQELLE, where E is glutamic acid, Q is glutamine, and L is leucine), such as an amino acid sequence that is 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 aspects, the lytic peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:30.

[0491] In some aspects of the present disclosure, the lytic peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:153 (QLLEQLLQQLLE, where E is glutamic acid, Q is glutamine, and L is leucine), such as 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 aspects, the lytic peptide group has an amino acid sequence that comprises or consists of SEQ ID NO:153.

[0492] In some aspects of the present disclosure, the lytic peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:92 (RLLRRLLRRLLR, where R is arginine and L is leucine), such as 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 aspects, the lytic peptide group has an amino acid sequence that comprises or consists of SEQ ID NO:92.

[0493] C. Cleavable linker

[0494] According to some aspects of the present disclosure, the linker of the disclosed delivery agent can be a cleavable linker that is capable of separating the lytic peptide group from the remainder of the delivery agent (including the masking peptide group) within a membrane-bound compartment (such as an endosome, endolysosome, or lysosomal compartment) after the delivery agent has been internalized into the endosome of a cell. Cleavage of the cleavable linker can dissociate the lytic peptide group from the masking peptide group, thereby allowing the lytic activity of the lytic peptide group to act within the membrane-bound compartment.

[0495] In some aspects of the present disclosure, the cleavable linker group can be cleaved by an enzyme present in an endosome, endolysosome, or lysosome. In certain aspects of the present disclosure, the cleavable linker group can be cleaved by cathepsin B.

[0496] In some aspects of the present disclosure, the cleavable linker comprises an amino acid sequence having a length of 2-10 (e.g., 4-10 or 6-10) amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In a particular non-limiting embodiment, the cleavable linker has a length of 7 amino acids. In another particular non-limiting embodiment, the cleavable linker has an amino acid sequence that is at least 70% identical to SEQ ID NO:3 (GFGFVGG, where G is glycine, F is phenylalanine, and V is valine), such as 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 a particular non-limiting aspect of the present disclosure, the cleavable linker has an amino acid sequence comprising or consisting of SEQ ID NO:3. In another particular non-limiting embodiment, the cleavable linker has an amino acid sequence that is at least 70% identical to SEQ ID NO:233 (GGGVXGG, where G is glycine, X is citrulline (or Cit), and V is valine), such as 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 a particular non-limiting aspect of the present disclosure, the cleavable linker has an amino acid sequence comprising or consisting of SEQ ID NO:233. In another particular non-limiting embodiment, the cleavable linker has an amino acid sequence that is at least 70% identical to SEQ ID NO:234 (GGGVKGG, where G is glycine, K is lysine, and V is valine), such as 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 a particular non-limiting aspect 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 that is at least 70% identical to SEQ ID NO: 227 (XGGXV, where G is glycine, each X is independently 6-aminohexanoic acid or homoarginine, and V is valine), such as having an amino acid sequence that is 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.

[0497] D. Masking peptide group

[0498] In some aspects of the present disclosure, the masking peptide group of the disclosed delivery agent can be a group that can reduce or prevent the exposure of the solubilizing peptide group of the delivery agent to biological components (such as a cell culture system or a subject) before the delivery agent is internalized into a cell. In a particular aspect of the present disclosure, the masking peptide group conceals the amino acid component of the solubilizing peptide group from serum, thereby reducing or avoiding the interaction between such an amino acid component and serum. In some aspects of the present disclosure, the hydrophobic residues (such as leucine) of the solubilizing peptide group sequence can have an adverse interaction with serum (such as serum albumin) and reduce the activity of the delivery agent. The masking peptide group can reduce and / or prevent these interactions by concealing the hydrophobic residues until the delivery agent reaches a target (such as a membrane-bound compartment or other biological target region).

[0499] In some cases, the masking peptide group can contain one or more pairs, preferably two or more pairs, of carbonyl-containing groups (such as amides), where the two carbonyl-containing groups in a pair are separated by 0, 1, 2 or 3 amino acids. Without being limited to a single theory, it is currently believed that the composition and positioning of the amino acids of the masking peptide group are such that in the presence of an aqueous phase, the masking peptide group forms a substantially α-helical structure, rendering it lipophilic through hydrogen bonding between paired amide side chains. Furthermore, it is believed that the α-helical nature of the masking peptide is beneficial for the masking of the solubilizing peptide. As described below, the masking peptide group of some aspects of the present disclosure contains one or more amino acids (such as glutamic acid) that can be negatively charged under physiological conditions in addition to one or more pairs, preferably two or more pairs, of carbonyl-containing groups (such as amides). In some cases, the solubility of the masking peptide in an aqueous environment may be higher, but it still retains sufficient α-helical characteristics in the masking peptide to facilitate the masking of the solubilizing peptide.

[0500] In some aspects of the present disclosure, the masking peptide group of the disclosed delivery agent is symmetric with the solubilizing peptide group of the delivery agent. However, in some independent aspects of the present disclosure, the masking peptide group of the disclosed delivery agent can be 1, 2, or 3 amino acids less than the solubilizing peptide group of the delivery agent. In a particular aspect of the present disclosure, the masking peptide group has the formula [X 2 Y 2 Y 2 X 2 m’ . In these aspects of the present disclosure, m' can be an integer selected from 2 to 8 or greater; each X 2 can independently be an acidic amino acid (such as glutamine, glutamic acid (and / or glutamate), aspartic acid (and / or aspartate)), a nonpolar amino acid (such as alanine), or a derivative thereof each time it appears; and each Y 2 can independently be a nonpolar amino acid or a derivative thereof each time it appears. In a particular aspect of the present disclosure, at least one X 2 is an acidic amino acid. In some aspects of the present disclosure, m' is 2, 3, 4, 5, 6, 7, 8, or greater than 8. In a particular non-limiting aspect of the present disclosure, m' is 3. In a particular non-limiting embodiment, each X 2 is independently glutamic acid (and / or glutamate), aspartic acid (and / or aspartate), glutamine, alanine, or a derivative thereof each time it appears, provided that at least one X 2 is not alanine. In a particular non-limiting embodiment, each X 2 is independently glutamic acid or a derivative thereof each time it appears. In another particular non-limiting embodiment, each X 2 is independently aspartic acid or a derivative thereof each time it appears. In another particular non-limiting embodiment, each X 2 is glutamine or a derivative thereof. In another particular non-limiting embodiment, at least one X 2 is alanine, provided that at least one other X 2 is not alanine. In some aspects of the present disclosure, each Y 2 is independently leucine, α-methyl leucine, methionine, alanine, or 2-aminobutyric acid, or a derivative thereof each time it appears. In a particular non-limiting embodiment, each Y 2 is leucine or a derivative thereof. In another particular non-limiting embodiment, each Y 2 is α-methyl leucine or a derivative thereof. In another particular non-limiting embodiment, each Y 2 is independently alanine or a derivative thereof each time it appears.

[0501] ​In some aspects of the present disclosure, the masking peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:4 (QLLQQLLQQLLQ, where Q is glutamine and L is leucine), for example, an amino acid sequence that is 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 aspects of the present disclosure, the masking peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:4.

[0502] In some aspects of the present disclosure, the masking peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:51 (QLLQQLLQQLLQQLLQ, where Q is glutamine and L is leucine), for example, an amino acid sequence that is 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 aspects of the present disclosure, the masking peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:51.

[0503] In some aspects of the present disclosure, the masking peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:154 (QLLQELLEQLLQ, where Q is glutamine, L is leucine, and E is glutamate), for example, an amino acid sequence that is 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 aspects of the present disclosure, the masking peptide group has an amino acid sequence comprising or consisting of SEQ ID NO:154.

[0504] In some aspects of the present disclosure, the masking peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:153 (QLLEQLLQQLLE, where Q is glutamine, L is leucine, and E is glutamate), such as 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 aspects of the present disclosure, the masking peptide group has an amino acid sequence that comprises or consists of SEQ ID NO:153.

[0505] In some aspects of the present disclosure, the masking peptide group has an amino acid sequence that is at least 85% identical to SEQ ID NO:148 (QLLAQLLAQLLQ, where Q is glutamine, L is leucine, and A is alanine), such as 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 aspects of the present disclosure, the masking peptide group has an amino acid sequence that comprises or consists of SEQ ID NO:148.

[0506] E. Anchoring group and / or targeting group

[0507] In some aspects of the present disclosure, endocytosis of the disclosed delivery agents can be facilitated by including one or more anchoring groups (such as 1, 2, 3, or 4 anchoring groups) in the structure of the delivery agent. In some aspects of the present disclosure, the anchoring group comprises a heteroaliphatic group, an antibody, a biotin group, an avidin group, a streptavidin group, or a neutravidin group. In certain aspects of the present disclosure, the anchoring group comprises a lipid anchoring group that comprises a tail group that facilitates localization to the cell surface.

[0508] The anchoring group of the delivery agent disclosed herein generally comprises an amino acid moiety that binds to an amino acid group of the peptide backbone of the delivery agent. In some aspects of the present disclosure, the amino acid moiety of the anchoring group binds to an amino acid of a masking peptide moiety, such as an amino acid of a masking peptide moiety located at the C-terminal group of the delivery agent. The anchoring group also comprises a tail group that aids in directing and accumulating the delivery agent at a desired location, such as on the cell surface (e.g., on the cell membrane). In certain aspects of the present disclosure, the amino acid moiety of the anchoring group also provides a C-terminal group that can be functionalized with different components described herein, such as the C-terminal groups shown in Formula IA or IB, which can comprise an amine-terminated glycine moiety, a fluorophore, etc., as described herein. In certain aspects of the present disclosure, the amino acid moiety of the anchoring group is lysine (K) and the tail group comprises an aliphatic tail. In some aspects of the present disclosure, the amino acid moiety of the anchoring group can bind to two separate tail groups, such as when the anchoring group caps the peptide backbone of the delivery agent or caps a branched group from the peptide backbone of the delivery agent. In some aspects of the present disclosure, the tail group can further comprise a functional group that facilitates the binding of the aliphatic tail to the amino acid moiety. In some aspects of the present disclosure, the functional group can be provided by a 2-aminoethyl bicarbonate group 4-aminobutyric acid group -CH(NH 2 )C(O)- group or -CH(N + Me 3 )C(O)- group. In some aspects of the present disclosure, the aliphatic tail comprises an alkyl chain having 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, or 12) carbon atoms. In further aspects of the present disclosure, the amino group of the 2-aminoethyl bicarbonate group and / or the 4-aminobutyric acid group can be functionalized to provide a quaternary amine. In certain aspects of the present disclosure, the amino group is functionalized with an aliphatic group such as a lower alkyl having 10 or fewer carbon atoms. In some exemplary aspects of the present disclosure, the amino group is functionalized with at least one methyl group, and some specific examples include two methyl groups. In certain aspects of the present disclosure, the tail portion can have a structure of any of the structures exemplified below (where the wavy line represents the point of attachment to the amino acid moiety of the anchoring group).

[0509]

[0510] In some aspects of the present disclosure, the anchoring group can have a structure as shown in any of Formulas IIA to IID below.

[0511]

[0512] In some aspects of the present disclosure, the delivery agent may include one or more anchoring groups. In some aspects of the present disclosure, two anchoring groups may be directly bound to each other, or may be indirectly bound through one or more amino acids. In some such aspects of the present disclosure, the two anchoring groups may be the same or different in terms of chemical structure. In some aspects of the present disclosure, where two (or more) anchoring groups are directly bound to each other, the amino acid moiety of one anchoring group may be bound to the amino acid moiety of the other anchoring group through a peptide bond. By way of example only, where the amino acid moiety of each of the two anchoring groups is lysine, the amino group of one lysine may be bound to a masking peptide group and its corresponding carboxylic acid group may be bound to the amino group of the second lysine. The carboxylic acid group of the second lysine may in turn be bound to the C-terminal group of the delivery agent. In some aspects of the present disclosure, the delivery agent may include two or more different anchoring groups, such as one or more groups having the structure of formula IIA, IIB, IIC or IID and one or more ligands. In receptor-mediated endocytosis, the anchoring groups attached or complexed to the composition to be absorbed are capable of binding (e.g., specifically binding) to receptors (e.g., receptors specific for the ligand) on the cell surface. Such anchoring groups can be used to enhance general endocytic uptake, or to target specific cell types.

[0513] The delivery agents disclosed herein may also include targeting groups, which may replace or supplement any of the anchoring groups included in the delivery agent. Targeting groups can be used to facilitate the specific delivery of the delivery agent (and any therapeutic agent conjugated thereto) to a specific target (e.g., a biological region, cell, or biological structure). The targeting moiety can include cells, antibodies (or fragments thereof), peptides, peptidomimetics, aptamers, sugars, small targeting molecules, or combinations thereof. Exemplary antibodies can include anti-CD33, anti-CD30, anti-HER2, anti-CD22, anti-nectin4, anti-nectin1, anti-AXL, anti-CD74, anti-ALK, anti-PTK7, anti-PSMA, anti-TM4SF1, anti-CD276, anti-CD20, anti-CD19, anti-CD3, anti-CD71. Exemplary antibody / target combinations can include the interferon-α / β cell surface receptor complex used to target type I interferons (IFNα, IFNβ), death receptors 4 and 5 (DR4 / 5) targeted by tumor necrosis factor-related apoptosis-inducing ligand, EGFRvIII targeted by an anti-EGFR antibody or nanobody (ENb), and CD36 targeted by thrombospondin. Exemplary peptides and / or peptidomimetics can 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, etc. 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, red blood cells, macrophages, neutrophils, monocytes, T cells, etc. Exemplary small targeting molecules can include, but are not limited to, folic acid (for targeting cancer), bisphosphonates (for targeting bone), dibenzocyclooctyne compounds (or “DBCO”) (for targeting cell surfaces or other cellular features), etc. In some embodiments, dibenzocyclooctyne compounds can also be used as anchoring groups because they can facilitate the binding of the delivery agent to the cell surface.

[0514] The targeting group can be attached to the delivery agent via an attachment point, which can include an amino acid of the delivery agent peptide backbone (e.g., a lysine or cysteine group) (or any such group present in a branch derived from the delivery agent peptide backbone). In some aspects of the present disclosure, the targeting group can be conjugated to the delivery agent by functionalizing one or more of these amino acids at an available amino, carboxyl, or side chain group of the amino acid. The attachment point can include the targeting group in the peptide backbone, a branch derived from the peptide backbone, and / or the C-terminal group of the peptide backbone. By way of example only, the targeting group can be attached to the delivery agent via the amino terminus of a lysine group or the amino group of a lysine side chain.

[0515] In some aspects of the present disclosure, the anchoring group and / or targeting group can be directly attached to the delivery agent by any of the above-described linking methods, or can be indirectly linked through a linking group. In some aspects of the present disclosure, the linking group can include a carbonyl group, an amide group, an alkenyl oxide group, or a combination thereof. In some aspects of the present disclosure, the linking group has a structure of Formula A, Formula B, or Formula C as exemplified below, wherein the circled wavy bonds of Formula A, Formula B, and Formula C represent the attachment points of the anchoring group and / or targeting group (or multiple independent anchoring groups and / or targeting groups, such as Formula A and C), and the other wavy lines of Formula A, Formula B, and Formula C represent attachment to the remainder of the delivery agent.

[0516]

[0517] In certain aspects of the present disclosure, the delivery agent has an amino acid sequence that is at least 85% identical to SEQ ID NO: 272, such as 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, where K at position 32 provides one or more DBCO groups that are bound to the Lys side chain through a linking group of any one of Formulas A, B, or C, X is naphthylalanine (Nal), the C-terminal G at position 35 is a modified glycine containing -C(O)-NH 2 group, and in some aspects, the N-terminal Q contains an N-terminal modified with an acetyl group. In certain non-limiting aspects, the delivery agent has an amino acid sequence that comprises or consists of SEQ ID NO: 272. In any of these aspects, K at position 32 provides (i) three DBCO groups that are bound to the Lys side chain through a linking group of Formula A; (ii) one DBCO group that is bound to the Lys side chain through a linking group of Formula B; or (iii) two DBCO groups that are bound to the Lys side chain through a linking group of Formula C.

[0518] In certain aspects of the present disclosure, the delivery agent has an amino acid sequence that is at least 85% identical to SEQ ID NO: 273, such as 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, where SEQ ID NO: 273 is ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKGXG, where K at position 32 provides one or more DBCO groups that are attached to the Lys side chain via a linking group of any one of Formulas A, B or C, X is naphthylalanine (Nal), the C-terminal G at position 35 is a modified glycine comprising -C(O)-NH 2 group, and in some aspects, the N-terminal Q comprises an N-terminal modified with an acetyl group. In certain non-limiting aspects, the delivery agent has an amino acid sequence comprising or consisting of SEQ ID NO: 273. In any of these aspects, K at position 32 provides (i) three DBCO groups that are attached to the Lys side chain via a linking group of Formula A; (ii) one DBCO group that is attached to the Lys side chain via a linking group of Formula B; or (iii) two DBCO groups that are attached to the Lys side chain via a linking group of Formula C.

