Peptide-based non-protein cargo delivery

Abstract

Described herein are methods, compositions, kits, and synthetic peptide shuttle agents related to transduction of protein and / or non-protein cargoes. The methods generally involve contacting a target eukaryotic cell with a non-protein cargo and a synthetic peptide shuttle agent at a concentration sufficient to increase the transduction efficiency of the non-protein cargo compared to the absence of the synthetic peptide shuttle agent. In embodiments, the non-protein cargo can be a drug, such as a small molecule drug for treating a disease. In other embodiments, novel synthetic peptide shuttle agents with transduction activity for protein and / or non-protein cargoes are described, as well as the use of propidium iodide or other membrane-impermeable fluorescent DNA intercalators as surrogate cargos to select for versatile synthetic peptide shuttle agents with transduction activity for both protein and non-protein cargoes.

Description

Technical Field

[0001] This disclosure relates to the intracellular delivery of non-protein cargos. More specifically, this disclosure relates to the use of synthetic peptide shuttle agents for the intracellular delivery of small molecules and other non-protein cargos, and to improved synthetic peptide shuttle agents having transduction activity for both proteins and small molecules.

[0002] This disclosure refers to a number of documents the content of which is incorporated herein by reference in its entirety.

Background Art

[0003] Most drugs are traditionally small molecule organic compounds and are small enough and lipophilic enough to pass through the cell membrane to engage intracellular targets. During conventional drug discovery processes, small molecule drug candidates are routinely selected based on their drug-like physicochemical properties that govern not only their affinity for biological targets but, especially, their ability to be delivered intracellularly and reach biological targets. Thus, under conventional drug discovery ideology, compounds identified in large-scale screening attempts as exhibiting high target binding affinity and specificity may ultimately be discarded as clinical drug candidates because of their reduced ability to be delivered intracellularly. Further, even cell membrane permeable compounds can benefit from improved intracellular / cytoplasmic delivery to, for example, increase uptake rates and / or reduce the concentration administered to obtain a desired biological effect. Accordingly, there is a need for technologies that can facilitate the intracellular / cytoplasmic delivery of small molecule cargos, provide greater flexibility in drug design, and perhaps open the door for the use of novel therapeutic compounds that otherwise may have been overlooked based on conventional small molecule drug design.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] [Non-Patent Document 1] Ilfeld and Yaksh (2009). “The End of Postoperative Pain - A Fast-Approaching Possibility? And, if So, Will We Be Ready?” Regional Anesthesia and Pain Medicine Vol. 34 (Issue 2): pp. 85-87 [Overview of the Initiative] [Means for solving the problem]

[0006] Synthetic peptide shuttles represent a recently defined family of peptides that have already been reported to rapidly and efficiently transduce protein cargoes into the cytoplasm and / or nucleus of a wide range of target eukaryotic cells. The first generation of such peptide shuttles was described in International Publication 2016 / 161516, in which the peptide shuttles comprise an endosomal leakage domain (ELD) manipulably linked to a cell membrane permeability domain (CPD). Subsequently, International Publication 2018 / 068135 further described synthetic peptide shuttles rationally designed based on a set of 15 design parameters with the aim of improving protein cargo transduction while reducing the toxicity of the first-generation peptide shuttles. This disclosure relates to the finding that such synthetic peptide shuttles, which have already been reported to transduce large protein cargoes, also generally have the ability to rapidly and efficiently transduce smaller non-protein cargoes (e.g., small molecule organic compounds). The experimental results shown in Example 2 demonstrate that synthetic peptide shuttles, including representative members of the shuttles described in International Publication No. 2016 / 161516 and International Publication No. 2018 / 068135, as well as additional rationally designed shuttles, can transduce propidium iodide (PI), a membrane-impermeable fluorescent dye that can be considered a small molecule organic compound cargo. Notably, negative control peptides that did not meet the key rational design parameters described in International Publication No. 2018 / 068135 for protein cargo delivery also did not transduce PI. This suggests that the rational design parameters of International Publication No. 2018 / 068135 for protein cargo delivery can also be generally applied to the design of peptide shuttles for non-protein cargo delivery. In Example 3, it is shown that representative synthetic peptide shuttles not only enable intracellular delivery of structurally unrelated small molecule inhibitors of the Hedgehog signaling pathway into cultured cells, but also that the delivered inhibitors freely bind to their intracellular targets and exert their inhibitory activity.Example 4 demonstrates that a representative synthetic peptide shuttle agent enables in vivo delivery and activity of a small molecule inhibitor of hedgehog signaling after topical application in shaved mice. Example 5 demonstrates that different representative synthetic peptide shuttle agents enable intracellular delivery of a membrane-impermeable small molecule compound, a sodium channel inhibitor (QX-314), resulting in a corresponding reduction in evoked current amplitude as measured by patch clamp. Finally, Examples 6 and 7 present the results of a large-scale screening of over 300 candidate peptide shuttle agents for PI and GFP-NLS transduction activity, showing a significant correlation between PI transduction efficiency and GFP-NLS transduction efficiency, suggesting that reliable PI transduction predicts shuttle agents with protein cargo transduction activity.

[0007] In some embodiments, this specification describes a method for transduction of a non-protein cargo, the method comprising contacting a target eukaryotic cell with a concentration of the synthetic peptide shuttle agent sufficient to increase the transduction efficiency of the non-protein cargo compared to the absence of the non-protein cargo and the synthetic peptide shuttle agent.

[0008] In some embodiments, this specification describes compositions for use in transducing a non-protein cargo into target eukaryotic cells, the compositions comprising a synthetic peptide shuttle formulation with pharmaceutically appropriate excipients, the concentration of the synthetic peptide shuttle in the composition being sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the non-protein cargo to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle.