[0519] In certain aspects of the present disclosure, the delivery agent has an amino acid sequence that is at least 85% identical to SEQ ID NO: 274, such as 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, where SEQ ID NO: 273 is ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKGXG, where K at position 32 provides one or more DBCO groups that are attached to the Lys side chain via a linking group of any one of Formulas A, B or C, X is naphthylalanine (Nal), the C-terminal G at position 35 is a modified glycine comprising -C(O)-NH 2Modified glycine of the group, and in some aspects, the N-terminal Q comprises an N-terminal modified with an acetyl group. In certain non-limiting aspects, the delivery agent has an amino acid sequence comprising or consisting of SEQ ID NO: 273. In any of these aspects, the K at position 32 provides (i) three DBCO groups that are attached to the Lys side chain via a linking group of formula A; (ii) one DBCO group that is attached to the Lys side chain via a linking group of formula B; or (iii) two DBCO groups that are attached to the Lys side chain via a linking group of formula C.

[0520] F. C-terminal group

[0521] In some aspects of the present disclosure, the C-terminal group of the delivery agent comprises an amine-terminated glycine moiety, a fluorophore, or both. In certain aspects of the present disclosure, where the C-terminal group comprises a fluorophore, the fluorophore can be naphthylalanine or tryptophan and can be flanked on one or both sides by a glycine moiety. In certain aspects of the present disclosure, the amine-terminated glycine moiety has a structure selected from -Nal-K-G’, -G-K-G’, -Nal-C-G’, -G-Nal-C-G’, -G-C-G’, -Nal-G’, -G-Nal-G’, -G-Nal-K-G’ (SEQ ID NO: 5), -W-G’, -G-W-G’, or -G-W-K-G’, where Nal is naphthylalanine, G is glycine, G’ is modified glycine comprising -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 of the C-terminal group can be used as an attachment point and can thus be used to attach a targeting group, such as those described herein. In certain non-limiting embodiments, the amine-terminated glycine moiety is G’ or -G-Nal-G’, where G’ is modified glycine comprising -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic (e.g., methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, n-butyl, etc.). In other aspects of the present disclosure, the amine-terminated glycine moiety is -G-Nal-K-G’, where G’ is modified glycine comprising -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic (e.g., methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, n-butyl, etc.).

[0522] In some aspects of the present disclosure, one or more other types of fluorophores can be attached to one or more amino acids (e.g., non-naturally occurring amino acids) present in the C-terminal group of the disclosed delivery agent. In such instances, one or more fluorophores can be attached to cysteine or amine-containing amino acids (e.g., lysine or other amine-containing amino acid derivatives) present in the C-terminal group of the disclosed delivery agent. In some such aspects of the present disclosure, the fluorophore is selected from 3-hydroxyisonicotinylaldehyde, 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, acridone-2-ylalanine, L-leucine 7-amide-4-methylcoumarin, trans-4-hydroxy-L-proline 7-amide-4-methylcoumarin, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 633, Alexa 647, Atto 465, Atto488, 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, or tetramethylrhodamine. In an exemplary aspect of the present disclosure, the fluorophore is selected from naphthylalanine, tryptophan, 3-hydroxyisonicotinylaldehyde, and / or azulene.

[0523] In some aspects of the present disclosure, a therapeutic agent or targeting group may be conjugated to a delivery agent covalently or non-covalently through a C-terminal group. In some aspects of the present disclosure, the therapeutic agent may be conjugated to the delivery agent through a disulfide bond, which can be cleaved to separate the delivery agent from the therapeutic agent. In some aspects of the present disclosure, the therapeutic agent may be conjugated to the delivery agent through a second cleavable linker, which has a similar or identical sequence, or a different sequence, compared to other cleavable linkers described herein present in the peptide backbone of the delivery agent. In these aspects of the present disclosure, the cleavable linker is attached to an amino-terminated glycine moiety. For example, the cleavable linker may covalently bind to the glycine of the amino-terminated glycine moiety, or may bind to a different amino acid of the amino-terminated glycine moiety, such as lysine or cysteine in aspects of the present disclosure that include an amino-terminated glycine moiety having the structure described above. In some aspects of the present disclosure, the therapeutic agent is conjugated to the delivery agent, which may indirectly bind to the cleavable linker group through a chemical linker group. In some such aspects of the present disclosure, the chemical linker group may have the structure of any of Formulas IIIA-IIID. In still other aspects of the present invention, the chemical linker group may be selected from a pyrophosphate diester linker (such as a pyrophosphate diester linker that can be cleaved by phosphatases and pyrophosphates in lysosomes, as 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 in these documents are incorporated herein by reference); a β-glucuronide linker (such as a β-glucuronide linker that can be cleaved by β-glucuronidase, the enzyme present in lysosomes and overexpressed in some tumors, as described in Lu et al., Int J Mol Sci. 2016, 17(4):561, the structure of such β-glucuronide linkers is incorporated herein by reference); a β-galactose linker (such as a β-galactose linker cleaved by β-galactosidase, the enzyme located 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 structures of such β-galactose linkers disclosed in these documents are incorporated herein by reference); an arylsulfate linker (such as an arylsulfate linker that can be cleaved by sulfatase, the enzyme located in lysosomes and overexpressed in some cancers) and / or a photo-responsive cleavable linker, both in Zheng et al.,described in Table 1 of Acta Pharmaceutica Sinica B. 2021, 11(12): 3889 - 3907 (the structure of Table 1 is hereby incorporated by reference herein); or another linker cleavable in a low pH environment, such as but not limited to orthoesters (Srinivasachar et al., Biochemistry, 1989, 28(6): 2501–2509, the orthoesters described therein are incorporated by reference herein), silyl ethers (Parrott et al., J. Am. Chem. Soc. 2010, 132(50): 17928–17932, the silyl ethers described therein are incorporated by reference herein), acetal linkers (Liu et al., J. Am. Chem. Soc. 2010, 132(5): 1500–1501, the acetal linkers described therein are incorporated by reference herein), β-thiopropionate linkers (Oishi et al., Am. Chem. Soc. 2005, 127(6): 1624–1625, the β-thiopropionate linkers described therein are incorporated by reference herein), aminophosphonate bonds (Jeong et al., Bioconjugate Chemistry, 2003, 14(2): 473 - 479, the aminophosphonate bonds described therein are incorporated by reference herein), imine linkers (Zhao et al., J. Am. Chem. Soc. 2010, 132(37): 13016–13025, the imine bonds described therein are incorporated by reference herein), vinyl ethers (Shin et al., J. Controlled Release, 2003, 91: 187 - 200, the vinyl ether bonds described therein are incorporated by reference herein), or hydrazones (Kale et al., Bioconjugate Chem. 2007, 18(2): 363–370, the hydrazone bonds described therein are incorporated by reference herein). With respect to Formulas IIIA - IIID, each p is independently an integer selected from 0 to 20, such as 1 to 15, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 5, or 1 to 4; and q is an integer selected from 0 or 1, and when q is 0, then there is no second cleavable linker and the chemical linker group is directly attached to the delivery agent.

[0524]

[0525] G. Exemplary Delivery Agent Embodiments

[0526] In certain non - limiting embodiments, the delivery agent comprises (1) an N - terminal group (such as an acetyl group); (2) having the formula [X 1 Y 1 Y 1 X1 m The lysing peptide group of the structure, where 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; (4) Having the formula [X 2 Y 2 Y 2 X 2 m’ The masking peptide group of the structure, where m' is 3, and each X 2 is glutamine, and each Y 2 is leucine; (5) An anchoring group having the structure of any one of formulas IIA - IID; and (6) A C-terminal group, such as an amine-terminated glycine moiety having the structure -G-Nal-G', where G is glycine, Nal is naphthylalanine, and G' is a modified glycine containing a -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic.

[0527] In another specific non-limiting embodiment, the delivery agent comprises (1) an N-terminal group (such as acetyl); (2) Having the formula [X 1 Y 1 Y 1 X 1 m The lysing peptide group of the structure, where 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; (4) Having the formula [X 2 Y 2 Y 2 X 2 m’ The masking peptide group of the structure, where m' is 3, and each X 2 is glutamine, and each Y 2 is leucine; (5) An anchoring group having the structure of any one of formulas IIA - IID; and (6) A C-terminal group, such as an amine-terminated glycine moiety having the structure -G-Nal-G', where G is glycine, Nal is naphthylalanine, and G' is a modified glycine containing a -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic.

[0528] ​​​​In some aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, and the masking peptide group comprise any one of SEQ ID NO: 6 or 9. In some aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, and the masking peptide group have an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 6 or 9, such as 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 aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, and the masking peptide group have an amino acid sequence that comprises or consists of any one of SEQ ID NO: 6 or 9.

[0529] In some aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, the masking peptide group, and the amino acid moiety of the anchoring group comprise any one of SEQ ID NO: 7 or 10. In some aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, the masking peptide group, and the amino acid moiety of the anchoring group have an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 7 or 10, such as 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: 7 or 10. In certain non-limiting aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, the masking peptide group, and the amino acid moiety of the anchoring group have an amino acid sequence that comprises or consists of any one of SEQ ID NO: 7 or 10.

[0530] In some aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, the masking peptide group, the amino acid portion of the anchoring group, and the C-terminal portion comprise any one of SEQ ID NO:8, 11, 13-15, 39, 149, or 171. In some aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, the masking peptide group, the amino acid portion of the anchoring group, and the C-terminal portion have an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:8, 11, 13-15, 39, 149, or 171, such as 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 aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, the masking peptide group, the amino acid portion of the anchoring group, and the C-terminal portion have an amino acid sequence that comprises or consists of any one of SEQ ID NO:8, 11, 13-15, 39, 149, or 171.

[0531] SEQ ID NO:6: ELLEELLEELLEGFGFVGGQLLQQLLQQLLQ, wherein in some aspects of the present disclosure, the N-terminal E comprises an N-terminal modified with an acetyl group.

[0532] SEQ ID NO:7: ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKK, wherein each K provides an anchoring group, and in some aspects of the present disclosure, the N-terminal E comprises an N-terminal modified with an acetyl group.

[0533] SEQ ID NO:8: ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGXG, wherein each K provides an anchoring group, X is naphthylalanine (Nal), the C-terminal G at position 36 is a modified glycine comprising a -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal E comprises an N-terminal modified with an acetyl group.

[0534] SEQ ID NO:9: DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQ, wherein in some aspects of the present disclosure, the N-terminal D comprises an N-terminal modified with an acetyl group.

[0535] SEQ ID NO:10: DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQKK, wherein each K provides an anchoring group, and in some aspects of the present disclosure, the N-terminal D comprises an N-terminal modified with an acetyl group.

[0536] SEQ ID NO:11: DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQKKGXG, wherein each K provides an anchoring group, X is Nal, the C-terminal G at position 36 is a modified glycine comprising a -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal D comprises an N-terminal modified with an acetyl group.

[0537] SEQ ID NO:13: ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKG, wherein each K provides an anchoring group, the C-terminal G at position 34 is a modified glycine comprising a -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal E comprises an N-terminal modified with an acetyl group or a fluorophore.

[0538] SEQ ID NO:14: ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGXG, wherein each K provides an anchoring group, X is Nal, and the C-terminal G at position 36 is a modified glycine comprising a -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal E comprises an N-terminal modified with an acetyl group.

[0539] SEQ ID NO:15: ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGWG, wherein each K provides an anchoring group, the C-terminal G at position 36 is a modified glycine comprising a -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal E comprises an N-terminal modified with an acetyl group.

[0540] SEQ ID NO:39: ELLEELLEELLEELLEGFGFVGGQLLQQLLQQLLQQLLQKKG, wherein each K provides an anchoring group, the C-terminal G at position 42 is a modified glycine comprising a -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal E comprises an N-terminal modified with an acetyl group.

[0541] SEQ ID NO:149: ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKG, where K provides an anchoring group, the C-terminal G at position 33 is a modified glycine containing -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal E contains an N-terminal modified with an acetyl group.

[0542] SEQ ID NO:171: QLLEQLLQQLLEGFGFVGGQLLEQLLQQLLEKG, where K provides an anchoring group, the C-terminal G at position 33 is a modified glycine containing -C(O)-NH 2 group, and in some aspects of the present disclosure, the N-terminal Q contains an N-terminal modified with an acetyl group.

[0543] Regarding all SEQ ID NO:6 - 11, 13 - 15, 39, 149, and 171, the N-terminal E, D, or Q amino acids of these sequences contain an N-terminal amine functionalized with an acetyl group.

[0544] Further exemplary non-limiting instances of the delivery agent are shown below, which further comprise a conjugated therapeutic agent linked by a chemical linker group and / or a second cleavable linker group.

[0545]

[0546]

[0547] Regarding delivery agents 1 and 2, SEQ ID NO:12 (DLLDDLLDDLLEGFGFVGGQLLQQLLQQLLQKKGXKG, where X is Nal, the Ks at positions 32 and 33 each provide an anchoring group, the K at position 36 is functionalized with a second cleavable linker that is conjugated to the therapeutic agent via a chemical linker group, and the C-terminal G at position 37 is functionalized with NH 2 group) is the combined amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, amino acid portions of two anchoring groups, and the C-terminal portion of delivery agents 1 and 2 as shown above.

[0548]

[0549] Regarding delivery agent 3, SEQ ID NO:108 (ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGXKG, where X is Nal, the Ks at positions 32 and 33 each provide an anchoring group, the K at position 36 is functionalized with a chemical linker group attached to the therapeutic agent, and the C-terminal G at position 37 is functionalized with NH 2The group functionalization) is the combined amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, amino acid portions of the two anchoring groups, and C-terminal portion of the delivery agent 3 as shown above.

[0550]

[0551] Regarding delivery agent 4, R 1 、R 2 、R 3 and R 4 are each independently H, CH 3 or cyclobutyl. SEQ ID NO:108 is the combined amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, amino acid portions of the two anchoring groups, and C-terminal portion of the delivery agent 4 as shown above.

[0552]

[0553] Regarding delivery agent 5, SEQ ID NO:235 (ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKG KG, where the Ks at positions 32 and 33 each provide an anchoring group, the K at position 35 is functionalized with a second cleavable linker that is conjugated to the therapeutic agent via a chemical linker group, and the C-terminal G at position 36 is functionalized with NH 2 group functionalization) is the combined amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, amino acid portions of the two anchoring groups, and C-terminal portion of the delivery agent 5 as shown above.

[0554]

[0555] Regarding delivery agent 6, R 1 、R 2 、R 3 and R 4 are each independently H, CH 3 or cyclobutyl. SEQ ID NO:270 (ELLEELLEELLEGFGFVGGQLLQQLLQQLLQKKGKG, where the Ks at positions 32 and 33 each provide an anchoring group, the K at position 35 is functionalized with a chemical linker group attached to the therapeutic agent, and the C-terminal G at position 36 is functionalized with NH 2 group functionalization) is the combined amino acid sequence of the solubilizing peptide group, cleavable linker, masking peptide group, amino acid portions of the two anchoring groups, and C-terminal portion of the delivery agent 6 as shown above.

[0556]

[0557] Regarding delivery agent 7, SEQ ID NO: 236 (ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKKG KG, where the Ks at positions 32 and 33 each provide an anchoring group, the K at position 35 is functionalized with a second cleavable linker that is conjugated to the therapeutic agent via a chemical linker group, and the C-terminal G at position 36 is functionalized with an NH 2 group) is the combined amino acid sequence of the solubilizing peptide group, the cleavable linker, the masking peptide group, the amino acid portions of the two anchoring groups, and the C-terminal portion of delivery agent 7 as shown above.

[0558]

[0559] Regarding delivery agent 8, R 1 、R 2 、R 3 and R 4 are each independently H, CH 3 or cyclobutyl, and SEQ ID NO: 237 (ELLEELLEELLEGFGFVGGQLLAQLLAQLLQKGKG, where the K at position 32 provides an anchoring group, the K at position 34 is functionalized with a chemical linker group attached to the therapeutic agent, and the C-terminal G at position 35 is functionalized with an NH 2 group) is the combined amino acid sequence of the solubilizing peptide group, the cleavable linker, the masking peptide group, the amino acid portions of the two anchoring groups, and the C-terminal portion of delivery agent 8 as shown above.