[0009] In some embodiments, this specification describes compositions for therapeutic use, comprising a synthetic peptide shuttle agent formulated with a non-protein cargo (e.g., a therapeutically or biologically active non-protein cargo) to be transduced into target eukaryotic cells by the synthetic peptide shuttle agent, wherein the concentration of the synthetic peptide shuttle agent in the composition is sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the non-protein cargo to the target eukaryotic cells at administration compared to the absence of the synthetic peptide shuttle agent.

[0010] In some embodiments, this specification describes synthetic peptide shuttle agents having transduction activity for both protein and non-protein cargoes, wherein the shuttle agent comprises or consists of one amino acid sequence from Sequence ID No. 1 to 50. In some embodiments, this specification describes synthetic peptide shuttle agents having transduction activity for both protein and non-protein cargoes, wherein the shuttle agent is one of SEQ ID NOs: 1-50 and at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% It contains or consists of amino acid sequences that are 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (calculated by excluding any linker domains, such as flexible serine / glycine-rich linker domains).

[0011] In some embodiments, this specification describes synthetic peptide shuttle agents having transduction activity for both protein cargo and non-protein cargo in target eukaryotic cells, wherein the shuttle agent is (1) Having a length of at least 17, 18, 19, or 20 amino acids, (2) An amphiphilic alpha helix motif, (3) Positively charged hydrophilic outer surface and hydrophobic outer surface It is a peptide containing an amphiphilic alpha-helix motif having, The following parameters (4)~(15): (4) The hydrophobic outer surface includes a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids, corresponding to 12-50% of the peptide's amino acids, based on an open cylinder representation of an alpha helix with 3.6 residues per turn; (5) The peptide has a hydrophobic moment (μ) of 3.5 to 11; (6) The peptide has a predicted net charge of at least +4 at physiological pH; (7) The peptide has an isoelectric point (pI) of 8 to 13; (8) The peptide is composed of 35% to 65% of any combination of amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (9) The peptide is composed of 0% to 30% of any combination of amino acids: N, Q, S, and T; (10) The peptide consists of 35% to 85% of any combination of amino acids: A, L, K, or R; (11) A peptide is composed of any combination of amino acids A and L, with at least 5% L present in the peptide; (12) The peptide is composed of 20% to 45% of any combination of amino acids: K and R; (13) The peptide is composed of any combination of amino acids D and E, from 0% to 10%; (14) The difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R) is 10% or less; and (15) The peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H. At least five of the following conditions are met. The shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA inserts by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to the corresponding negative control lacking the shuttle agent, and / or in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in the target eukaryotic cells, at least 10%, 11%, 12%, 13% of propidium iodide or other membrane-impermeable fluorescent DNA inserts. This enables transduction efficiencies of 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% (for example, as determined by flow cytometry).

[0012] In some embodiments, this specification describes synthetic peptide shuttle agents having transduction activity for both protein cargo and non-protein cargo in target eukaryotic cells, wherein the shuttle agent is (a) SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 3 (b)(a) contains or consists of an amino acid sequence that differs from (a) only by conservative amino acid substitutions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer conservative amino acid substitutions, preferably excluding any linker domain such as a flexible serine / glycine-rich linker domain); and the shuttle agent is less than the corresponding negative control lacking the shuttle agent. In a eukaryotic cell line model (e.g., HeLa) suitable for increasing the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents, and / or for evaluating cargo transduction in the target eukaryotic cells, This enables transduction efficiencies of 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% (for example, as determined by flow cytometry).

[0013] In some embodiments, this specification describes synthetic peptide shuttle agents having protein cargo transduction activity in target eukaryotic cells, wherein the shuttle agent comprises or consists of (a) any one amino acid sequence of SEQ ID NOs. 52, 57, 79, 108, 140, 147, 148, 173, 241, 261, 286, 295, 301, 309, 312, 325, 333-337, or 343; or (b) an amino acid sequence that differs from (a) by only conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer conservative amino acid substitutions, preferably excluding any linker domain such as a flexible serine / glycine-rich linker domain); The Toll agent increases the transduction efficiency of GFP-NLS by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to the corresponding negative control lacking the shuttle agent, and / or enables transduction efficiency of at least 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% (as determined, for example, by flow cytometry) of GFP-NLS in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in the target eukaryotic cells.

[0014] In some embodiments, this specification describes synthetic peptide shuttle agent variants having transduction activity for protein cargo and / or non-protein cargo in target eukaryotic cells, wherein the synthetic peptide shuttle agent variant is identical to any one of the synthetic peptide shuttle agents defined herein, except that at least one amino acid is replaced by a corresponding synthetic amino acid having a side chain with similar physicochemical properties (e.g., structure, hydrophobicity, or charge) to the amino acid being replaced, and the shuttle agent variant increases the transduction efficiency of the cargo in target eukaryotic cells compared to the absence of the shuttle agent variant.

[0015] In some embodiments, the present specification describes in vitro or in vivo methods for the transduction of protein cargos and / or non-protein cargos, the method comprising contacting a target eukaryotic cell with a synthetic peptide shuttle agent or a synthetic peptide shuttle agent variant as defined herein at a concentration sufficient to increase the transduction efficiency of the cargo into the target eukaryotic cell as compared to the absence of the cargo and the synthetic peptide shuttle agent.

[0016] In some embodiments, the present specification describes a composition for use in therapy, the composition comprising a synthetic peptide shuttle agent or a synthetic peptide shuttle agent variant as defined herein formulated with a protein cargo and / or a non-protein cargo to be transduced into a target eukaryotic cell by the synthetic peptide shuttle agent, wherein the concentration of the synthetic peptide shuttle agent or the synthetic peptide shuttle agent variant in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the cargo to the target eukaryotic cell upon administration as compared to the absence of the synthetic peptide shuttle agent.

[0017] In some embodiments, the present specification describes a kit comprising a synthetic peptide shuttle agent or a synthetic peptide shuttle agent variant as defined herein, and a protein cargo and / or a non-protein cargo to be transduced by the synthetic peptide shuttle agent or the synthetic peptide shuttle agent variant.