[0560] In some aspects of the present disclosure, the amino acid sequences of the solubilizing peptide group, the cleavable linker, the masking peptide group, the amino acid moiety of the anchoring group, and the C-terminal moiety (collectively, all or part of the amino acid sequence of the disclosed delivery agent) include any of the following sequences: SEQ ID 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 some aspects of the present disclosure,All or part of the amino acid sequence of the disclosed delivery agent has an amino acid sequence that is at least 85% identical to any one of the following sequences: SEQ ID 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,An amino acid sequence that is 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 the following sequences: SEQ ID 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 a specific non-limiting aspect of the present disclosure,All or part of the amino acid sequence of the disclosed delivery agent has an amino acid sequence comprising any one of the following sequences: SEQ ID 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,The amino acid sequences of the disclosed delivery agents have an amino acid sequence consisting of any one of the following sequences: SEQ ID 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 aspects of the present disclosure, all or part of the amino acid sequence of the disclosed delivery agent has an amino acid sequence comprising or consisting of any one of the sequences SEQ ID NO:8, 11, 39, 149 and 171.,

[0561] IV. Compositions and Administration Methods

[0562] The present disclosure provides compositions comprising the delivery agents disclosed herein. In some instances, the compositions further comprise one or more other therapeutic agents, adjuvants, carriers, buffers, detergents (such as deoxycholic acid), salts, lipids, stabilizers, emulsifiers, solubilizers, or any combination thereof. In some aspects of the present disclosure, the compositions comprise a therapeutic agent covalently or non-covalently bound to the peptide-based delivery agent, or a therapeutic agent associated only with the delivery agent. In still other aspects of the present disclosure, two separate compositions can be administered such that one composition comprises the therapeutic agent and the other composition comprises the delivery agent.

[0563] Methods of administering the disclosed delivery agents are conventional and can be determined by a clinician. The disclosed delivery agents can be administered using a variety of dosing regimens, such as in combination with one or more therapeutic agents. The disclosed delivery agents are typically administered locally, intranasally, intravenously, orally, intracranially, intramuscularly, parenterally, or by implant, but in principle can even be administered rectally or vaginally. In some aspects of the present disclosure, the disclosed delivery agents are administered intraperitoneally, intratumorally, intravitreally, intracerebrally, transcorneally or intravitreally, intracerebrally, epidermally, intradermally, subcutaneously, transdermally, intramuscularly, or by intraventricular injection. The disclosed delivery agents and one or more therapeutic agents can also be administered to a subject using a combination of these techniques.

[0564] The administration of a therapeutically effective amount can be a single administration or multiple administrations. The administration can be a dose once a day, more than once a day, or less than once a day (such as once a week, once a month, etc.) over a period of days to weeks or months or even years. In certain non-limiting instances, the administration includes a monthly dose, a dose every three weeks, a dose every two weeks, a weekly dose, a twice-weekly dose, or a daily dose, or a combination thereof. A laboratory technician, attending physician, or veterinarian (in the case of administration to a non-human animal subject) will select a specific mode / manner of administration and dosing regimen taking into account the specific circumstances of the case (such as the subject, the disease, the disease state / severity involved, the specific administration, and whether the treatment is prophylactic).

[0565] The delivery agent (such as in combination with one or more therapeutic agents) can be administered using a variety of routes such as intratumorally, intravenously, intraperitoneally, intramuscularly, subcutaneously, orally, or locally, and a particular route can provide a more direct and effective response than other routes. The disclosed delivery agents and one or more therapeutic agents can be administered by the same or different routes. In some aspects of the present disclosure, the delivery agent and one or more therapeutic agents are administered using any suitable route of administration, such as intravenous or intratumoral administration. In an exemplary aspect of the present disclosure, the delivery agent and one or more therapeutic agents can be administered by intratumoral injection. Alternatively or additionally, the delivery agent and one or more therapeutic agents can be administered intravenously.

[0566] In some aspects of the present disclosure, each of the delivery agent and one or more therapeutic agents may be administered to a subject one or more times at one or more different time intervals at different concentrations. In some aspects of the present disclosure, the disclosed delivery agent and one or more therapeutic agents are administered such that the effective time period of the delivery agent overlaps with the administration of the one or more therapeutic agents. In some aspects of the present disclosure, the delivery agent and one or more therapeutic agents are administered such that the delivery agent and one or more therapeutic agents can be internalized into a cell (e.g., a target cell) within the same endosome of the cell.

[0567] The pharmaceutical compositions can be used in the methods of the present disclosure and include a therapeutically effective amount of the disclosed delivery agent and / or one or more therapeutic agents. These pharmaceutical compositions can include any suitable carrier. For example, formulations suitable for intratumoral, intravenous, intramuscular, subcutaneous, intraperitoneal or topical administration can contain a sterile aqueous solution of the active ingredient. Such formulations can be prepared by dissolving the therapeutic agent and / or other active and / or inactive ingredients in water containing a physiologically compatible substance such as sodium chloride (e.g., 0.1 - 2.0 M), glycine, etc. (with a buffered pH compatible with physiological conditions to produce an aqueous solution) and rendering the solution sterile. The suitable carrier can be the same for some (e.g., two or more) delivery agents and all of the one or more therapeutic agents administered, or can be different for the delivery agent and all of the one or more therapeutic agents.

[0568] Suitable solid or liquid pharmaceutical preparation forms such as aerosols, (micro)capsules, creams, drops, drops in ampoules or injection solutions, emulsions, granules, powders, suppositories, suspensions, syrups, tablets, coated tablets, and formulations with sustained release of the active compound, usually use excipients and additives and / or adjuvants such as binders, coating agents, disintegrants, flavoring agents, lubricants, solubilizers, sweetening agents or swelling agents, as described above. The pharmaceutical agents are suitable for various drug delivery systems. A brief review of various drug delivery methods can be found in Langer, “New Methods of Drug Delivery,” Science 249:1527 - 1533 (1990), which is incorporated herein by reference as long as it does not conflict with the present disclosure.

[0569] The delivery agents of the present disclosure can be formulated as therapeutically active pharmaceutical agents that can be administered to a subject parenterally or orally. Parenteral administration routes include but are not limited to epidural, intra - arterial, intramuscular (IM and long - acting IM), intraperitoneal (IP), intravenous (IV), intracardiac injection or infusion techniques, intranasal (inhalation), intrathecal, intragastric injection, subcutaneous injection (subcutaneous (SQ and long - acting SQ), transdermal, topical and ocular.

[0570] The disclosed delivery agents, such as in combination with one or more therapeutic agents, can be mixed or combined with suitable pharmaceutically acceptable excipients to prepare pharmaceutical agents. Pharmaceutically acceptable excipients include, but are not limited to, aluminum oxide, aluminum stearate, buffers (such as phosphates), glycine, ion exchange agents (such as those that help control the 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 those based on cellulose materials, colloidal silica, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols such as polyethylene glycol, polyethylene-polypropylene-block polymers, polyvinylpyrrolidone, potassium hydrogen phosphate, protamine sulfate, group 1 halide salts such as sodium chloride, sodium carboxymethylcellulose, waxes, lanolin, and zinc salts. Liposome suspensions can also be used as pharmaceutically acceptable carriers.

[0571] After mixing or adding one or more of the disclosed delivery agents, the resulting mixture can be a solid, solution, suspension, emulsion, etc. These formulations can be prepared according to suitable methods. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the pharmaceutical agent in the selected carrier. Pharmaceutical carriers suitable for administering the disclosed delivery agents include any such carriers known to be suitable for a particular mode of administration. In addition, the disclosed delivery agents can also be mixed with other inactive or active materials (such as one or more therapeutic agents) that do not impair the intended effect, or with materials that supplement the intended effect or have another effect.

[0572] When the solubility of the delivery agent in the carrier is insufficient, dissolution methods can be used. Such methods exist, including but not limited to dissolution in an aqueous solution of sodium bicarbonate, use of solubilizing agents such as dimethyl sulfoxide (DMSO), and use of surfactants such as (ICI Americas, Inc., Wilmington, DE).

[0573] The disclosed delivery agents can be prepared with carriers that protect them from rapid elimination from the body, such as coated or sustained-release formulations. Such carriers include controlled-release formulations, such as but not limited to microencapsulated delivery systems. The disclosed delivery agents are contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect on the subject being treated, typically an amount that avoids adverse side effects. The therapeutically effective concentration can be determined empirically by testing the compound in known in vitro and in vivo model systems for the disorder or disease being treated. For example, a mouse model of the disorder or disease of interest, such as a genetic disorder, rare disease, cancer, immune disorder, or infectious disease, can be used to determine the effective amount or concentration, which can then be translated to a dosage for other subjects, such as humans.

[0574] Injectable 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, saline solution or water; or suitable dispersing or wetting agents and suspending agents, such as sterile, mild fixed oils, including synthetic monoglycerides or diglycerides, and fatty acids, including oleic acid; naturally occurring vegetable oils, such as coconut oil, cottonseed oil, peanut oil, sesame oil, etc.; glycerol; polyethylene glycol; propylene glycol; or other synthetic solvents; antimicrobial agents, such as benzyl alcohol and methylparaben; antioxidants, such as ascorbic acid and sodium bisulfite; buffering agents, such as acetate, citrate and phosphate; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); tonicity adjusting agents, such as sodium chloride and glucose; and combinations thereof. Parenteral preparations can be packaged in ampoules, disposable syringes or multi-dose vials made of glass, plastic or other suitable materials. Buffering agents, preservatives, antioxidants, etc. can be added as needed. When administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof. Liposome suspensions, including tissue-targeted liposomes, can also be used as pharmaceutically acceptable carriers.

[0575] For topical application, the disclosed delivery agents, for example in combination with one or more therapeutic agents, can be formulated into creams, lotions, ointments, solutions or suspensions in a suitable aqueous or non-aqueous carrier. Topical application can also be achieved through transdermal patches or bandages containing the therapeutic substance. Additives can also be included, such as buffering agents, such as sodium bisulfite or disodium ethylenediaminetetraacetate; preservatives, such as bactericides and fungicides, including phenylmercuric acetate or nitrate, benzalkonium chloride or chlorhexidine; and thickening agents, such as hypromellose.

[0576] If the disclosed delivery agents (e.g., in combination with one or more therapeutic agents) are administered orally in suspension form, the pharmaceutical agents can be prepared according to suitable pharmaceutical formulation techniques and can contain suspending agents, such as alginic acid or sodium alginate, swelling agents, such as microcrystalline cellulose, viscosity enhancing agents, such as methylcellulose, and sweetening / flavoring agents. Oral liquid preparations can contain conventional additives, such as suspending agents, such as gelatin, glucose syrup, hydrogenated edible fat, methylcellulose, sorbitol and syrup; emulsifying agents, such as gum arabic, lecithin or sorbitan monooleate; non-aqueous carriers (including edible oils), such as almond oil, fractionated coconut oil, oily esters such as glycerol, propylene glycol or ethanol; preservatives, such as methylparaben or propylparaben or sorbic acid; and, if desired, conventional flavoring or coloring agents. When formulated into immediate-release tablets, these pharmaceutical agents can contain dibasic calcium phosphate, lactose, magnesium stearate, microcrystalline cellulose and starch and / or other binders, diluents, disintegrants, excipients, fillers and lubricants.

[0577] If oral administration is desired, the disclosed delivery agents (e.g., in combination with one or more therapeutic agents) can be provided in a composition that protects them from the gastric acid environment. For example, the disclosed delivery agents can be formulated with an enteric coating that maintains their integrity in the stomach and releases the active compound in the intestine. The disclosed delivery agents can also be formulated in combination with antacids or other such ingredients.

[0578] Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For oral therapeutic administration, the disclosed delivery agents can be mixed with excipients and used in the form of capsules, tablets, or lozenges. Pharmaceutically compatible adjuvant materials or binders can be part of the composition.

[0579] Capsules, pills, tablets, lozenges, etc. can contain any of the following ingredients or compounds of similar nature: binders such as, but not limited to, gum arabic, 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; glidants such as, but not limited to, colloidal silica; sweeteners such as sucrose or saccharin; disintegrants such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and flavoring agents such as peppermint, methyl salicylate, or fruit flavors.

[0580] When the dosage unit form is a capsule, in addition to the materials of the above types, it can also contain a liquid carrier such as a fatty oil. In addition, the dosage unit form can contain various other materials that alter the physical form of the dosage unit, such as coatings of sugar and other enteric solvents. The disclosed delivery agents can also be administered as components of dosage forms such as elixirs, suspensions, syrups, wafers, teas, chewing gums, etc. In addition to the active compound, syrups can also contain sucrose or glycerin as sweeteners and some preservatives, dyes, and coloring agents as well as flavors.

[0581] When administered orally, the compounds can be in the common dosage forms for oral administration. These dosage forms include common solid unit dosage forms such as tablets and capsules and liquid dosage forms such as solutions, suspensions, and elixirs. When solid dosage forms are used, they can be of the sustained-release type, whereby the frequency of administration of the compounds is lower.

[0582] In some instances, the disclosed delivery agents (e.g., in combination with one or more therapeutic agents) are injected into the stomach of a subject or systemically incorporated into the subject, e.g., into different muscle groups. Examples of methods and compositions for administering therapeutic substances including proteins are discussed in Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems 2ed. (2005); Mahato, Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids (2004); McNally, Protein Formulation and Delivery, 2ed. (2007); and Kumar et al., “Novel Delivery Technologies for Protein and Peptide Therapeutics,” Current Pharm. Biotech., 7:261-276 (2006); each of which is incorporated herein by reference, so long as it does not conflict with the present disclosure.

[0583] In some aspects of the present disclosure, the delivery agent and / or therapeutic agent can be administered via a nanoparticle carrier. Nanoparticles (NPs) are synthetic particles with a size range from one nanometer to several hundred nanometers, comprising an inorganic core surrounded by an organic layer. Nanoparticles with an inorganic core such as gold, silica, superparamagnetic iron oxide (SPIO) can be obtained. For example, in cancerous tissue, NPs extravasate from leaky cancer blood vessels to a greater extent than in healthy tissue and remain in the area via the enhanced permeability and retention (EPR) effect. In some aspects of the present disclosure, the delivery agent and / or therapeutic agent can be delivered via lipid nanoparticles, peptide-based nanoparticles (Bioactive Materials Volume 11, May 2022, Pages 268-282), nanotubes (e.g., NanoPortal, NanoPrecision Medical, Emeryville, California, USA), subcutaneous implantable reservoirs (e.g., Delpore, Brisbane, California, USA), long-acting injections (e.g., or CLOUD TM, administered by Durect, Cupertino, California, USA) and / or a transdermal microneedle system (Zosano Pharma, Fremont, California, USA). In certain aspects of the present disclosure, the nanoparticles can be combined with a delivery agent and / or a therapeutic agent as follows: 1) encapsulating the delivery agent and / or the therapeutic agent within the nanoparticle carrier; and / or 2) covalently attaching, binding, linking, or associating the delivery agent and / or the therapeutic agent to the exterior of the nanoparticle carrier. To evaluate the delivery performance of embodiments that combine the use of nanoparticles with a delivery agent and / or a therapeutic agent, the HeLa Luc / 705 cell luciferase assay discussed herein can be used. Alternatively or additionally, a biological cell assay can be used to measure (i) a change in the phenotype of a cell or biomolecule or (ii) a change in the expression of a molecular marker due to the delivery of a therapeutic agent or the delivery of an exogenous biomolecule, such as measuring the fluorescence of GFP expression after delivering GFP RNA to a cell. By way of example only, if GFP RNA with and without a delivery agent is encapsulated in a nanoparticle carrier and if biological cells are treated with the nanoparticle carrier in growth medium for 24 hours or longer, the average increase in fluorescence of the cells treated with the nanoparticle carrier containing the delivery agent is significantly greater compared to the cells treated with the nanoparticle carrier without the delivery agent.

[0584] The dose of each delivery agent and / or one or more therapeutic agents administered to a subject should be sufficient to induce a beneficial therapeutic response over time in the subject, such as reducing the burden of a genetic disease, rare disease, cancer, immune disorder, or infectious disease in the subject, increasing the survival rate of the subject, reducing the recurrence rate of a genetic disease, rare disease, cancer, immune disorder, or infectious disease in the subject, or a combination thereof. A beneficial therapeutic response may require administering one or more doses of one or more of the disclosed delivery agents and / or one or more therapeutic agents at the same or different times, such as administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 or more doses. In some embodiments, an effective amount of the disclosed delivery agent (e.g., in combination with a therapeutic agent) is administered as a single dose per time period (e.g., every three or four months, one month, one week, or one day), or it can be divided into at least two unit doses for administration over a period of time. Treatment can continue until the desired result is achieved. For example, treatment can continue for 3 or 4 weeks up to 12 to 24 months or longer, including continuous treatment. The compound can also be administered intermittently in several doses, such as every few days (e.g., at least every two days, three days, four days, five days, or ten days) or every few weeks (e.g., at least every two weeks, three weeks, four weeks, five weeks, or ten weeks).

[0585] The dosage can vary among subjects or can be the same. For example, appropriate dosages can be determined using routine experimentation. Generally, compared to a therapeutic agent without a delivery agent, the administration of the disclosed delivery agent and / or one or more therapeutic agents can provide a powerful synergistic therapeutic effect for treating a subject's genetic disease, rare disease, cancer, immune disorder, or infectious disease. Accordingly, a specific dosing regimen can be customized for a particular subject, the disorder to be treated, or the desired outcome. For example, when treating a target disease or disorder (such as a genetic disease (e.g., muscular dystrophy), rare disease, cancer, immune disorder, or infectious disease) using the methods disclosed herein, an initial treatment regimen can be applied to suppress the disorder. This initial treatment regimen can include administering a higher dosage of the disclosed delivery agent, or administering such agent more frequently, such as once daily. After the desired therapeutic effect is obtained, a second treatment regimen can be applied, such as administering a lower dosage of the delivery agent or administering such agent less frequently, such as once a month, once every two months, once every quarter, or once every six months. In such cases, the second regimen can serve as a "booster" to restore or maintain the desired level of muscle regeneration. Similar treatment regimens can be used for other subjects with reduced or impaired muscle regenerative capacity, such as elderly subjects.

[0586] The effective amount of the various treatments of the present disclosure may depend on the severity of the disorder or disease, the weight and general condition of the subject, as well as the absorption, inactivation, and excretion rates of the delivery agent (and any one or more therapeutic agents administered to the subject together with the delivery agent), the dosing schedule and the amount administered, and other factors recognized in the art, particularly those beneficial to the present disclosure. It should also be clear that the exact dosage and frequency of administration will depend on the particular delivery agent or other therapeutic agent being administered, the particular disorder being treated, the severity of the disorder being treated, the age, weight, general physical condition of the particular subject, and other medications the subject may be taking. Generally, the dosage used in vitro can provide useful guidance for the amount available for in vivo administration of the pharmaceutical composition, and animal models can be used to determine the effective dose for treating a particular disorder. For example, a mouse model of a particular disorder or disease (such as a genetic disorder, a rare disease, cancer, an immune disorder, or an infectious disease) can be used to determine the effective dose, which can then be translated into a dose for other subjects (such as humans). Various considerations in dose determination have been 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, provided that it does not conflict with the present disclosure.

[0587] In certain instances, the disclosed delivery agent is administered to a subject in an amount sufficient to provide a dosage range of the delivery agent from 10 nmol / g to 500 nmol / g, such as between 2 nmol / g and 20 nmol / g or between 2 nmol / g and 10 nmol / g. In other instances, the delivery agent is administered to the subject in an amount sufficient to provide a dosage range between 0.01 μg / kg and 1000 mg / kg or between 0.1 mg / kg and 1000 mg / kg, and in certain instances, the amount is provided daily or weekly. In another instance, the disclosed delivery agent is administered to the subject in an amount sufficient to provide a dosage range of 0.2 mg / kg to 2 mg / kg. In another instance, the disclosed delivery agent is administered to the subject in an amount sufficient to provide a pharmaceutical dosage between 1.0 mg / kg and 30 mg / kg, such as 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 further instances, the delivery agent is administered to the subject in an amount sufficient to provide a concentration of the delivery agent in the administered material between 5 nM and 500 nM, such as between 50 nM and 200 nM, or 100 nM. In further instances, the delivery agent is administered to the subject in an amount sufficient to provide a concentration of the delivery agent in the administered material between 0.1 μM and 500 μM, such as between 5 μM and 30 μM, or 10 μM. In other instances, the concentration of the delivery agent administered to the subject is from 25 mg / ml to 1 mg / ml, such as from 5 mg / ml to 1 mg / ml, from 1000 μg / ml to 500 μg / ml, from 500 μg / ml to 50 μg / ml, including 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.

[0588] The present disclosure provides exemplary therapeutic agents that can be co-administered with the disclosed delivery agents. In some aspects of the present disclosure, any therapeutic agent that is active within the cell cytosol can be used with the disclosed delivery agents and methods. In certain aspects of the present disclosure, the therapeutic agents used herein are formulations that, when combined with the disclosed delivery agents, are internalized into cells (e.g., target cells in a subject or cell culture) at a higher rate compared to the absence of the delivery agent and / or have a greater therapeutic effect in a subject compared to the absence of the delivery agent. In some aspects of the present disclosure, the one or more therapeutic agents are chemotherapeutic agents, therapeutic antibodies, immunotherapeutic agents, antibiotics, agents for treating mental disorders, agents for treating substance use disorders, or combinations thereof. In certain non-limiting aspects 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 octadecene-1-amine hydrofluoride, 1-hexadecylamine hydrofluoride, gefitinib, lapatinib, olaparib, mitomycin C, sunitinib, geftinib, nintedanib, PD173074, erdafitinib, sorafenib, or combinations thereof.

[0589] In certain instances, the amount of therapeutic agent administered to a subject is sufficient to provide a dosage range of the therapeutic agent from 10 nmol / g to 500 nmol / g, such as between 2 nmol / g and 20 nmol / g or between 2 nmol / g and 10 nmol / g. In other instances, the amount of therapeutic agent administered to a subject is sufficient to provide a dosage between 0.01 μg / kg and 1000 mg / kg or between 0.1 mg / kg and 100 mg / kg or between 100 mg / kg and 1000 mg / kg, and in certain instances, this amount is provided daily or weekly. In another instance, the amount of the disclosed therapeutic agent administered to a subject is sufficient to provide a dosage of the agent between 0.2 mg / kg and 2 mg / kg. In a further instance, the amount of therapeutic agent administered to a subject is sufficient to provide a concentration of the therapeutic agent in the administered material between 5 nM and 500 nM, such as between 50 nM and 200 nM, or 100 nM. In a further instance, the amount of therapeutic agent administered to a subject is sufficient to provide a concentration of the therapeutic agent in the administered material between 0.1 μM and 500 μM, such as between 0.1 μM and 50 μM, or 20 μM. In other instances, a therapeutic agent between 500 μg / ml and 1 μg / ml is administered to a subject, such as between 300 μg / ml and 3 μg / ml, between 200 μg / ml and 20 μg / ml, including 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.

[0590] Therapeutic antisense and ribozyme molecules can also be used in combination with the delivery agents and 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 the antisense or ribozyme molecules, thereby improving the efficacy of the antisense or ribozyme molecules, for example, in subjects suffering from a related disorder or disease (such as a genetic disorder, a rare disease, cancer, an immune disorder or an infectious disease). 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, 1990W). In cells, the antisense nucleic acid hybridizes with the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acid interferes with the translation of the mRNA because the cell does not translate double-stranded mRNA. In some aspects of the present disclosure, the antisense molecules used herein are mRNA, miRNA, siRNA or CRISPR-related molecules. In one embodiment, a therapeutic antisense oligomer of 10-25 nucleotides is administered to a subject in combination with the disclosed delivery agent. The use of antisense methods to inhibit gene translation has been described, for example, in Marcus-Sakura, Anal. Biochem. 172:289, 1988.

[0591] Generally, the principle behind antisense technology is that an antisense compound hybridizes with a target nucleic acid and affects the regulation of gene expression activity or function, such as transcription, translation or splicing. Regulation of gene expression can be achieved, for example, by target RNA degradation or occupancy-based inhibition. An example of regulating target RNA function by degradation is RNase H-based degradation when the target RNA hybridizes with a DNA-like antisense compound, such as an antisense oligonucleotide. Antisense oligonucleotides can also be used to regulate gene expression (such as splicing) by occupancy-based inhibition (such as by 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 aspects of the present disclosure, the 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.

[0592] Any type of antisense compound that specifically targets and modulates the expression of one or more genes of interest can be used with the disclosed delivery agents and methods. Such antisense compounds include single-stranded compounds, such as antisense oligonucleotides, and double-stranded compounds, including compounds having at least a partially double-stranded structure, including siRNA, miRNA, shRNA, and ribozymes. The antisense molecule can also have flanking sequences (such as regulatory sequences). Ribozymes can have any general structure, including but not limited to hairpin, hammerhead, or axehead structures, so long as the molecule is capable of cleaving RNA. In some aspects of the present disclosure, a therapeutically effective amount of the disclosed delivery agent and a viral vector comprising a nucleic acid inhibitor are administered to the cells of a subject or cell culture.

[0593] The length of the antisense oligonucleotides used with the disclosed delivery agents and methods can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. Antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions. For example, antisense nucleic acid molecules can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides, which are designed to increase the biological stability of the molecule or the physical stability of the duplex formed between the antisense and sense nucleic acids. For example, phosphorothioate derivatives and acridine-substituted nucleotides can be used. 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-galactosylqueosine, inosine, and the like. Methods for screening the specificity of antisense compounds are available (see, for example, U.S. Patent Application Publication No. 2003 / 0228689).

[0594] Ribozymes can also be used, which are RNA molecules that have the ability to specifically cleave other single-stranded RNAs in a manner similar to DNA restriction endonucleases. By modifying the nucleotide sequences encoding these RNAs, molecules can be designed that recognize and cleave specific nucleotide sequences in RNA molecules (Cech, J. Amer. Med. Assn. 260:3030, 1988). One advantage of this method is that, because it is sequence-specific, only mRNAs having a specific sequence are inactivated.

[0595] There are two basic types of ribozymes, namely the Tetrahymena type (Hasselhoff, Nature 334:585, 1988) and the "hammerhead" type. The Tetrahymena type ribozyme recognizes a sequence of four bases, while the "hammerhead" type ribozyme recognizes a base sequence of 11 - 18 bases. The longer the recognition sequence, the greater the likelihood that the sequence is unique among the target mRNA species. Therefore, the hammerhead type ribozyme is more preferably used to inactivate specific mRNA species than the Tetrahymena type ribozyme, and an 18-base recognition sequence is more preferred compared to shorter recognition sequences.

[0596] There are various delivery systems that can be used to administer siRNA and other inhibitory nucleic acid molecules as therapeutic agents. These systems include, for example, encapsulation in liposomes, microparticles, microcapsules, nanoparticles, in recombinant cells capable of expressing the therapeutic molecule (see, for example, Wu et al., J. Biol. Chem. 262, 4429, 1987), construction of the therapeutic nucleic acid as part of a retrovirus or other vector, and so on.

[0597] In other non-limiting aspects of the present disclosure, the therapeutic agent is a peptide nucleic acid (PNA) (such as those described in Nielsen and Egholm, Current Issues Molec. Biol. (1999) 1(2):89 - 104, the relevant PNAs of which are incorporated herein by reference). Like DNA, synthetic PNA binds to complementary nucleic acid strands and can specifically target and regulate the expression of one or more genes of interest.

[0598] In certain non-limiting embodiments, the therapeutic agent is a morpholino. Representative morpholinos are discussed in Moulton et al., J Drug Discov Develop and Deliv, 3(2):1023, 2016, the relevant morpholinos of which are incorporated herein by reference. Morpholinos, such as phosphorodiamidate morpholino oligomers, are oligomeric molecules that can be used to block sites on nucleic acids such as target RNA molecules and have the potential for a wide range of therapeutic uses, such as for targeting pathogens and genetic diseases. Morpholinos are specific, soluble, non-toxic, stable, and effective antisense therapeutic agents. They can target a variety of RNAs, such as blocking the translation of RNA molecules (e.g., mRNA), modifying the splicing of precursor mRNA, inhibiting the maturation and / or activity of miRNAs, etc. Although morpholinos can be delivered into a range of cultured cells, embryos, and multicellular subjects (e.g., mammals, such as humans and non-human animals), co-administering the disclosed delivery agent with one or more therapeutic morpholinos can enhance the internalization and therapeutic efficacy 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 following sequences can be used: 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).

[0599] V. Methods

[0600] A. Methods of Contacting Cells

[0601] The present disclosure provides methods of contacting cells with the disclosed compositions comprising the delivery agents described herein (e.g., contacting cells with an effective amount of the disclosed delivery agents). In aspects of the present disclosure, the delivery agents of the compositions deliver one or more therapeutic agents to the cytosol of the cells. The disclosed delivery agents can contact the cells with one or more therapeutic agents in the same composition or in different compositions, and can contact the cells with one or more therapeutic agents simultaneously or at different times. In some aspects of the present disclosure, the cells are contacted with the disclosed delivery agents (e.g., in combination with one or more therapeutic agents) for at least 1 to 60 minutes, such as at least 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, or 50 to 60 minutes, such as 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, or 60 minutes. In some aspects of the present disclosure, the cells are contacted with the disclosed delivery agents (e.g., in combination with one or more therapeutic agents) for at least 1 to 40 days, such as 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 to 30 days, 10 to 20 days, 20 to 30 days, 15 to 25 days, such as 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 aspects of the present disclosure, exosomes are added to the cell culture medium in one addition. In other aspects of the present disclosure, exosomes are added to the cell culture medium multiple times during the culture, such as 2 to 5 times, such as 2, 3, 4, or 5 times.

[0602] In certain instances, cells are contacted with the disclosed delivery agent at a dose of the delivery agent between 1 nM and 500 nM, such as between 1 nM and 50 nM, between 50 nM and 100 nM, between 100 nM and 150 nM, between 150 nM and 200 nM, between 200 nM and 250 nM, between 250 nM and 300 nM, between 300 nM and 350 nM, between 350 nM and 400 nM, between 400 nM and 450 nM, or between 450 nM and 500 nM. In additional instances, the cells are contacted with the disclosed delivery agent at a dose of the delivery agent between 0.5 μM and 100 μM, such as 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, such as 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 instances, the cells are contacted with the disclosed delivery agent at a dose of the delivery agent between 500 μg / ml and 1 μg / ml, such as between 300 μg / ml and 3 μg / ml, between 200 μg / ml and 20 μg / ml, including 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 instance, the cells are contacted with the disclosed delivery agent at a dose of the delivery agent between 0.5 ng / kg and 500 ng / kg, such as between 0.5 μg / kg and 500 μg / kg, such as between 0.5 mg / kg and 500 mg / kg.

[0603] In certain instances, cells are contacted with one or more therapeutic agents at a dose between 1 nM and 500 nM, such as between 1 nM and 50 nM, between 50 nM and 100 nM, between 100 nM and 150 nM, between 150 nM and 200 nM, between 200 nM and 250 nM, between 250 nM and 300 nM, between 300 nM and 350 nM, between 350 nM and 400 nM, between 400 nM and 450 nM, or between 450 nM and 500 nM. In additional instances, cells are contacted with one or more therapeutic agents at a dose between 0.5 μM and 100 μM, such as 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, such as 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 instances, cells are contacted with one or more therapeutic agents at a dose between 500 μg / ml and 1 μg / ml, such as between 300 μg / ml and 3 μg / ml, between 200 μg / ml and 20 μg / ml, including 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 instance, cells are contacted with one or more therapeutic agents at a dose between 0.5 ng / kg and 500 ng / kg, such as between 0.5 μg / kg and 500 μg / kg, such as between 0.5 mg / kg and 500 mg / kg.

[0604] In some instances, contacting cells with the disclosed delivery agent and one or more therapeutic agents can enhance the activity of the one or more therapeutic agents (e.g., 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, such as 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 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., as compared to a method in which cells are contacted with one or more therapeutic agents but without the delivery agent).

[0605] In some instances, contacting cells with the disclosed delivery agents and one or more therapeutic agents can reduce cell viability (e.g., 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, such as 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 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 a method in which cells are contacted with one or more therapeutic agents but without a delivery agent).

[0606] In certain aspects of the present disclosure, after the therapeutic agent and the delivery agent are internalized into the cell, contacting the cell with the composition induces endosomal membrane lysis, thereby delivering the therapeutic agent to the cytosol of the cell. In other particular aspects of the present disclosure, after one or more therapeutic agents and the disclosed delivery agent are internalized into the cell, contacting the cell with the composition induces the formation of pores in the endosomal membrane, thereby delivering one or more therapeutic agents to the cytosol of the cell. In other particular aspects of the present disclosure, after the therapeutic agent and the delivery agent are internalized into the cell, contacting the cell with the composition results in local membrane instability and / or permeability, thereby delivering the therapeutic agent to the cytosol of the cell (Pei and Buyanova, Bioconjug Chem. 30(2):273–283, 2019).

[0607] In aspects of the present disclosure, the cells can be contacted in vitro or in vivo. In some aspects of the present disclosure, the cells are non-human mammalian cells (e.g., cells from laboratory animals, including but not limited to zebrafish or Xenopus laevis, or cells from veterinary animals, including but not limited to dogs and cats, as well as mice, rats, rabbits, sheep, horses, cattle, and non-human primates) or human cells.

[0608] The present disclosure also discloses methods for identifying therapeutic agents using cells (such as cells in a cell culture system). In some aspects of the present disclosure, the methods include contacting the cells with the disclosed delivery agents 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, for example, compared to a control. In some aspects of the present disclosure, a decrease in cell viability indicates that the one or more compounds (or combination of compounds) are therapeutic agents (or combination of therapeutic agents) that can be used to treat a specific disorder or disease in a subject. In some instances, the methods include determining the IC 50 value.