[0018] In one aspect, the present specification describes a method for producing a candidate synthetic peptide shuttle agent predicted to have transduction activity for a cargo of interest in a target eukaryotic cell. The method includes the step of synthesizing a peptide that: (1) is at least 17, 18, 19, or 20 amino acids long; (2) is an amphipathic alpha-helical motif; (3) has a positively charged hydrophilic outer surface and a hydrophobic outer surface, and at least 5 of the parameters (4)-(15) defined herein are satisfied. The shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalating agents by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold compared to the corresponding negative control lacking the shuttle agent, and / or enables a transduction efficiency of at least 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% (e.g., when determined by flow cytometry) of propidium iodide or other membrane-impermeable fluorescent DNA intercalating agents in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in the target eukaryotic cell.

[0019] In some embodiments, this specification describes in vitro or in vivo methods for identifying, qualifying, or selecting synthetic peptide shuttles expected to have transduction activity for both protein and non-protein cargoes in target eukaryotic cells, the methods comprising: providing a model eukaryotic cell or model organism suitable for evaluating cargo transduction in target eukaryotic cells; providing a candidate synthetic peptide shuttle (e.g., as defined herein); and measuring the transduction activity (e.g., transduction efficiency, e.g., by flow cytometry) of the candidate synthetic peptide shuttle for transducing propidium iodide or other membrane-impermeable fluorescent DNA insertors into the model eukaryotic cell or model organism, wherein the candidate shuttle exhibits less transduction activity (e.g., transduction efficiency) of propidium iodide or other membrane-impermeable fluorescent DNA insertors than the corresponding negative control lacking the candidate synthetic peptide shuttle. If the amount increases by 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times, and / or if the amount of propidium iodide or other membrane-impermeable fluorescent DNA insertion agent increases by at least 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% If transduction efficiencies of %, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% (e.g., measured by flow cytometry) occur in model eukaryotic cells or model organisms, it is expected that the substance will have transduction activity for both protein and non-protein cargoes in target eukaryotic cells.

[0020] general definition Titles and other identifiers, such as (a), (b), (i), (ii), etc., are provided solely to facilitate the reading of this specification and the claims. The use of titles and other identifiers in this specification and the claims does not necessarily require that the processes or elements be carried out in alphabetical or numerical order, or in the order in which they are presented.

[0021] The use of the words “a” or “an” in the claims and / or specification may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.”

[0022] The term "approximately" is used to indicate that a value includes the standard deviation of the error of the device or method used to determine the value. Generally, the technical term "approximately" specifies a possible variation of up to 10%. Therefore, variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of the value are included within the term "approximately." Unless otherwise indicated, the use of the term "approximately" prior to a range applies to both ends of the range.

[0023] As used herein and in the claims, the words “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “includes” and “include”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are comprehensive or open-ended and do not preclude any further unlisted elements or process steps.

[0024] As used herein, “protein,” “polypeptide,” or “peptide” means any peptide-linked chain of amino acids, which may or may not include any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfation, SUMOylation, prenylation, ubiquitination, etc.). For further clarity, protein / polypeptide / peptide modifications are assumed as long as the modifications do not disrupt the cargo transduction activity of the shuttle agents described herein. For example, the shuttle agents described herein may be linear or cyclic, may be synthesized from one or more D- or L-amino acids, and / or may be conjugated to fatty acids (e.g., their N-terminus). The shuttle agents described herein may also have at least one amino acid which is replaced by a corresponding synthetic amino acid which has a side chain with similar physicochemical properties (e.g., structure, hydrophobicity, or charge) to the amino acid being replaced.

[0025] As used herein, “domain” or “protein domain” generally refers to a portion of a protein having a specific functional group or function. Some domains preserve their function when isolated from the rest of the protein and can therefore be used modularly. The modular nature of many protein domains can provide flexibility through their arrangement within the shuttle agent described herein. However, some domains may function better when genetically engineered at specific locations within the shuttle agent (e.g., in or between the N-terminal and C-terminal regions). The location of a domain within its endogenous protein may also serve as an indicator of where the domain should be genetically engineered within the shuttle agent and what type / length of linker should be used. Standard recombinant DNA techniques may be used by those skilled in the art to manipulate the arrangement and / or number of domains within the shuttle agent described herein in consideration of this disclosure. Furthermore, assays disclosed herein and others known in the art may be used to evaluate the functionality of each domain in terms of the shuttle agent (e.g., its ability to facilitate cell penetration across the cell membrane, endosomal escape, and / or access to the cytoplasm). Furthermore, standard methods can be used to evaluate whether the domains of the shuttle agent affect the activity of the cargo to be delivered into the cell. In this context, the expression “operatably linked,” as used herein, refers to the ability of a domain to perform its intended function (e.g., cell penetration, endosomal escape, and / or intracellular targeting) in the context of the shuttle agent described herein. For clarity, the expression “operatably linked” means defining a functional linkage between two or more domains, not limited to a specific order or distance between them.

[0026] As used herein, the term “synthetic” in expressions such as “synthetic peptide,” “synthetic peptide shuttle,” or “synthetic polypeptide” is intended to refer to molecules that do not exist in nature and can be produced in vitro (e.g., chemically synthesized and / or produced using recombinant DNA technology). The purity of various synthetic preparations can be evaluated, for example, by high-performance liquid chromatography analysis and mass spectrometry. Chemical synthesis approaches may be advantageous over cell expression systems (e.g., yeast or bacterial protein expression systems) because they can eliminate the need for large-scale recombinant protein purification steps (e.g., required for clinical use). In contrast, longer synthetic polypeptides may be more complex and / or more expensive to produce by chemical synthesis approaches, and such polypeptides may be more advantageously produced using cell expression systems. In some embodiments, the peptides or shuttles described herein may be chemically synthesized (e.g., solid-phase or liquid-phase peptide synthesis) as opposed to being expressed from recombinant host cells. In some embodiments, the peptides or shuttles described herein may lack an N-terminal methionine residue. Those skilled in the art can adapt the synthetic peptides or shuttle agents described herein to meet specific stability requirements or other needs by using one or more modified amino acids (e.g., amino acids that do not occur naturally) or by chemically modifying the synthetic peptides or shuttle agents described herein.