[0609] In some instances, the methods are high-throughput screening methods. Using automation, data processing / control software, liquid handling devices, and sensitive detectors, high-throughput screening can rapidly test a large number (such as thousands or millions) of molecules (such as test compounds) to determine the potency of each molecule for a desired purpose, such as the potency of each molecule for treating a disorder or disease of interest (such as a genetic disorder, a rare disease, cancer, an immune disorder, or an infectious disease). In some instances, the identification of therapeutic agents using cells is performed in a microplate, such as a 24-well, 48-well, 96-well, or 384-well microtiter plate. In some aspects of the present disclosure, a microplate reader is used to detect result metrics, such as metrics of cell survival or a specific cell phenotype (such as an increase or decrease in the expression of a molecule of interest (such as a protein of interest)), such as a fluorescence or bioluminescence signal. The microplate reader detects biological, chemical, or physical events in the microplate. A high-intensity lamp delivers light into the microplate wells, and then the light emitted by the reaction in the wells is quantified by a detector. The detection modes of microplate assays include absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. In some aspects of the present disclosure, fluorescence intensity is measured using a microplate reader, such as M5 (Molecular Devices), ELX800 TM Absorbance Microplate Reader (BioTek), SpectraFluor (Tecan), or VICTOR3 TM (Perkin Elmer). Wavelengths suitable for excitation and the corresponding emission wavelengths can be used. Such wavelength ranges can be from 425 nm to 800 nm, such as from 450 nm to 500 nm, or from 450 nm to 495 nm, or from 485 nm to 505 nm, such as from 495 nm to 500 nm, or from 620 nm to 800 nm, such as from 620 nm to 750 nm. In a specific instance, fluorescence intensity is measured using a Tecan SpectraFluor microplate reader, with excitation at 485 nm and emission measured at 535 nm.

[0610] In some aspects of the present disclosure, the method includes contacting cells with the disclosed delivery agent and one or more test compounds, for example, adding the delivery agent and one or more test compounds to intact cells representative of a particular disorder or disease of interest (such as a genetic disorder, 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 the compounds to enter the cells. The delivery agent and one or more test compounds can be incubated with the cells for 1 to 120 minutes, such as 10 minutes to 100 minutes, 20 minutes to 90 minutes, 30 minutes to 80 minutes, 40 minutes to 70 minutes, 50 minutes to 60 minutes, for example, at least five minutes, such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, or 120 minutes. In some aspects of the present disclosure, the delivery agent and one or more test compounds can be incubated with the 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 carried out at a temperature such that the delivery agent can cause one or more test compounds to be internalized into the cells and released from endolysosomes into the cytosol of the cells. For example, the incubation of the delivery agent and one or more test compounds with the intact cells can be carried out at about room temperature, such as at a temperature of about 20°C to about 25°C. In other aspects 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 at 37°C for 60 minutes. In another particular example, the delivery agent and one or more test compounds are incubated with the intact cells at 37°C for 18 hours.

[0611] In some aspects of the present disclosure, after incubation with the delivery agent and one or more test compounds, the cells are washed to remove any delivery agent and test compound material that has not been internalized into the cells. The washing can be carried out by standard methods, such as by 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 (such as RPMI-1640 or DMEM). The buffer can contain a small amount of solvent (such as 0.5% to 2% ethanol or methanol) or a carrier molecule (such as 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 particular example, the cells are washed three times by centrifuging at 400×g for 2 to 10 minutes, the resulting supernatant is removed, and the cells are resuspended in phosphate-buffered saline.

[0612] In some instances, the screening method further comprises selecting test compounds identified as having therapeutic activity (e.g., those that reduce the expression of a protein of interest, induce a phenotype of interest in a cell, and / or reduce or enhance cell viability). In some instances, the screening method further comprises administering such selected compounds, either alone or in combination with a disclosed delivery agent, to a research animal (e.g., a mammalian subject such as a rabbit, non-human primate, cat, dog, mouse, or rat) to further analyze the efficacy of the delivery agent and one or more test compounds in treating or inhibiting a disorder or disease of interest (e.g., a genetic disorder, rare disease, cancer, immune disorder, or infectious disease).

[0613] B. Method of Treating a Subject

[0614] The present disclosure also provides methods of administering a therapeutically effective amount of the disclosed compositions to a subject. In certain aspects of the present disclosure, the therapeutically effective amount of the composition treats, ameliorates, or prevents the onset of a disorder or disease in a subject, such as a genetic disorder, a rare disease, cancer, an immune disorder, an infectious disorder, a mental disorder, or any combination thereof. Accordingly, methods of treating a subject having a disorder or disease by administering a delivery agent (e.g., a therapeutically effective amount of the disclosed delivery agent) disclosed herein are provided. In some aspects of the present disclosure, the delivery agent is administered in combination with a therapeutically effective amount of one or more therapeutic agents. In some aspects of the present disclosure, the disorder or disease treated using the disclosed methods is a genetic disorder, a rare disease, cancer, an immune disorder, an infectious disorder, a mental disorder. In some aspects of the present disclosure, the disorder or disease is cancer (e.g., solid cancers such as sarcomas (e.g., rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, and alveolar soft part sarcoma); carcinomas (e.g., colorectal cancer); and lymphomas, such as Hodgkin lymphoma or non-Hodgkin 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, or central nervous system lymphoma); neuroblastoma; gynecological cancers (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; myxoid round cell tumor; endocrine tumor; and / or blood cancers (e.g., myelomas, such as multiple myeloma; lymphomas, e.g., Hodgkin lymphoma or non-Hodgkin 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, or central nervous system lymphoma; or leukemias, e.g., acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML))), immune disorders (e.g., autoimmune disorders or transplant rejection), or infectious diseases (e.g., cytomegalovirus, adenovirus, respiratory syncytial virus, Epstein-Barr virus, HIV infection or hepatitis virus infection (e.g., hepatitis C virus or hepatitis B virus), coronavirus (e.g., COVID-19), Ebola hemorrhagic fever virus, rabies, West Nile virus, yellow fever, herpes type I and II, hantavirus pulmonary syndrome, Marburg virus infection, Lassa virus infection, and dengue).

[0615] In some instances, the method includes treating or inhibiting a genetic disorder by administering to a subject a delivery agent disclosed herein (e.g., in combination with one or more therapeutic agents) using the methods disclosed herein. In some instances, the genetic disorder is selected from abetalipoproteinemia; acid maltase deficiency / Pompe disease, ADCY5-induced dyskinesia; adenosine aminohydrolase deficiency (ADA); X-linked agammaglobulinemia type 1; Alagille syndrome; all hypertrophic and dilated cardiomyopathies; congenital alopecia (ALUNC); Alpers syndrome; alpha-mannosidosis; alpha-1-antitrypsin deficiency; alpha-thalassemia - Southeast Asian; amyotrophic lateral sclerosis - Lou Gehrig's disease; androgen insensitivity syndrome; aniridia; ankylosing spondylitis; APC-related polyposis; argininosuccinate lyase deficiency; arrhythmogenic right ventricular dysplasia / cardiomyopathy; aspartylglucosaminuria; ataxia with oculomotor apraxia type 2; ataxia with vitamin E deficiency; ataxia-telangiectasia; autoimmune polyendocrine syndrome; beta-hydroxyisobutyryl-CoA dehydratase deficiency (HIBCH deficiency); beta-mannosidosis; biotinidase deficiency; blepharophimosis-ptosis-epicanthus inversus syndrome; Bloom syndrome; brachydactyly; brachydactyly-hypertension syndrome; brachydactyly type B1; branchio-oto-renal spectrum disorders; BRCA1; brachydactyly dysplasia; Canavan disease; cerebrotendinous xanthomatosis; neuronal ceroid lipofuscinosis - Batten syndrome; Charcot-Marie-Tooth disease type 2B; hereditary motor and sensory neuropathy type 1B; hereditary motor and sensory neuropathy type 2A2; CHARGE syndrome; cherubism; choroideremia; Citrin deficiency; citrullinemia type I; Coffin-Lowry syndrome; Cohen syndrome; Collagen 4A5 syndrome; common variable immunodeficiency; congenital adrenal hyperplasia; congenital cataract, facial dysmorphism, and neuropathy; congenital disorder of glycosylation type 1a; congenital myasthenic syndrome; Cornelia de Lange syndrome; Crohn's disease; cystic fibrosis; cystinosis; Darier disease; desmin storage myopathy; DFNA2 nonsyndromic hearing impairment; Diamond-Blackfan anemia; DNMT1 complex disorder; double cortex syndrome; Duane syndrome; Duchenne / Becker muscular dystrophy; limb-girdle muscular dystrophy; dyskeratosis congenita; early-onset familial Alzheimer's disease; early-onset primary dystonia (DYT1); Ehlers Danlos syndrome; Ehlers Danlos syndrome, classical type; Ehlers Danlos syndrome, hypermobile type; Ehlers-Danlos syndrome, kyphoscoliotic type;Emery-Dreifuss muscular dystrophy, X-linked; simplex hereditary epidermolysis bullosa; Fabry disease; facioscapulohumeral muscular dystrophy; familial dysautonomia (HSAN III); familial hyperinsulinism (FHI); familial hypertrophic cardiomyopathy; familial transthyretin amyloidosis; Fanconi anemia; fatal familial insomnia; progressive fibrodysplasia ossificans; fragile X syndrome; Friedreich ataxia; FRMD7-related infantile nystagmus; Fryns syndrome; fucosidosis; galactosemia; galactosialidosis; Gaucher disease; Gerstmann-Straussler-Scheinker syndrome; Glut1 deficiency syndrome; glycine encephalopathy; glycogen storage disease type VI; hemophagocytic lymphohistiocytosis; hemophilia A; hemophilia B; hepatic veno-occlusive disease with immunodeficiency; hereditary hemorrhagic telangiectasia; hereditary neuropathy with liability to pressure palsies; hereditary nonpolyposis colorectal cancer; hexosaminidase A deficiency; HFE-related hereditary hemochromatosis; Holt-Oram syndrome; Huntington disease; hydroxymethylbilane synthase (HMBS) deficiency; hypophosphatasia; inclusion body myopathy 2; incontinentia pigmenti; juvenile polyposis syndrome; Kallmann syndrome; Krabbe disease; Leber congenital amaurosis; Leber congenital amaurosis 10; leukodystrophy with brainstem and spinal cord involvement and elevated lactate (LBSL); Li-Fraumeni syndrome; limb-girdle muscular dystrophy type 2A Calpainopathy; LIS1-related lissencephaly; long QT syndrome; Lowe syndrome; lysosomal storage diseases (such as Gaucher disease) malignant hyperthermia susceptibility; maple syrup urine disease; MAPT-related diseases; mast cell diseases; McKusick-Kaufman syndrome; MECP2-Rett syndrome; Menkes; metachromatic leukodystrophy; methylmalonic acidemia; mucolipidosis II; multiple endocrine neoplasia type 1; mucolipidosis I; mucolipidosis II; multiple endocrine neoplasia type 2; myotonia congenita; myotonic dystrophy type 1; myotonic dystrophy type 2; nail-patella syndrome; nemaline myopathy; neurofibromatosis type 1; neurofibromatosis type 2; Noonan syndrome; ocular albinism, X-linked; oculocutaneous albinism type 1; oculocutaneous albinism type 2; oculopharyngeal muscular dystrophy; optic atrophy type 1; ornithine transcarbamylase deficiency; osteogenesis imperfecta; Parkinson disease; Pendred syndrome; peroxisome biogenesis disorders, Zellweger syndrome; polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC) disease; phenylketonuria; polycystic kidney disease; Pompe disease - GSD II; porphyria; primary ciliary dyskinesia;Progeria / HGPS, retinitis pigmentosa; retinoblastoma; Saethre-Chotzen syndrome; Schindler disease; SCN9A-related hereditary erythermalgia; SHOX-related haploinsufficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; Smith-Magenis syndrome; Sotos syndrome; spastic paraplegia type 3A; spastic paraplegia type 7; spastic paraplegia type 8; spastic paraplegia type 1; spastic paraplegia type 4; spinal muscular atrophy; spinocerebellar ataxia type 2; spinocerebellar ataxia type 3; spinocerebellar ataxia type 7; spinocerebellar ataxia type 1; Stickler syndrome; lethal osteogenesis imperfecta; thoracic aortic aneurysm and aortic dissection; Treacher Collins syndrome; trimethylaminuria; tuberous sclerosis; Udd distal myopathy; Usher syndrome type 1; LBSL disease; very long-chain acyl-CoA dehydrogenase deficiency; von Hippel-Lindau syndrome; Waardenburg syndrome type 1; Werner syndrome; Wilms tumor; Wilson disease; Wiskott-Aldrich syndrome; X-linked congenital adrenal hypoplasia; X-linked adrenoleukodystrophy; X-linked dystonia-parkinsonism; X-linked juvenile retinoschisis; X-linked myotubular myopathy; X-linked SCIDS; and Zellweger syndrome.

[0616] In some instances, a delivery agent disclosed herein and one or more therapeutic agents for a genetic disorder are administered to a subject having the genetic disorder. Exemplary therapeutic agents for treating or inhibiting genetic disorders can be found, 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, glycerol phenylbutyrate (ravicti), metronidazole, cysteamine, chlorophyllin, growth hormone, insulin, testosterone, etidronate, antihypertensive agents, metreleptin, lumasiran, N-carbamylglutamate, nitisinone, bezafibrate, phenylbutyric acid, sirolimus, diazoxide, somatostatin analogs, nifedipine, glucagon, insulin-like growth factor-1, mTOR inhibitors, GLP-1 receptor antagonists, oral antidiabetic agents, glibenclamide, leflunomide, rapamycin, emapalumab, TNF inhibitors, corticosteroids, platelet transfusions, red blood cell transfusions, antibacterial 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 vaccine, pneumococcal vaccine, purified C1 inhibitor concentrate, ecallantide, icatibant, lanadelumab, ezogabine, memantine, dextromethorphan, carbamazepine, stiripentol, 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, Methylxanthine, Valproic Acid, Cyclopyranopterin Monophosphate, Epalrestat, Burosumab, Isotretinoin, Patisiran, Yegsedi, Tafamidis, Miglustat, Ambroxol, Factor VIII, Factor IX, Fibrinogen Concentrate, Factor XIII A Subunit, Fresh Frozen Plasma, Cryoprecipitate, Factor XIII Concentrate, Eteplirsen, Vyondys 53, Deferoxamine, Mitapivat, Growth Hormone Receptor Antagonists, Estrogen, Thiazide Diuretics, Plerixafor, Ruxolitinib, Abatacept, Leniolisib, Rituximab, Adalimumab, Tacrolimus, Non-Steroidal Anti-Inflammatory Drugs, Ofatumumab, Angiotensin-Converting Enzyme Inhibitors, Febuxostat, Volanesorsen, Indomethacin, Alfacalcidol, Oxcarbazepine, DDAVP, Anakinra, Rilonacept, Canakinumab, Colchicine, Infliximab, Tocilizumab, Etanercept, Ustekinumab, Secukinumab, Mesalazine, Cyclosporine, Methotrexate, Azathioprine, Hydroxychloroquine, Selumetinib, Imatinib, Sunitinib, Tolvaptan, Levothyroxine, Tetrabenazine, Omeprazole,Ranirestat, chloroquine, inclisiran, evolocumab, "lomitapide", desferrioxamine, baricitinib, deoxycytidine, deoxythymidine, benzoates and phenylacetates, sodium phenylbutyrate, Carglumic acid, betaine, Pegvaliase, Eliglustat, Velaglucerase α, Imiglucerase, Taliglucerase, Agalsidase β, Agalsidase α, Migalastat, Sebelipase α, α-glucosidase, Velmanase α, Laronidase, Idursulfase, Elosulfase α, Galsulfase, Vestronidase α, Cerliponase α, cysteamine (enteric-coated), cysteamine hydrochloride eye drops, rosuvastatin calcium, Lomitapide, cholic acid, chenodeoxycholic acid, Asfotase α, Burosumab-twza, calcium acetate, alendronate sodium, ascorbic acid, thiamine, sodium citrate, Levocarnitine, Triheptanoin, riboflavin, uridine triacetate, potassium citrate, Tiopronin, penicillamine, trientine hydrochloride, zinc acetate, hydroxocobalamin, Inotersen, Patisiran sodium, Teriflunomide, Fingolimod HCl, Siponimod, Rasagiline, Selegiline, Carbidopa / Levodopa, Pitolisant, sodium oxybate, Deutetrabenazine, Baclofen, Everolimus, folic acid, biotin, Rufinamide, Cannabidiol, Midazolam, Levetiracetam, Clobazam, TopiramateGabapentin, Riluzole, Edaravone, Pyridostigmine Bromide, Amifampridine, Mexiletine Hydrochloride, Nusinersen Sodium, Octocog alpha, Rurioctocog alfa pegol, Lonoctocogα, Emicizumab, Damoctocog alfa pegol, Turoctocog α, Simoctocog alfa, Moroctocog α, Desmopressin Acetate, Recombinant Factor VIII, Efmoroctocog α, Factor VIII / von Willebrand Factor, Vonicog alfa, Eftrenonacog α, Albutrepenonacog alfa, Nonacog α, Human Coagulation Factor IX, Nonacog β pegol, Nonacog γ, Recombinant Factor IX, Eptacog α (activated), Recombinant Factor VIIa, Human Coagulation Factor X, Catridecacog, Human Protein C, Hydroxycarbamide, Epoetin alfa, Eltrombopag, Deferasirox, Methylene Blue Injection, Siltuximab, Anagrelide Hydrochloride, Ravulizumab, Macapegfilgrastim, Busulfan, Thiotepa, Deferiprone, Caplacizumab, Romiplostim, Romipleron alpha-2b, Immunoglobulin for Infusion, Methylprednisolone, Golimumab, Mesalamine, 5-Aminosalicylate, Obeticholic acid, Tocofersolan, C1 Inhibitor (human), Icatibant acetate, Danazol, Tranexamic acid, C1-Esterase Inhibitor, Human Conestat alfa, IL-1 Receptor Antagonist Anakinra, Cenegermin, Ciclosporin, Dexamethasone, Somatropin for InjectionOctreotide, Lanreotide, Pegvisomant, Pasireotide, Osilodrostat, Ketoconazole, Hydrocortisone, Human Chorionic Gonadotropin, Gonadotropin-Releasing Hormone, Mecasermin, Injectable Human Calcitonin, Parathyroid Hormone, Tasimelteon, Macitentan, Tadalafil, Ambrisentan, Nitric Oxide, Sildenafil, Bosentan monohydrate, Selexipag, Iloprost, Treprostinil, Riociguat, Mannitol, Tezacaftor / ivacaftor, Tobramycin, Aztreonam, Polymyxin E Sodium Methanesulfonate, Lumacaftor / ivacaftor, Levofloxacin, Pirfenidone, Nintedanib, Caffeine Citrate, Pegademase, CD34+ cells transduced with ADA cDNA, Interferon γ1-b, Sodium Cromoglicate, Amiodarone, Autologous Human Corneal Stem Cells, Voretigeneneparvovec, Teduglutide, Defibrotide, Ibuprofen, or Dexrazoxane.