[0027] As used herein, the term “independent” is intended to generally refer to molecules or agents that are not covalently bonded to each other. For example, the expression “independent cargo” is intended to refer to a cargo delivered into a cell (transduced) that is not covalently bonded (e.g., not fused) to the shuttle agent described herein. In some embodiments, having a shuttle agent that is independent of (and not fused to) the cargo may be advantageous by increasing the versatility of the shuttle agent, for example, by allowing the ratio of the shuttle agent to the cargo to be easily varied (in contrast to the case of covalent bonding between the shuttle agent and the cargo, where the ratio is limited to a fixed ratio).

[0028] As used herein, the expressions “is or derived from” or “derived from” include conservative amino acid substitutions, deletions, modifications, and functional variants of a given protein domain (e.g., CPD or ELD) that do not inhibit the activity of the protein domain.

[0029] Other purposes, advantages, and features described herein will become more apparent upon reading the following non-restrictive description of the particular embodiments given for illustrative purposes only, with reference to the accompanying drawings. [Brief explanation of the drawing]

[0030] [Figure 1A]This figure shows the delivery and survival results of HeLa cells co-incubated for 1 minute with various categories of synthetic peptide shuttles combined with non-protein cargo (propidium iodide, PI; Figures 1A and 1B) or protein cargo (GFP-NLS protein; Figures 1C and 1D). Results were obtained by flow cytometry 2 hours after cargo delivery and are expressed as the percentage of fluorescent cells (%PI+ cells or %GFP+ cells). Categories of peptides shown (from left to right): Synthetic peptide shuttles containing an endosomal leakage domain (ELD) operably linked to a cell membrane permeability domain (CPD) as described in International Publication 2016 / 161516, etc.; rationally designed synthetic peptide shuttles as described in International Publication 2018 / 068135; additional rationally designed synthetic peptide shuttles as described herein; cyclic peptides as described herein; and negative control peptides that do not adhere to some rational design parameters as described in International Publication 2018 / 068135. In Figure 1A, "FS, then PI" indicates that PI was added 1 hour after treatment with the synthetic peptide shuttle agent to ensure that the PI-positive signal was not due to cell death. "Negative control" refers to cells incubated with cargo alone ("PI" in Figures 1A and 1B, or "GFP-NLS" in Figures 1C and 1D), or untreated cells not exposed to cargo or the peptide shuttle agent ("NT," Figures 1A-1D). [Figure 1B]This figure shows the delivery and survival results of HeLa cells co-incubated for 1 minute with various categories of synthetic peptide shuttles combined with non-protein cargo (propidium iodide, PI; Figures 1A and 1B) or protein cargo (GFP-NLS protein; Figures 1C and 1D). Results were obtained by flow cytometry 2 hours after cargo delivery and are expressed as the percentage of fluorescent cells (%PI+ cells or %GFP+ cells). Categories of peptides shown (from left to right): Synthetic peptide shuttles containing an endosomal leakage domain (ELD) operably linked to a cell membrane permeability domain (CPD) as described in International Publication 2016 / 161516, etc.; rationally designed synthetic peptide shuttles as described in International Publication 2018 / 068135; additional rationally designed synthetic peptide shuttles as described herein; cyclic peptides as described herein; and negative control peptides that do not adhere to some rational design parameters as described in International Publication 2018 / 068135. "Negative controls" are cells incubated with the cargo alone ("PI" in Figures 1A and 1B, or "GFP-NLS" in Figures 1C and 1D), or untreated cells that were not exposed to the cargo or peptide shuttle agent ("NT" in Figures 1A-1D). [Figure 1C]This figure shows the delivery and survival results of HeLa cells co-incubated for 1 minute with various categories of synthetic peptide shuttles combined with non-protein cargo (propidium iodide, PI; Figures 1A and 1B) or protein cargo (GFP-NLS protein; Figures 1C and 1D). Results were obtained by flow cytometry 2 hours after cargo delivery and are expressed as the percentage of fluorescent cells (%PI+ cells or %GFP+ cells). Categories of peptides shown (from left to right): Synthetic peptide shuttles containing an endosomal leakage domain (ELD) operably linked to a cell membrane permeability domain (CPD) as described in International Publication 2016 / 161516, etc.; rationally designed synthetic peptide shuttles as described in International Publication 2018 / 068135; additional rationally designed synthetic peptide shuttles as described herein; cyclic peptides as described herein; and negative control peptides that do not adhere to some rational design parameters as described in International Publication 2018 / 068135. "Negative controls" are cells incubated with the cargo alone ("PI" in Figures 1A and 1B, or "GFP-NLS" in Figures 1C and 1D), or untreated cells that were not exposed to the cargo or peptide shuttle agent ("NT" in Figures 1A-1D). [Figure 1D]This figure shows the delivery and survival results of HeLa cells co-incubated for 1 minute with various categories of synthetic peptide shuttles combined with non-protein cargo (propidium iodide, PI; Figures 1A and 1B) or protein cargo (GFP-NLS protein; Figures 1C and 1D). Results were obtained by flow cytometry 2 hours after cargo delivery and are expressed as the percentage of fluorescent cells (%PI+ cells or %GFP+ cells). Categories of peptides shown (from left to right): Synthetic peptide shuttles containing an endosomal leakage domain (ELD) operably linked to a cell membrane permeability domain (CPD) as described in International Publication 2016 / 161516, etc.; rationally designed synthetic peptide shuttles as described in International Publication 2018 / 068135; additional rationally designed synthetic peptide