[0617] In some particular aspects of the present disclosure, the genetic disorder is muscular dystrophy. In certain instances, 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-Dreifuss muscular dystrophy. In some aspects of the present disclosure, the muscular dystrophy is Duchenne muscular dystrophy. In some aspects of the present disclosure, the muscular dystrophy is a demyelinating disease. In certain further aspects of the present disclosure, the demyelinating disease is multiple sclerosis, Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, and / or Guillain-Barré syndrome. In some instances, the method is used to treat a subject having one or more signs or symptoms of a muscle disease such as muscular dystrophy such as, but not limited to, DMD.

[0618] In certain instances, an effective amount of the disclosed delivery agent combination with one or more therapeutic agents is administered to a subject to reduce or inhibit one or more signs or symptoms associated with muscular dystrophy. Exemplary therapeutic agents can include an α7β1 modulator or laminin-111 protein therapy, which functions to stabilize the sarcolemma and reduce muscle degeneration. Therapeutic agents used herein can also include components of the extracellular matrix, such as integrin, dystrophin, dystroglycan, utrophin, or growth factors. In some instances, other therapeutic agents reduce or enhance the expression of substances that enhance extracellular matrix formation or maintenance. In some instances, the other substances can include proteoglycans, 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, glycosaminoglycan (such as heparin), glycoprotein (such as dystroglycan), proteoglycan (such as heparan sulfate), and combinations thereof can also be administered. In a particular non-limiting instance, a morpholino combination with the disclosed delivery agent is administered to a subject having DMD. Exemplary morpholinos for treating DMD include (Sarepta Therapeutics).

[0619] In some instances, the disclosed methods can be used to administer the following growth stimulants in combination with a delivery agent, such as cytokines, polypeptides, and growth factors such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNF), epidermal growth factor (EGF), fibroblast growth factor (FGF), glial growth factor (GGF), glial maturation factor (GMF), glial cell line-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, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and combinations thereof.

[0620] In some instances, the methods include treating or inhibiting cancer, such as hematological malignancies or solid tumors. In certain aspects of the present disclosure, administering a therapeutically effective amount of the disclosed combination therapy to a subject reduces the cancer burden of the subject, increases the survival rate of the subject, decreases the incidence of cancer recurrence in the subject, induces the maturation of dendritic cells in the subject, or a combination thereof. The methods can include selecting a subject having cancer. Examples of hematological malignancies include leukemia, including acute leukemia (such as 11q23-positive acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic leukemia, promyelocytic leukemia, granulomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), T-cell large granular lymphocyte leukemia, polycythemia vera, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (indolent and advanced forms; including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocyte lymphoma, 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 myeloproliferative disorders.

[0621] Examples of solid tumors are, for example, sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoma (including indolent and high-grade forms; Hodgkin lymphoma; and non-Hodgkin lymphoma, such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma or central nervous system lymphoma), pancreatic cancer, breast cancer (including basal-like breast cancer, ductal carcinoma and lobular carcinoma of the breast), 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, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma).

[0622] In certain instances, hematological malignancies that can 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 myeloid leukemia, prolymphocytic / myelocytic leukemia, plasma cell leukemia, NK cell leukemia, Waldenstrom macroglobulinemia, Hodgkin lymphoma and non-Hodgkin lymphoma (indolent and high-grade forms; including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma or central nervous system lymphoma). In other certain instances, solid tumors that can be treated or inhibited by the methods disclosed herein include lung cancer, prostate cancer, pancreatic cancer (such as insulinoma), breast cancer, colorectal adenocarcinoma or squamous cell carcinoma, neuroblastoma, testicular cancer (such as seminoma) and ovarian cancer. In certain non-limiting instances, the subject has chronic myeloid leukemia, acute monocytic leukemia or non-Hodgkin lymphoma (indolent and high forms; including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma or central nervous system lymphoma).

[0623] In some instances, a delivery agent and one or more chemotherapeutic agents and / or radiation therapy disclosed herein are administered to a subject (e.g., a subject having cancer). Chemotherapeutic agents useful in the disclosed methods include chemical or biological agents having therapeutic utility in treating diseases characterized by abnormal cell growth. Specific examples of chemotherapeutic agents that can be used include microtubule-binding agents, DNA intercalating 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 provided in: Sausville and Longo, Principles of Cancer Treatment, Chapter 69 in Harrison’s Principles of Internal Medicine (20 th ed.), McGraw-Hill, 2018; Niederhuber et al., Cancer Pharmacology, Ch. 25 in Abeloff’s Clinical Oncology (6 th ed.), Elsevier, 2019; Gullatte et al., Clinical Guide to Antineoplastic Therapy: A Chemotherapy Handbook (4 th(ed.), Oncology Nursing Society, 2020; Chabner and Longo, Cancer Chemotherapy, Immunotherapy and Biotherapy: Principles and Practice (6th ed.), Lippincott Williams & Wilkins, 2018; Skeel, Handbook of Cancer Chemotherapy (9th ed.), Lippincott Williams & Wilkins, 2016. The disclosed methods can include administering more than one chemotherapeutic agent, such as in combination with the delivery agents disclosed herein. Such chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin or dacarbazine); antimetabolites such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine) and purine analogs such as mercaptopurine or thioguanine; or natural products such as vinca alkaloids (e.g., vinblastine, vincristine or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., actinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin or mitomycin C) and enzymes (e.g., L-asparaginase). Other agents include platinum coordination complexes (e.g., cis-diamminedichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine) and adrenocortical inhibitors (e.g., mitotane and aminoglutethimide); hormones and antagonists such as adrenocortical steroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen) and androgens (e.g., testosterone propionate and fluoxymesterone). The most commonly used chemotherapeutic drugs include doxorubicin, melphalan Ara-C (cytarabine), carmustine, busulfan, lomustine, carboplatin, cisplatin, cyclophosphamide daunorubicin, dacarbazine, 5-fluorouracil, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, plicamycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes such as docetaxel), vinblastine, vincristine, VP-16, and newer drugs include gemcitabine trastuzumab Irinotecan (CPT-11), cladribine (leustatin), navelbine, rituximab Imatinib (STI-571), topotecan Capecitabine, ibrutinib and calcitriol.

[0624] Treatment of the disorders and diseases described herein generally begins after the onset of the disorders described herein or after the onset of prodromal disorders (such as dysplasia or the development of a benign tumor). Treatment can begin in the early stages of cancer. For example, treatment can begin before a subject exhibits symptoms of the disorder, such as during a stage I diagnosis or when dysplasia is diagnosed. However, treatment can begin at any stage of the disease, such as, but not limited to, stage I, II, III, and IV cancers. Treatment before the onset of the disorder, such as treatment based on the detection of dysplasia or early (benign) prodromal disorders, is referred to herein as treatment of "subjects at risk of disease". In some aspects of the present disclosure, combination therapy can be administered during or after the onset of the disorders described herein.

[0625] Thus, subjects having or at risk of having cancer, such as the cancers described herein, such as colorectal cancer, renal cancer, or melanoma, can be selected for treatment. Typical subjects for administration of the combination therapies disclosed herein include humans, as well as non-human primates and other animals. To identify relevant subjects, accepted screening methods are employed to determine risk factors associated with a targeted or suspected cancer (such as colorectal cancer, renal cancer, or melanoma) in the subject and / or to diagnose the cancer, or to determine the status of an existing cancer in the subject. These screening methods include, for example, routine examinations to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected cancer, and diagnostic methods, such as, but not limited to, various histopathological, morphological, and / or cytological analyses to identify or diagnose the targeted cancer. These and other routine methods allow a clinician to select patients in need of treatment. In accordance with these methods and principles, the combination therapies disclosed herein can be administered as a stand-alone prophylactic or therapeutic regimen in accordance with the teachings herein or other routine methods, or as a subsequent, adjunctive, or coordinating therapeutic regimen to other treatments.

[0626] In some instances, the method includes treating or inhibiting an immune disease or disorder by administering to a subject a delivery agent disclosed herein (e.g., in combination with one or more therapeutic agents) using the methods disclosed herein. The immune disease or disorder can be any type of immune system disorder, such as a cytokine storm, an immune system disorder (e.g., an inflammatory or autoimmune disorder), or can be an immune system disorder associated with another disorder and / or disease (e.g., HIV infection or exposure to microgravity). In some non-limiting instances, the immune system disease or disorder is an inflammatory disorder. In certain aspects of the present disclosure, the inflammatory disorder can be rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel disease, or systemic lupus erythematosus. In other instances, the immune system disease or disorder is an autoimmune disorder. In certain aspects of the present disclosure, the autoimmune disease is type I diabetes, multiple sclerosis, lupus, myasthenia gravis, ankylosing spondylitis, celiac disease, Crohn's disease, Graves' disease, Hashimoto's thyroiditis, transplant rejection, or autoimmune uveitis.

[0627] In some instances, one or more immunomodulatory therapies (e.g., immunomodulatory biologics such as 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, e.g., Yao et al., Nat Rev Drug Discov, 12(2):130–146, 2013 and Kamphorst et al.) are also administered to a subject (e.g., a subject suffering from an immune disease or disorder such as an autoimmune disease, transplant rejection, or inflammatory disease)., Vaccine, 33(0 2):B21–B28, 2015, both of which are incorporated herein by reference in their entirety; regulatory cytokines such as IL-7; mTOR modulators such as rapamycin; antimicrobial therapies such as vaccination, antifungal agents, and / or antibiotics), anti-inflammatory agents (NSAIDs; anti-leukotrienes; immunoselective anti-inflammatory derivatives, ImSAIDs; bioactive compounds with anti-inflammatory activity such as plumbagin and plumericin; and / or steroids), disease-modifying antirheumatic drugs (DMARDs such as methotrexate, sulfasalazine, leflunomide, hydroxychloroquine, tofacitinib, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, anakinra, abatacept, and / or rituximab), antimalarial drugs such as chloroquine and hydroxychloroquine, medical procedures including surgery and stem cell transplantation; immunosuppressive agents (e.g., for preventing rejection of transplanted organs or tissues, treating autoimmune diseases, and / or inflammatory diseases; e.g., glucocorticoids such as prednisone, dexamethasone, and hydrocortisone; cytostatic agents such as alkylating agents and antimetabolites; antibodies such as Atgam, thymoglobulin, and T cell receptor and IL-2 receptor-directed antibodies; immunophilin-targeting agents such as cyclosporine, tacrolimus, sirolimus, and everolimus; interferons (IFNs) such as IFNλ and IFNβ; opioids; TNF-binding proteins such as infliximab, etanercept, and adalimumab; mycophenolate mofetil; and small biologics such as fingolimod and myriocin), immunotolerance therapies (e.g., for treating subjects at risk of organized or organ transplant rejection, allergic subjects, and / or subjects with autoimmune diseases; e.g., T cell- or B cell-targeting or T cell- or B cell-inhibiting drugs such as CAMPATH-1H, calcineurin inhibitors, rituximab, epratuzumab, belimumab, and atacicept; anti-cluster of differentiation (CD) 3 antibodies; abatacept; inducing hematopoietic chimerism such as mixed hematopoietic chimerism, where the bone marrow of an organ or tissue recipient is replaced with the bone marrow of a donor or a mixture of donor and recipient bone marrow to reduce organ or tissue transplant rejection; antigen desensitization; see Nepom et al.,Immunol Rev; 241(1):49–62, 2011, incorporated herein by reference), antihistamines, parasitic therapies (e.g., deliberately infecting a subject with a parasite or parasite eggs to treat immune diseases).

[0628] In some instances, the methods include treating or inhibiting an infectious disease by administering to a subject a delivery agent disclosed herein (e.g., in combination with one or more therapeutic agents) using the methods disclosed herein. In some instances, the infectious disease is selected from arthropod-borne viral infections, botulism, brucellosis, candidiasis, campylobacteriosis, chickenpox, chlamydial infections, cholera, coronavirus infections, staphylococcal infections, coxsackievirus infections, Creutzfeldt-Jakob disease, cryptosporidiosis, cyclospora infections, cytomegalovirus infections, Epstein-Barr virus infections, dengue, diphtheria, ear infections, encephalitis, influenza virus infections, parainfluenza virus infections, giardiasis, gonorrhea, Haemophilus influenzae infections, hantavirus infections, viral hepatitis, herpes simplex virus infections, HIV / AIDS, Helicobacter pylori infections, human papillomavirus (HPV) infections, infectious mononucleosis, legionellosis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphocytic choriomeningitis, malaria, measles, Marburg hemorrhagic fever, meningitis, monkeypox, mumps, mycobacterial infections, mycoplasma infections, norovirus infections, pertussis, pinworm infections, pneumococcal disease, Streptococcus pneumoniae infections, Mycoplasma pneumoniae infections, Moraxella catarrhalis infections, Pseudomonas aeruginosa infections, rotavirus infections, psittacosis, rabies, respiratory syncytial virus infections (RSV), ringworm, Rocky Mountain spotted fever, rubella, salmonellosis, SARS, scabies, sexually transmitted diseases, shigellosis, shingles, sporotrichosis, streptococcal infections, syphilis, tetanus, trichinosis, tuberculosis, tularemia, typhoid, viral meningitis, bacterial meningitis, West Nile virus infections, yellow fever, adenovirus-mediated infections and diseases, retrovirus-mediated infectious diseases, and Yersinia zoonoses. For example, the infectious disease can be influenza, parainfluenza virus, respiratory syncytial virus infection. In certain instances, the infectious disease is human immunodeficiency virus (HIV) infection, hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, tuberculosis (TB), malaria, dental caries, or Helicobacter pylori infection.

[0629] In some instances, one or more anti-infective agents (such as antibodies, antifungals, antivirals, and / or antiparasitics) are also administered to a subject (such as a subject suffering from an infectious disease, such as HIV). In some instances, 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 instances, the anti-infective agent is an antiviral agent, such as tilorone, vicriviroc, arbidol, or nelfinavir. In certain instances, the infectious disease is HIV, and an antiretroviral drug, 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 broad-spectrum inhibitor, such as a natural antiviral drug, is also administered to the subject. Other exemplary drugs include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine, and efavirenz.

[0630] In some instances, the method includes treating or inhibiting a mental disorder by administering to a subject a delivery agent disclosed herein (such as in combination with one or more therapeutic agents) using the methods disclosed herein. The mental disorder can be any type of mental disorder, such as an anxiety disorder, attention deficit hyperactivity disorder (ADHD), childhood behavioral and / or emotional disorders, bipolar disorder, depression, dissociative and dissociative disorders, eating disorders, obsessive-compulsive disorder, panic disorder, paranoia, post-traumatic stress disorder, psychosis, and / or schizophrenia. In some non-limiting instances, the mental disorder is depression.

[0631] In some instances, one or more antidepressants, such as, but not limited to, selective serotonin reuptake inhibitors (SSRI), tricyclic antidepressants, and / or monoamine oxidase inhibitors (MAOI), are also administered to a subject (such as a subject suffering from a mental disorder, such as a subject suffering from depression). Tricyclic antidepressants include imipramine, amitriptyline, nortriptyline, and desipramine. MAOIs approved for the treatment of depression include phenelzine tranylcypromine and isocarboxazid SSRIs mainly affect the neurotransmitter serotonin, including escitalopram hydrobromide Fluoxetine Sertraline Fluvoxamine Paroxetine and citalopram Other drugs can affect both norepinephrine and serotonin, such as venlafaxine and nefazodone or phenelzine Tranylcypromine Mirtazapine Nefazodone Triazolopyridine and bupropion

[0632] C. Embodiments of methods for preparing delivery agents

[0633] The present disclosure also discloses methods for preparing the delivery agents of the present disclosure. The delivery agents can be prepared by coupling the components of the delivery agent as described herein, for example, by performing one or more chemical couplings. In some aspects of the present disclosure, the chemical coupling can include suitable peptide bond formation reactions, conditions, and reagents. The chemical coupling can be carried out in any suitable order. For example, the chemical coupling can be carried out sequentially such that the C-terminal group is used as a building block for the remaining peptide backbone of the synthetic delivery agent. In some aspects of the present disclosure, a peptide bond is formed outside the C-terminal group to provide an anchoring group attached to the C-terminal group, followed by a masking peptide sequence attached to the anchoring group, followed by a cleavable linker group attached to the masking peptide sequence, followed by a solubilizing peptide group attached to the cleavable linker group. The solubilizing peptide can be functionalized to provide an N-terminal group as disclosed herein. Other components that can be coupled to the delivery agent can be added during or after the synthesis of the delivery agent. For example, a targeting group and / or a therapeutic agent 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 its branches) can be functionalized with a targeting group and / or a therapeutic agent using a chemical linker group that can covalently bind to the side chain or C- or N-terminus of lysine or cysteine. Suitable coupling conditions and reagents can be used to attach such components. In further aspects of the present disclosure, click chemistry can be used to attach a therapeutic agent and / or a targeting group to a chemical linker group. For example, click chemistry using azide-alkyne cycloaddition coupling conditions recognized by those skilled in the art can be used to couple a chemical linker and a therapeutic agent or a targeting group. In other aspects of the present disclosure, the therapeutic agent can be covalently coupled 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 (such as active carbonates, thiocarbonyls, active esters, α,β-unsaturated ketones, olefins, etc.), particularly taking advantage of the benefits of the present disclosure.

[0634] In some aspects of the present disclosure, suitable peptide bond formation conditions may include the use of coupling reagents, including but not limited to: 2-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethylammonium hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-N,N,N’,N’-hexafluorophosphate, 2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethylammonium hexafluorophosphate, 1-hydroxybenzotriazole, dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide·HCl, benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate, benzotriazol-1-yloxy-tris(pyrrolidino)-phosphonium hexafluorophosphate, bromo-tris(pyrrolidino)-phosphonium hexafluorophosphate, etc., or combinations thereof; and bases, such as amine bases (e.g., diisopropylethylamine, isopropylamine, n-methylmorpholine, etc.).

[0635] VI. Kit

[0636] Kits are provided herein that can be used for various aspects of the present disclosure described herein. The kits can contain various materials and reagents (e.g., for carrying out the methods described herein). For example, the kits can contain reagents, including but not limited to the disclosed delivery agents, one or more therapeutic agents, cells (e.g., cells representative of a particular disorder or disease and suitable for the methods of screening one or more test compounds described herein), cell culture media, serum, and other solutions or buffers that can be used to perform the assays and other methods provided herein. The kits can also include control samples, materials that can be used in the methods described herein, and containers, syringes, vials, test tubes, ampoules, capsules or bottles, microtiter plates, etc. in which the assay reactions can be carried out. The kits can be packaged in a container that can include compartments for receiving the kit contents and can include instructions for carrying out the methods described herein.

[0637] The kits can include labels or package inserts on or associated with any of the containers of the kit. The labels or package inserts can generally further include instructions for the use of the delivery agents, one or more therapeutic agents, and / or cells provided by the kit, e.g., for the methods disclosed herein. The instructional materials can be in written, electronic, or visual (e.g., video file) form.

[0638] For example, the kit may include (1) a peptide-based delivery agent as described herein (e.g., a composition comprising the delivery agent described herein); (2) one or more therapeutic agents (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 the one or more therapeutic agents of (2); (4) one or more administration devices; or (5) any combination of the delivery agent of (1) and (2) to (4).

[0639] VII. Overview of Some Examples

[0640] Examples of peptide-based delivery agents are disclosed herein, which comprise: a solubilizing peptide group having the structure of [X 1 Y 1 Y 1 X 1 m where each X 1 is independently a basic amino acid, an acidic amino acid, a nonpolar amino acid, or a derivative thereof each time it appears, provided that at least one X 1 is a basic amino acid or an acidic amino acid; each Y 1 is independently a nonpolar amino acid or a derivative thereof each time it appears; and m is an integer selected from 2 to 8; a cleavable linker group; a masking peptide group having the structure of [X 2 Y 2 Y 2 X 2 m’ where each X 2 is independently an acidic amino acid, a nonpolar amino acid, or a derivative thereof each time it appears, provided that at least one X 2 is an acidic amino acid; each Y 2 is independently a nonpolar amino acid or a derivative thereof each time it appears; and m' is an integer selected from 2 to 8; and an anchoring 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 peptidomimetic, an aptamer, a sugar, or a small targeting molecule; or a combination thereof.

[0641] In any or all of the above examples, the delivery agent has the structure of Formula IA or IB:

[0642] [N-terminal group]-[X 1 Y 1 Y 1 X 1 m -[Cleavable linker]-[X 2 Y 2 Y​​​2 X 2 m’ -[Anchoring / targeting group]-[C-terminal group],

[0643] Formula IA

[0644] [N-terminal group]-[X 2 Y 2 Y 2 X 2 m -[Anchoring / targeting group]-[Cleavable linker]-[X 1 Y 1 Y 1 X 1 m’ -[C-terminal group],

[0645] Formula IB

[0646] wherein each X 1 is independently, each time it appears, a basic amino acid, an acidic amino acid, a non-polar amino acid or a derivative thereof, provided that at least one X 1 is a basic amino acid or an acidic amino acid; each Y 1 is independently, each time it appears, a non-polar amino acid or a derivative thereof; each m and m' are independently, each time they appear, an integer selected from 2 to 8; each X 2 is independently, each time it appears, an acidic amino acid, a non-polar amino acid or a derivative thereof, provided that at least one X 2 is an acidic amino acid; each Y 2 is independently, each time it appears, a non-polar amino acid or a derivative thereof; the cleavable linker is an amino acid sequence having a length of 2 to 10 amino acids; the anchoring 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 peptidomimetic, an aptamer, a sugar or a small targeting molecule; or a combination thereof; the C-terminal group comprises an amino-terminated glycine moiety; and the N-terminal group comprises a capping group or a fluorophore.

[0647] In any or all of the above instances, each X 1 is independently, each time it appears, glutamic acid, glutamine, arginine, alanine, aspartic acid or a derivative thereof, provided that if X 1 is alanine, then at least one additional X 1 is glutamine, glutamic acid, aspartic acid, arginine or a derivative thereof.

[0648] In any or all of the above instances, each X 1 ​​​Independently upon each occurrence, it is glutamic acid, aspartic acid or a derivative thereof.

[0649] In any or all of the above instances, each X1 independently upon each occurrence is (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 solubilizing peptide sequence of DLLE or ELLD; or (iv) any combination of (i), (ii) and (iii).

[0650] In any or all of the above instances, each Y 1 independently upon each occurrence and each Y 2 independently upon each occurrence is leucine, α-methyl leucine, alanine or a derivative thereof.

[0651] In any or all of the above instances, each Y 1 independently upon each occurrence and each Y 2 independently upon each occurrence is leucine or a derivative thereof.

[0652] In any or all of the above instances, each Y 1 independently upon each occurrence and each Y 2 independently upon each occurrence is α-methyl leucine or a derivative thereof.

[0653] In any or all of the above instances, each X 2 independently upon each occurrence is glutamine, glutamic acid, aspartic acid, alanine or a derivative thereof, provided that if X 2 is alanine, then at least one additional X 2 is glutamine, glutamic acid, aspartic acid or a derivative thereof.

[0654] In any or all of the above instances, m is 3 and m' is 3; or m is 4 and m' is 4.

[0655] In any or all of the above instances, each X 1 is glutamic acid; each Y 1 and each Y 2 is leucine; each X 2 is glutamine or alanine; m is 3; and m' is 3; provided that if X 2 is alanine, then at least one other X 2 is glutamine.

[0656] In any or all of the above instances, the cleavable linker can be cleaved by cathepsin B.

[0657] In any or all of the above instances, the cleavable linker comprises an amino acid sequence having a length of 2 to 10 amino acids.

[0658] In any or all of the above instances, the length of the cleavable linker is 7 amino acids.

[0659] In any or all of the above instances, the cleavable linker comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:3.

[0660] In any or all of the above instances, the cleavable linker comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:3.

[0661] In any or all of the above instances, the cleavable linker comprises the amino acid sequence of SEQ ID NO:3.

[0662] In any or all of the above instances, the cleavable linker consists of the amino acid sequence of SEQ ID NO:3.

[0663] In any or all of the above instances, the anchoring group comprises a lysine moiety, wherein the side chain of the lysine moiety is functionalized with a heteroaliphatic group of the formula -C(O)-X-[CH 2 p Z, where X is oxygen or CH 2 ; Z is -N(R) 2 or -N + (R) 3 , where each R is independently hydrogen or aliphatic; and p is an integer in the range of 1 to 3.

[0664] In any or all of the above instances, X is oxygen or CH 2 ; Z is -N(R) 2 , where one R group is hydrogen and the other R group is C 6 -C 12 alkyl; and p is 2.

[0665] In any or all of the above instances, X is oxygen or CH 2 ; Z is -N + (R) 3 , where two R groups are methyl and the other R group is C 6 -C 12 alkyl; and p is 2.

[0666] In any or all of the above instances, the anchoring group is selected from:

[0667]

[0668] In any or all of the above instances, there are multiple anchoring groups.

[0669] In any or all of the above instances, there is one, two, or three anchoring groups. ​

[0670] In any or all of the above instances, the solubilizing peptide group provides the N-terminal group of the peptide-based delivery agent, while the anchoring group provides the C-terminal group of the peptide-based delivery agent.

[0671] In any or all of the above instances, (i) the N-terminal group binds to a carbonyl group-containing group; and (ii) the C-terminal group binds to an amine-terminated glycine moiety, a fluorophore, or a combination thereof.

[0672] In any or all of the above instances, the carbonyl group-containing group is an acetyl group or a fluorophore.

[0673] In any or all of the above instances, the fluorophore comprises 2-(methylamino)benzamide.

[0674] In any or all of the above instances, the amine-terminated glycine moiety has a structure selected from -Nal-K-G’, -G-Nal-K-G’, -G-K-G’, -Nal-C-G’, -G-Nal-C-G’, -G-C-G’, -Nal-G’, -G-Nal-G’, -W-G’, -G-W-G’ or -G-W-K-G’, where Nal is naphthylalanine, G is glycine, G’ is a modified glycine containing -C(O)-amine, W is tryptophan, K is lysine, and C is cysteine.

[0675] In any or all of the above instances, the amine-terminated glycine moiety is -G-Nal-G’, where G’ is a modified glycine containing -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic.

[0676] In any or all of the above instances, the solubilizing peptide group has the structure [ELLE] 3 ; the cleavable linker has the amino acid sequence of SEQ IDNO:3; the masking peptide group has the structure [QLLQ] 3 ; and the anchoring group has the structure according to any or all of the above instances; and wherein (i) the solubilizing peptide group provides the N-terminus that binds to the acetyl group, and (ii) the anchoring group binds to an amine-terminated glycine moiety having the structure -G-Nal-G’, where G’ is a modified glycine containing -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic.

[0677] In any or all of the above instances, the solubilizing peptide group has the structure [ELLE] 4; The cleavable linker has the amino acid sequence of SEQ ID NO:3; The masking peptide group has the structure [QLLQ] 4 ; There are two anchoring groups that bind directly to each other, and each anchoring group has the structure described in any or all of the above examples; and wherein (i) the solubilizing peptide group provides an N-terminus that binds to an acetyl group, and (ii) the anchoring group binds to a modified glycine containing a -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic.

[0678] In any or all of the above examples, the solubilizing peptide group has the structure [QLLE]-[QLLQ]-[QLLE]; the cleavable linker has the amino acid sequence of SEQ ID NO:3; the masking peptide group has the structure [QLLE]-[QLLQ]-[QLLE]; and the anchoring group has the structure described in any or all of the above examples; and wherein (i) the solubilizing peptide group provides an N-terminus that binds to an acetyl group, and (ii) the anchoring group binds to a glycine moiety that is amino-terminated with a structure -G-K-G', where G is glycine, K is lysine, and G' is a modified glycine containing a -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic.

[0679] In any or all of the above examples, the solubilizing peptide group has the structure [ELLE] 3 ; The cleavable linker has the amino acid sequence of SEQ ID NO:1; the masking peptide group has the structure [QLLA]-[QLLA]-[QLLQ]; the anchoring group has the structure described in any or all of the above examples; and wherein (i) the solubilizing peptide group provides an N-terminus that binds to an acetyl group, and (ii) the anchoring group binds to a glycine moiety that is amino-terminated with a structure -G-K-G', where G is glycine, K is lysine, and G' is a modified glycine containing a -C(O)-N(R a ) 2 group, where each R a is independently hydrogen or aliphatic.

[0680] In any or all of the above embodiments, the peptide-based delivery agent has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of the sequences of SEQ ID NO:272, 273 or 274, and wherein the 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.

[0681] The present disclosure also provides an example of a composition comprising: a peptide-based delivery agent according to any or all of the above examples; and a therapeutic agent.

[0682] In any or all of the above examples, the therapeutic agent is covalently or non-covalently bound to the peptide-based delivery agent.

[0683] 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.

[0684] In any or all of the above examples, the therapeutic agent is saponin, cisplatin, methotrexate, fluorouracil, doxorubicin, cyclophosphamide, chlorambucil, vinblastine, vincristine, docetaxel, paclitaxel, chlorhexidine, triclosan, xylitol, octadec-1-en-1-amine hydrofluoride, 1-hexadecylamine hydrofluoride, gefitinib, lapatinib, olaparib, mitomycin C, sunitinib, gefitinib, nintedanib, PD173074, erdafitinib, sorafenib, or a combination thereof.

[0685] In any or all of the above examples, the therapeutic agent is a morpholino.

[0686] In any or all of the above examples, the morpholino comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences of SEQ ID NO: 229, 240, 241, 242, 243, and 244.

[0687] In any or all of the above examples, (i) the peptide-based delivery agent comprises an antibody as an anchoring group or a targeting group, (ii) the therapeutic agent is a morpholino and wherein the morpholino is indirectly covalently bound to the peptide-based delivery agent via a linker group.

[0688] In any or all of the above examples, the composition further comprises an adjuvant, a carrier, a buffer, a detergent, or a combination thereof.

[0689] 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.

[0690] The present disclosure also provides an example of a method comprising contacting a cell with a delivery agent and / or a composition according to any or all of the above examples.

[0691] 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.

[0692] In any or all of the above instances, the cell is a non-human mammalian cell or a human cell.

[0693] In any or all of the above instances, the cell is contacted in vitro or in vivo.

[0694] In any or all of the above instances, contacting the cell with the composition induces endosomal membrane lysis after the therapeutic agent and the peptide-based delivery agent are internalized into the cell; or induces pore formation in the endosomal membrane after the therapeutic agent and the peptide-based delivery agent are internalized into the cell; or induces local disruption / destabilization of the endosomal membrane after the therapeutic agent and the peptide-based delivery agent are internalized into the cell; thereby delivering the therapeutic agent into the cytosol of the cell.

[0695] In any or all of the above instances, 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.

[0696] Also disclosed herein are instances of a method, including administering to a subject a therapeutically effective amount of a delivery agent and / or composition according to any or all of the above instances.

[0697] In any or all of the above instances, the subject is a human or a non-human mammalian.

[0698] In any or all of the above instances, the therapeutically effective amount of the composition treats, ameliorates or prevents a disorder or disease selected from genetic diseases, rare diseases, cancers, immune diseases, infectious diseases, mental disorders, substance use disorders or any combination thereof or the occurrence of the disorder or disease.