shuttles as described herein; cyclic peptides as described herein; and negative control peptides that do not adhere to some rational design parameters as described in International Publication 2018 / 068135. "Negative controls" are cells incubated with the cargo alone ("PI" in Figures 1A and 1B, or "GFP-NLS" in Figures 1C and 1D), or untreated cells that were not exposed to the cargo or peptide shuttle agent ("NT" in Figures 1A-1D). [Figure 2] This table summarizes the results shown in Figure 1A-1D. [Figure 3] This figure shows the activity of small molecule inhibitors of hedgehog signaling (Gant61, HPI-4, itraconazole, or ATO) transduced into NIH3T3 Gli-luciferase reporter cells using the peptide shuttle agent FSD250D. Successful small molecule transduction in the presence of the peptide shuttle agent ("+FSD250D"; SEQ ID NO: 36) resulted in reduced fluorescence intensity in NIH3T3 Gli-luciferase reporter cells stimulated with recombinant mouse sonic hedgehog protein (+mShh) compared to the absence of the peptide shuttle agent ("-FSD250D"). [Figure 4]This figure shows the successful in vivo transduction of small molecule inhibitors of Hedgehog signaling (Gant61 and itraconazole) into dermal cells of shaved mice using the peptide shuttle agent FSD250D. Hair loss in mouse skin induces hair growth associated with potent induction of the Hedgehog pathway. This experiment consisted of activating the Hedgehog pathway in mice by hair loss, and then measuring the delay of hair regrowth by delivering dermal cell small molecule Hedgehog pathway inhibitors (Gant61 or itraconazole) that bind to intracellular targets. The results show that mice treated with the small molecule hedgehog inhibitor Gant61 or itraconazole ("FSD250D + Gant61 100 μM" and "FSD250D + itraconazole 100 μM") in the presence of FSD250D showed delayed hair regrowth 10 days (*) after treatment, compared to mice in the absence of FSD250D ("Gant61 100 μM" and "itraconazole 100 μM") or in the presence of shuttle peptide alone ("FSD250D"). [Figure 5A] Figures 5A-5C show representative patch-clamp electrophysiological whole-cell current traces of HEK293 cells stably expressing sodium channel Nav1.7 upon exposure to the membrane-impermeable sodium channel inhibitor QX-314 with or without FSD194. When cells were transiently exposed to QX-314 and GFP-NLS in the presence of FSD194 (i.e., 1 mM QX-314 + 15 μM GFP-NLS + 5 μM FSD194), a reduction in current amplitude was observed, consistent with the presence of QX-314 in the cells (Figure 5C). This same reduction in current amplitude was not observed in the absence of QX-314 (i.e., 15 μM GFP-NLS + 5 μM FSD194 +; Figure 2A) or FSD194 (i.e., 2.5 mM QX-314 + 15 μM GFP-NLS; Figure 2B). Furthermore, GFP-NLS-positive cells were identified under the QX-314+GFP-NLS+FSD194 and FSD194+GFP-NLS conditions, but not under the QX-314+GFP-NLS condition, indicating that GFP-NLS was indeed co-transduced with QX-314 by the peptide shuttle agent. [Figure 5B] Figures 5A-5C show representative patch-clamp electrophysiological whole-cell current traces of HEK293 cells stably expressing sodium channel Nav1.7 upon exposure to the membrane-impermeable sodium channel inhibitor QX-314 with or without FSD194. When cells were transiently exposed to QX-314 and GFP-NLS in the presence of FSD194 (i.e., 1 mM QX-314 + 15 μM GFP-NLS + 5 μM FSD194), a reduction in current amplitude was observed, consistent with the presence of QX-314 in the cells (Figure 5C). This same reduction in current amplitude was not observed in the absence of QX-314 (i.e., 15 μM GFP-NLS + 5 μM FSD194 +; Figure 2A) or FSD194 (i.e., 2.5 mM QX-314 + 15 μM GFP-NLS; Figure 2B). Furthermore, GFP-NLS-positive cells were identified under the QX-314+GFP-NLS+FSD194 and FSD194+GFP-NLS conditions, but not under the QX-314+GFP-NLS condition, indicating that GFP-NLS was indeed co-transduced with QX-314 by the peptide shuttle agent. [Figure 5C]Figures 5A-5C show representative patch-clamp electrophysiological whole-cell current traces of HEK293 cells stably expressing sodium channel Nav1.7 upon exposure to the membrane-impermeable sodium channel inhibitor QX-314 with or without FSD194. When cells were transiently exposed to QX-314 and GFP-NLS in the presence of FSD194 (i.e., 1 mM QX-314 + 15 μM GFP-NLS + 5 μM FSD194), a reduction in current amplitude was observed, consistent with the presence of QX-314 in the cells (Figure 5C). This same reduction in current amplitude was not observed in the absence of QX-314 (i.e., 15 μM GFP-NLS + 5 μM FSD194 +; Figure 2A) or FSD194 (i.e., 2.5 mM QX-314 + 15 μM GFP-NLS; Figure 2B). Furthermore, GFP-NLS-positive cells were identified under the QX-314+GFP-NLS+FSD194 and FSD194+GFP-NLS conditions, but not under the QX-314+GFP-NLS condition, indicating that GFP-NLS was indeed co-transduced with QX-314 by the peptide shuttle agent. [Figure 6-1] Figure 6 shows the results of a large-scale screening of over 300 candidate peptide shuttles for PI and GFP-NLS transduction activity. Figure 6 shows the results for all screened candidate peptide shuttles with an average PI transduction efficiency of 10% or higher, selected based on the level of average PI transduction efficiency. [Figure 6-2] Continuation of Figure 6. [Figure 6-3] Continuation of Figure 6. [Figure 6-4] Continuation of Figure 6. [Figure 6-5] Continuation of Figure 6. [Figure 6-6] Continuation of Figure 6. [Figure 6-7] Continuation of Figure 6. [Figure 6-8] Continuation of Figure 6. [Figure 7]This figure shows the results of a large-scale screening of over 300 candidate peptide shuttles for PI and GFP-NLS transduction activity. Figure 7 shows the results for all screened candidate peptide shuttles with an average PI transduction efficiency of less than 10% and an average GFP-NLS transduction efficiency of at least 7%, selected based on the level of average GFP-NLS transduction efficiency. [Modes for carrying out the invention]

[0031] Sequence List This application includes a computer-readable sequence listing prepared on April 15, 2020, having a size of approximately 122 kb. The computer-readable format is incorporated herein by reference.

[0032] [Table 1A]

[0033] [Table 1B]

[0034] [Table 1C]

[0035] Detailed explanation In some embodiments, this specification describes methods for transduction of non-protein cargoes and / or protein cargoes. These methods generally involve contacting target eukaryotic cells with non-protein cargoes and / or protein cargoes, as well as with a concentration of the synthetic peptide shuttle agent sufficient to increase the transduction efficiency of the cargoes, compared to the absence of the synthetic peptide shuttle agent. This specification also describes versatile synthetic peptide shuttle agents having dual transduction activity for both protein and non-protein cargoes, and the use of PI or other membrane-impermeable fluorescent DNA insertion agents as "surrogate" cargoes for selecting such dual transduction activity synthetic peptide shuttle agents.

[0036] Non-protein cargo In some embodiments, the non-protein cargo may be a compound (e.g., an organic compound) having a molecular weight of 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or less than 1,000 Da. In some embodiments, the non-protein cargo may be a compound (e.g., an organic compound) having a molecular weight of 50-5,000, 50-4,000, 50-3,000, 50-2,000, or 50-1,000 Da. In some embodiments, the non-protein cargo may be a small molecule, such as a small molecule drug that binds to a biological or therapeutic target within a cell. In some embodiments, the non-protein cargo may not be a biomacromolecule such as a polynucleotide or polysaccharide, and in particular, not a biomacromolecule having a uniform negative charge, such as a polynucleotide with a length exceeding 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides. In some embodiments, the non-protein cargo may have a cationic net charge in aqueous solution. In some embodiments, the non-protein cargo is not covalently bonded to the synthetic peptide shuttle agent (e.g., during transduction) (i.e., is independent of it).

[0037] In some embodiments, the non-protein cargo may be impermeable to the cell membrane or have low membrane permeability (e.g., due to the physicochemical properties of the cargo, it cannot freely diffuse across the cell membrane), and the peptide shuttle agents described herein facilitate or increase its intracellular delivery and / or access to the cytoplasm. In some embodiments, the non-protein cargo may be permeable to the cell membrane, and the peptide shuttle agents described herein nevertheless increase its intracellular delivery and / or access to the cytoplasm. In some embodiments, the peptide shuttle agents described herein can reduce the amount or concentration of cargo required to be administered to achieve its intended biological effect compared to the administration of the cargo alone.

[0038] In some embodiments, the transduced non-protein cargo may be a drug for treating any disease or condition having an intracellular biological or therapeutic target. In some embodiments, the non-protein cargo may be a drug for treating cancer (e.g., skin cancer, basal cell carcinoma, nevus basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye disease, atrophic or exudative age-related macular degeneration, finger ulcers, actinic keratosis, idiopathic pulmonary fibrosis), pain (e.g., chronic or acute), or diseases affecting the lungs (e.g., cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), or idiopathic pulmonary fibrosis).

[0039] In certain embodiments, the non-protein cargo to be transduced may be or include hedgehog inhibitors (e.g., itraconazole, posaconazole, arsenic trioxide (ATO), Gant61, PF-4708671, HPI-1, HPI-4). In certain embodiments, the non-protein cargo to be transduced may be or include pain inhibitors such as voltage-gated sodium (Nav) channel inhibitors (e.g., QX-314). In certain embodiments, the non-protein cargo to be transduced may be inflammation inhibitors such as inhibitors of pathways leading to the production of inflammatory cytokines (e.g., NF-κB pathway inhibitors).

[0040] In some embodiments, the shuttle agents described herein may have the ability to transduce both non-protein cargo and protein cargo into the cytoplasm of target eukaryotic cells.

[0041] Rational design parameters and peptide shuttle agents In some embodiments, the shuttle agents described herein may be peptides having transduction activity to protein cargo, non-protein cargo, or both protein and non-protein cargo in target eukaryotic cells. In some embodiments, the shuttle agents described herein preferably satisfy one or more of the following 15 rational design parameters.

[0042] (1) In some embodiments, the shuttle agent is a peptide having a length of at least 17, 18, 19, or 20 amino acids. For example, the peptide may have a minimum length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues and a maximum length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues. In some embodiments, shorter peptides (e.g., in the range of 17–50 or 20–50 amino acids) may be particularly advantageous because they can be more easily synthesized and purified by chemical synthesis methods and are more suitable for clinical use (in contrast to recombinant proteins that must be purified from cell expression systems). While the numbers and ranges described herein are often listed as multiples of 5, this description is not to be limited in that way. For example, the maximum length described herein includes lengths such as 56, 57, 58…61, 62, etc., and it should be understood that the non-listing of such lengths is simply for the sake of brevity. The same reasoning applies to the percentage of identity described herein.