[0699] In any or all of the above instances, the administration includes injection, aerosol delivery, intranasal administration, oral administration, topical administration or a combination thereof.

[0700] In any or all of the above instances, the administration includes providing the therapeutic agent and the peptide-based delivery agent to the subject simultaneously or at different times.

[0701] In any or all of the above instances, the administration includes providing the therapeutic agent and the peptide-based delivery agent to the subject using the same administration route or different administration routes.

[0702] Also disclosed herein are instances of a method for identifying a therapeutic compound, including: contacting a cell with the peptide-based delivery agent and one or more compounds in any or all of the above instances; determining the effect of the one or more compounds on the contacted cell; 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.

[0703] In any or all of the above instances, the method further comprises determining the IC 50 value of the one or more compounds.

[0704] In any or all of the above instances, the method is a quantitative high-throughput screening method.

[0705] In any or all of the above instances, the method further comprises selecting one or more compounds that have a differential effect on the contacted cells relative to a control.

[0706] In any or all of the above instances, the effect of the one or more compounds on the contacted cells includes: a decrease in the viability of the contacted cells compared to the control; an increase in the viability of the contacted cells compared to the control; induction of a phenotype of interest in the contacted cells compared to the control; an increase in the expression of one or more genes in the contacted cells compared to the control; and / or a decrease in the expression of one or more genes in the contacted cells compared to the control.

[0707] Examples of kits are disclosed herein that comprise a container, wherein the container comprises a peptide-based delivery agent of any or all of the above instances or a composition of any one of the above instances, and wherein the container is selected from a syringe, vial, tube, ampoule, capsule, or bottle.

[0708] In any or all of the above instances, the kit further comprises: (i) instructions for administering or using the peptide-based delivery agent of any or all of the above instances or a composition of any one of the above instances; (ii) a container comprising a therapeutic agent; (iii) one or more administration devices; or (iv) any combination of (i) to (iii).

[0709] VIII. EXAMPLES

[0710] The following examples are provided to give a complete disclosure and description of examples of how to prepare and use the peptide-based delivery agents and methods described herein. They are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments conducted. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0711] Unless otherwise indicated, the following examples include a delivery agent backbone having the following structure:

[0712] [N-terminal group]-[Lytic]-[CL]-[Mask]-[K(LA) n -[C-terminal group],

[0713] Wherein:

[0714] · The N-terminal group contains a fluorophore or a capping group, as follows:

[0715] Fluorophore (“Fluor”): wherein R represents the connection to the delivery agent;

[0716] Capping group (“Cap”): wherein R represents the connection to the delivery agent.

[0717] · The lytic peptide group (“Lytic”) is ELLEELLEELLE (SEQ ID NO:1);

[0718] · The cleavable linker (“CL”) is GFGFVGG (SEQ ID NO:3);

[0719] · The masking peptide group (“Mask”) is QLLQQLLQQLLQ (SEQ ID NO:4);

[0720] · n is the number of anchoring groups;

[0721] · The anchoring group (“LA”) structure contains the specified lysine and a tail group having the structure, unless otherwise specified;

[0722] Each tail group of the anchoring group is independently covalently bound to the side chain of the specified lysine in the peptide chain to form a neutral amide bond, such that “K(LA)” has the structure unless otherwise specified.

[0723] · The C-terminal group is G’, G-Nal-G’ or G-W-G’, where Nal is naphthylalanine and G’ is a modified glycine that contains -C(O)NH 2 at the C-terminus instead of -C(O)OH, unless otherwise specified.

[0724] Unless otherwise specified, the morpholino used (also referred to as the standard morpholino in the examples) is:

[0725]

[0726] Moreover, for the examples that include a control, unless otherwise specified, the control sample may consist of morpholino (alone), or may include a delivery agent that does not contain any morpholino.

[0727] For in vivo mouse studies, the morpholino sequence (the "morpholino" in the above structure, which is "Morpholino 1" having the sequence 5'-GGCCAAACCTCGGCTTACCTGAAAT-3'; SEQ ID NO: 229) is complementary to the mouse dystrophin pre-mRNA and is used for targeted deletion of exon 23. For in vitro cell culture studies, the morpholino sequence (the "morpholino" in the above structure, which is "Morpholino 2" having the sequence 5'-GCAATATGAAACCTCTTACCTCAGT-3'; SEQ ID NO: 230) targets and disrupts an aberrant splicing site of the firefly luciferase gene stably transfected in HeLa cells (Kang et al. Biochemistry 1998, 37, 18, 6235–6239).

[0728] In some embodiments, Endo-Porter (Gene Tools, Philomath, Oregon) is used as a positive control. Endo-Porter is a peptide designed to facilitate the delivery of morpholinos into cultured cells. Endo-Porter is an endosomal delivery agent; it is uncharged at neutral pH but becomes cationic upon protonation at the low pH of late endosomes. Endo-Porter releases the contents of acidic endosomes into the cytosol; this has been shown for morpholinos and oxygen-responsive fluorescent probes. Endo-Porter is sparingly soluble in water but soluble in DMSO. Cultured cells are incubated with fresh medium containing the cargo to be delivered (e.g., morpholino), and then the Endo-Porter solution (usually formulated in DMSO) is added to the medium. When Endo-Porter enters the aqueous medium, it aggregates into particles; the culture should be immediately spun to disperse the Endo-Porter and limit the size of the aggregates. The Endo-Porter particles slowly sediment to the bottom of the culture vessel, bringing them into contact with adherent cells. If Endo-Porter is to be used for delivery to suspension cultures, the culture should be gently and continuously rotated (such as on a slow orbital shaker) to keep the particles suspended (Moulton et al., J Drug Discov Develop and Deliv, 3(2):1023, 2016).

[0729] Unless otherwise specified, the luciferase reporter assay was used to measure the activity of the compounds applied to cell cultures in the following examples. 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 the firefly luciferase gene interrupted by a mutated human β-globin intron. The mutation (IVS2-705) results in aberrant splicing of the luciferase pre-mRNA and prevents translation. Treatment with a morpholino oligonucleotide targeting the splice site corrects the splicing and restores luciferase activity.

[0730] For the assay, cells were cultured in DMEM containing glucose, glutamine, sodium pyruvate (from Corning 10-013-CV), 10% fetal bovine serum (from Corning 35-010-CV), and penicillin / streptomycin. In some cases, the medium was supplemented with glutamine (MP Biomedicals Cat#1680149). Cells were trypsinized with 0.25% trypsin-EDTA (from Corning 25-053-CI) for cell passage. Morpholinos (sequences provided herein, some examples 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 wells of a 60-90% confluent Luc / 705 HeLa cell plate containing serum-containing medium; the final solution concentrations of the test / control compounds and the different percentages of serum used were described in each example. After incubation for a period of time (16-22 hours), 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 in the lysate supernatant was evaluated using a luciferase detection system (E1501, Promega, Madison WI) in a luminometer (Turner Biosystems). In some cases, the Bright-Glo TM luciferase detection system (E2610, Promega, Madison WI) was used to evaluate luciferase activity according to the manufacturer's protocol and luminescence was measured using a Spectramax M3 microplate reader (Molecular Devices). In some examples, protein levels were quantified using a protein colorimetric assay reagent (e.g., Bio-Rad Laboratories Inc. kit #5000001, USA) for normalization.

[0731] To evaluate cytotoxicity and saporin delivery, HeLa cells in 96-well clear-bottom microplates (from Corning, Corning NY, USA) were treated with the delivery agent, saporin protein dissolved in water (Millipore Sigma, S9896), or both diluted in DMEM medium only, and then added to the plate wells in the amounts shown in the examples and treated for 1 hour. The treatment agent was removed and replaced with DMEM supplemented medium containing 10% FBS (the above medium). After 24 hours, the medium was removed and the cells were washed with 1×PBS. Then the cells were treated with 2 μM Calcein AM (Biotium, San Francisco CA, USA) for 30 minutes and the fluorescence was measured in a SpectraMax fluorescence microplate reader (Molecular Devices) according to the manufacturer's protocol.

[0732] Methods for evaluating in vivo delivery of morpholinos have been previously published and adapted (Morcos et al. Biotechniques 2008 45:613-623). Successful morpholino delivery resulted in deletion of dystrophin exon 23 sequences from the mRNA. Gene Tools LLC conducted studies 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 combined with the delivery agent in water, and the mixture was injected intravenously once daily for 2 or 3 days, and then the animals were sacrificed 1 day or more after the last injection. RNA was isolated from the excised muscle tissue using the MELT Total Nucleic Acid Isolation System (Ambion, Inc., Austin, TX, USA). RT-PCR was performed on the total RNA 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 exons 21 and 24. The RT-PCR react...

Claims

1. A peptide-based delivery agent comprising: Having a dissolved peptide group with a structure according to the formula [X 1 Y 1 Y 1 X 1 m wherein​ Each X 1 is independently, upon each occurrence, a basic amino acid, an acidic amino acid, a nonpolar amino acid, or a derivative thereof, provided that at least one X 1 is a basic amino acid or an acidic amino acid; Each Y 1 is independently a nonpolar amino acid or a derivative thereof each time it appears; and m is an integer selected from 2 to 8; a cleavable linker group; A masking peptide group having a structure according to formula [X 2 Y 2 Y 2 X 2 m’ , wherein​ Each X 2 is independently an acidic amino acid, a nonpolar amino acid or a derivative thereof each time it appears, provided that at least one X 2 is an acidic amino acid; Each Y 2 is independently a nonpolar amino acid or its derivative each time it appears; and m' is an integer selected from 2 to 8; and an anchoring 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 peptidomimetic, an aptamer, a sugar, or a small targeting molecule; or a combination thereof.

2. The peptide-based delivery agent of claim 1, further comprising an N-terminal group and a C-terminal group, and 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’ –[Anchoring / targeting group]–[C - terminal group]​​ Formula IA [N-terminal group]–[X 2 Y 2 Y 2 X 2 m –[Anchoring / targeting group]–[Cleavable linker]–[X 1 Y 1 Y 1 X 1 m’ –[C-terminal group]​​ Formula IB wherein the cleavable linker is an amino acid sequence having a length of 2 to 10 amino acids; if an anchoring group is present, it 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; if a targeting group is present, it is selected from a cell, an antibody or antibody fragment, a peptide, a peptidomimetic, an aptamer, a sugar, or a small targeting molecule; or a combination thereof; the C-terminal group comprises an amino-terminated glycine moiety; and the N-terminal group includes a capping group or a fluorophore.

3. The peptide-based delivery agent of claim 1 or claim 2, wherein each X 1 is independently glutamic acid, glutamine, arginine, alanine, aspartic acid or a derivative thereof each time it appears, provided that if X 1 is alanine, then at least one additional X 1 is glutamine, glutamic acid, aspartic acid, arginine or a derivative thereof, optionally wherein each X 1 is independently glutamic acid, aspartic acid or a derivative thereof each time it appears; further optionally wherein each X 1 is independently (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 solubilizing peptide sequence of DLLE or ELLD; or (iv) any combination of (i), (ii) and (iii).

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

5. A peptide-based delivery agent according to any one of claims 1 to 4, wherein each X 2 is, independently each time it appears, glutamine, glutamate, aspartic acid, alanine or a derivative thereof, provided that if X 2 is alanine, then at least one additional X 2 is glutamine, glutamate, aspartic acid or a derivative thereof.

6. The peptide-based delivery agent of any one of claims 1 to 5, wherein m is 3 and m' is 3; or m is 4 and m' is 4.

7. The peptide-based delivery agent of claim 1 or claim 2, wherein each X 1 is glutamic acid; each Y 1 and each Y 2 is leucine; each X 2 is glutamine or alanine; m is 3; and m' is 3; provided that if X 2 is alanine, then at least one additional X 2 is glutamine.

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

9. The peptide-based delivery agent of any one of claims 1 to 8, wherein the cleavable linker comprises an amino acid sequence that is at least 70% identical or 85% identical to SEQ ID NO:3, optionally wherein the cleavable linker consists of the amino acid sequence of SEQ ID NO:

3.

10. A peptide-based delivery agent according to any one of claims 1 to 10, wherein the anchoring group comprises a lysine moiety, wherein the side chain of the lysine moiety is functionalized with a heteroaliphatic group having the formula -C(O)-X-[CH 2 p Z, where X is oxygen or CH 2 ; Z is -N(R) 2 or -N + (R) 3 , where each R is independently hydrogen or aliphatic; and p is an integer from 1 to 3, optionally wherein (i) X is oxygen or CH 2 ; Z is -N(R) 2 , where one R group is hydrogen and the other R group is C 6 -C 12 alkyl; and p is 2; or (ii) X is oxygen or CH 2 ; Z is -N + (R) 3 , where two R groups are methyl and the other R group is C 6 -C 12 alkyl; and p is 2.​ 11. The peptide-based delivery agent of claim 10, wherein the anchoring group is selected from 12. The peptide-based delivery agent of any one of claims 1 to 11, wherein multiple anchoring groups are present, optionally wherein one, two, or three anchoring groups are present.

13. A peptide-based delivery agent according to any one of claims 1 to 12, wherein the solubilizing peptide group provides the N-terminal group of the peptide-based delivery agent, and the anchoring group provides the C-terminal group of the peptide-based delivery agent, optionally wherein (i) the N-terminal group is bound to a carbonyl-containing group, optionally wherein the carbonyl-containing group is acetyl or a fluorophore comprising 2-(methylamino)benzamide; and (ii) the C-terminal group is bound to an amine-terminated glycine moiety, a fluorophore, or a combination thereof, optionally wherein the amine-terminated glycine moiety has a structure selected from -Nal-K-G’, -G-Nal-K-G’, -G-K-G’, -Nal-C-G’, -G-Nal-C-G’, -G-C-G’, -Nal-G’, -G-Nal-G’, -W-G’, -G-W-G’, or -G-W-K-G’, wherein Nal is naphthylalanine, G is glycine, G’ is a modified glycine comprising -C(O)-amine, W is tryptophan, K is lysine, and C is cysteine, further optionally wherein the amine-terminated glycine moiety is -G-Nal-G’, wherein G’ is a modified glycine comprising -C(O)-N(R a ) 2 group, wherein each R a is independently hydrogen or aliphatic.

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

15. The peptide-based delivery agent of claim 1, having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO:272, 273, or 274, and wherein the 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. A composition comprising the peptide-based delivery agent of any one of claims 1 to 15; and A therapeutic agent selected from chemotherapeutic agents, morpholinos, therapeutic antibodies, immunotherapeutic agents, antibiotics, antidepressants, or combinations thereof, optionally wherein (i) the therapeutic agent is covalently or non-covalently bound to a peptide-based delivery agent; and / or (ii) the therapeutic agent is saponarin, cisplatin, methotrexate, fluorouracil, doxorubicin, cyclophosphamide, chlorambucil, vinblastine, vincristine, docetaxel, or paclitaxel, chlorhexidine, triclosan, xylitol, or octadec-1-ylamine hydrofluoride, hexadec-1-ylamine hydrofluoride, gefitinib, lapatinib, olaparib, mitomycin C, sunitinib, gefitinib, nintedanib, PD173074, erdafitinib, sorafenib, or combinations thereof; optionally wherein the composition further comprises an adjuvant, a carrier, a buffer, a detergent, or combinations thereof.

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

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

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

20. A method comprising contacting a cell in vitro or in vivo with the 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 the therapeutic agent to the cytosol of the cell.

21. The method of claim 20, wherein contacting the cell with the composition: induces endosomal membrane lysis after the therapeutic agent and the peptide-based delivery agent have been internalized into the cell; or induces pore formation in the endosomal membrane after the therapeutic agent and the peptide-based delivery agent have been internalized into the cell; or induces local disruption / destabilization of the endosomal membrane after the therapeutic agent and the peptide-based delivery agent have been internalized into the cell; thereby delivering the therapeutic agent into the cytosol of the cell; optionally wherein 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. The composition of any one of claims 16 to 19, for treating, ameliorating, or preventing the occurrence of a disorder or disease in a subject, the disorder or disease selected from genetic diseases, rare diseases, cancers, immune disorders, infectious diseases, mental disorders, substance use disorders, or any combination thereof.

23. A compound for use in the said use, wherein the subject is a human or non-human mammal, and wherein the composition is administered using a technique selected from injection, aerosol delivery, intranasal administration, oral administration, topical administration or a combination thereof, optionally wherein administering the composition comprises providing the therapeutic agent and the peptide-based delivery agent to the subject either simultaneously or at different times.

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

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

26. The method of claim 24 or claim 25, wherein the effect of the one or more compounds on the contacted cell comprises: a decrease in the viability of the contacted cell as compared to a control; an increase in the viability of the contacted cell as compared to a control; induction of a phenotype of interest in the contacted cell as compared to a control; an increase in the expression of one or more genes in the contacted cell as compared to a control; and / or a decrease in the expression of one or more genes in the contacted cell as compared to a control.

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

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