[0043] (2) In some embodiments, the peptide shuttle agent comprises an amphiphilic alpha-helix motif. As used herein, the expression “alpha-helix motif” or “alpha-helix” means, unless otherwise specified, a right-handed coiled or spiral structure (helical body) having an alpha-helix with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn. As used herein, the expression “contains an alpha-helix motif” or “amphiphilic alpha-helix motif,” etc., means the three-dimensional structure that the peptide (or peptide segment) described herein is expected to adopt in a biological setting based on the peptide primary amino acid sequence, regardless of whether the peptide actually adopts it when used in cells as a shuttle agent. Furthermore, the peptide described herein may comprise one or more alpha-helix motifs at various peptide positions. For example, the shuttle agent FSD5 in International Publication No. 2018 / 068135 is expected to take the form of an alpha helix over its entire length (see Figure 49C in International Publication No. 2018 / 068135), while the shuttle agent FSD18 in International Publication No. 2018 / 068135 is expected to contain two separate alpha helices directed toward the N and C-terminal regions of the peptide (see Figure 49D in International Publication No. 2018 / 068135). In some embodiments, the shuttle agents described herein are not expected to contain beta sheet motifs, such as those shown in Figures 49E and 49F of International Publication No. 2018 / 068135. Methods for predicting the presence of alpha helices and beta sheets in proteins and peptides are well known in the art. For example, one such method is based on 3D modeling using PEP-FOLD®, an online resource for predicting novel peptide structures (http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD / ) (Lamiable et al., 2016; Shen et al., 2014; Thevenet et al., 2012). Other methods for predicting the presence of alpha helices in peptides and proteins are known and readily available to those skilled in the art.

[0044] As used herein, the expression “amphiphilic” means a peptide having both hydrophobic and hydrophilic elements (for example, based on the side chains of the amino acids that make up the peptide). For example, the expression “amphiphilic alpha-helix” or “amphiphilic alpha-helix motif” means a peptide that is expected to take an alpha-helix motif having a nonpolar hydrophobic surface and a polar hydrophilic surface, based on the properties of the side chains of the amino acids that form the helix.

[0045] (3) In some embodiments, the peptide shuttle agents described herein include an amphiphilic alpha-helix motif having a positively charged hydrophilic outer surface, such as one rich in R and / or K residues. As used herein, the expression “positively charged hydrophilic outer surface” means the presence of at least three lysine (K) and / or arginine (R) residues clustered on one side of the amphiphilic alpha-helix motif, based on an alpha-helix wheel projection (see, for example, the left panel of Figure 49A in International Publication No. 2018 / 068135). Such helical wheel projections can be prepared using various programs, such as the online helical wheel projection tool available at http: / / rzlab.ucr.edu / scripts / wheel / wheel.cgi. In some embodiments, the amphiphilic alpha-helix motif includes a positively charged hydrophilic outer surface which includes (a) at least two, three, or four adjacent positively charged K and / or R residues; and / or (b) a segment of six adjacent residues on a helical wheel projection, based on an alpha-helix having a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn, with K and / or R residues included in each segment.

[0046] In some embodiments, the peptide shuttle agent described herein comprises an amphiphilic alpha-helix motif including a hydrophobic outer surface, the hydrophobic outer surface comprising (a) at least two adjacent L residues on a helical wheel projection; and / or (b) a segment of 10 adjacent residues on a helical wheel projection, based on an alpha-helix having a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn, comprising at least five hydrophobic residues selected from L, I, F, V, W, and M.

[0047] (4) In some embodiments, the peptide shuttle agents described herein include an amphiphilic alpha-helix motif having a highly hydrophobic core composed of spatially adjacent highly hydrophobic residues (e.g., L, I, F, V, W, and / or M). In some embodiments, for example, as shown in Figure 49A, right panel of International Publication No. 2018 / 068135, the highly hydrophobic core may consist of L, I, F, V, W, and / or M amino acids that are spatially adjacent and, after calculation excluding histidine-rich domains (see below), correspond to 12-50% of the amino acids of the peptide. In some embodiments, the highly hydrophobic core may consist of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% to 25%, 30%, 35%, 40%, or 45% of the peptide's amino acids. More specifically, the highly hydrophobic core parameter may be calculated by first arranging the peptide's amino acids in an open cylinder representation, and then depicting regions of consecutive highly hydrophobic residues (L, I, F, V, W, M) as shown in Figure 49A, right panel, of International Publication No. 2018 / 068135. The number of highly hydrophobic residues in this depicted highly hydrophobic core is then divided by the total amino acid length of the peptide excluding histidine-rich domains (e.g., N and / or C-terminal histidine-rich domains). For example, in the case of the peptide shown in Figure 49A of International Publication No. 2018 / 068135, there are 8 residues in the depicted highly hydrophobic core and a total of 25 residues in the peptide (excluding the 12 terminal histidines). Therefore, the highly hydrophobic core is 32% (8 / 25).

[0048] (5) The hydrophobic moment is a measure of the amphiphilicity of a helix, peptide, or part thereof, calculated from the vector sum of the hydrophobicity of the side chains of amino acids (Eisenberg et al., 1982). An online tool for calculating the hydrophobic moment of a polypeptide is available at http: / / rzlab.ucr.edu / scripts / wheel / wheel.cgi. A high hydrophobic moment indicates strong amphiphilicity, while a low hydrophobic moment indicates insufficient amphiphilicity. In some embodiments, the peptide shuttle agents described herein may consist of or include peptides or alpha-helix domains having a hydrophobic moment of 3.5 to 11 (μ). In some embodiments, the shuttle agent is 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, The peptide may contain an amphiphilic alpha-helix motif having a hydrophobic moment between the lower limits of 6.7, 6.8, 6.9, and 7.0 and the upper limits of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0. In some embodiments, the shuttle agent may be a peptide having a hydrophobic moment between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5. In some embodiments, the hydrophobic moment is calculated excluding all histidine-rich domains that may be present in the peptide.

[0049] (6) In some embodiments, the peptide shuttle agents described herein may have a predicted effective charge of at least +4 at physiological pH, calculated from the side chains K, R, D, and E. For example, the effective charge of the peptide may be at least +5, +6, +7, at least +8, at least +9, at least +10, at least +11, at least +12, at least +13, at least +14, or at least +15 at physiological pH. These positive charges are generally conferred by the presence of more positively charged lysine and / or arginine residues, in contrast to negatively charged aspartic acid and / or glutamic acid residues.

[0050] (7) In some embodiments, the peptide shuttle agents described herein may have a predicted isoelectric point (pI) of 8 to 13, preferably 10 to 13. Programs and methods for calculating and / or measuring the isoelectric point of peptides or proteins are known in the art. For example, the pI can be calculated using Prot Param software, available at http: / / web.expasy.org / protparam / .

[0051] (8) In some embodiments, the peptide shuttle agents described herein may consist of 35-65% hydrophobic residues (A, C, G, I, L, M, F, P, W, Y, V). In certain embodiments, the peptide shuttle agents may consist of 36-64%, 37-63%, 38-62%, 39-61%, and 40-60% of any combination of amino acids: A, C, G, I, L, M, F, P, W, Y, and V.

[0052] (9) In some embodiments, the peptide shuttle agents described herein may consist of 0-30% neutral hydrophilic residues (N, Q, S, T). In certain embodiments, the peptide shuttle agents may consist of any combination of amino acids: N, Q, S, and T in amounts of 1%-29%, 2%-28%, 3%-27%, 4%-26%, 5%-25%, 6%-24%, 7%-23%, 8%-22%, 9%-21%, or 10%-20%.

[0053] (10) In some embodiments, the peptide shuttle agent described herein may consist of 35-85% amino acids: A, L, K and / or R. In certain embodiments, the peptide shuttle agent may consist of any combination of amino acids: A, L, K, or R in 36-80%, 37-75%, 38-70%, 39-65%, or 40-60%.

[0054] (11) In some embodiments, the peptide shuttle agent described herein may consist of 15-45% amino acids A and / or L, provided that at least 5% L is present in the peptide. In certain embodiments, the peptide shuttle agent may consist of any combination of amino acids A and L in 15-40%, 20-40%, 20-35%, or 20-30%, provided that at least 5% L is present in the peptide.

[0055] (12) In some embodiments, the peptide shuttle agent described herein may consist of 20-45% amino acids:K and / or R. In certain embodiments, the peptide shuttle agent may consist of any combination of 20-40%, 20-35%, or 20-30% amino acids:K and R.

[0056] (13) In some embodiments, the peptide shuttle agent described herein may consist of 0-10% amino acids D and / or E. In certain embodiments, the peptide shuttle agent may consist of any combination of 5-10% amino acids D and E.

[0057] (14) In some embodiments, the absolute difference between the percentage of A and / or L in the peptide shuttle and the percentage of K and / or R may be 10% or less. In certain embodiments, the absolute difference between the percentage of A and / or L in the peptide shuttle and the percentage of K and / or R may be 9%, 8%, 7%, 6%, or 5% or less.

[0058] (15) In some embodiments, the peptide shuttle agents described herein may consist of 10% to 45% of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, or H (i.e., other than A, L, K, or R). In certain embodiments, the peptide shuttle agents may consist of 15% to 40%, 20% to 35%, or 20% to 30% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[0059] In some embodiments, the peptide shuttle agents described herein satisfy at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or all of the parameters (1) to (15) described herein. In certain embodiments, the peptide shuttle agents described herein satisfy all of the parameters (1) to (3) and also satisfy at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all of the parameters (4) to (15) described herein.

[0060] In some embodiments, the peptide shuttle agent described herein contains only one histidine-rich domain, and the residues of the single histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein. In some embodiments, if the peptide shuttle agent described herein contains two or more histidine-rich domains, only the residues of the single histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein. For example, if the peptide shuttle agent described herein contains two histidine-rich domains, a first histidine-rich domain facing the N terminus and a second histidine-rich domain facing the C terminus, only the first histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein.

[0061] In some embodiments, machine learning or computer-aided design techniques may be used to generate peptides that satisfy one or more of the parameters (1) to (15) described herein. Some parameters, such as parameters (1) and (5) to (15), are more readily implemented by computer-aided design techniques, while structural parameters, such as parameters (2), (3), and (4), are more readily applicable by manual design techniques. Therefore, in some embodiments, peptides that satisfy one or more parameters (1) to (15) may be generated by combining computer-aided and manual design techniques. For example, multiple sequence alignment analyses of several peptides shown herein (or elsewhere) to function as effective shuttle agents have revealed the presence of several consensus sequences—i.e., patterns of commonly recognized alternations of hydrophobic, cationic, and hydrophilic alanine and glycine amino acids. The presence of these consensus sequences may induce the satisfying of structural parameters (2), (3), and (4) (i.e., amphiphilic alpha-helix formation, positively charged surfaces, and a highly hydrophobic core of 12% to 50%). Therefore, these and other consensus sequences can be employed in machine learning and / or computer-aided design methods to generate peptides that satisfy one or more parameters (1) to (15).

[0062] Therefore, in some embodiments, the peptide shuttle agents described herein may include or consist of the following amino acid sequences: (a)[X1]-[X2]-[Linker]-[X3]-[X4](Equation 1); (b)[X1]-[X2]-[Linker]-[X4]-[X3](Equation 2); (c)[X2]-[X1]-[Linker]-[X3]-[X4](Equation 3); (d)[X2]-[X1]-[Linker]-[X4]-[X3](Equation 4); (e)[X3]-[X4]-[Linker]-[X1]-[X2](Equation 5); (f)[X3]-[X4]-[Linker]-[X2]-[X1](Equation 6); (g) [X4] - [X3] - [Linker] - [X1] - [X2] (Formula 7); or (h) [X4] - [X3] - [Linker] - [X2] - [X1] (Formula 8); wherein, [X1] is selected from the following: 2[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; 2[Φ] - 1[+] - 2[Φ] - 2[+] -; 1[+] - 1[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; and 1[+] - 1[Φ] - 1[+] - 2[Φ] - 2[+] -; [X2] is selected from the following: -2[Φ] - 1[+] - 2[Φ] - 2[ζ] -; -2[Φ] - 1[+] - 2[Φ] - 2[+] -; -2[Φ] - 1[+] - 2[Φ] - 1[+] - 1[ζ] -; -2[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; -2[Φ] - 2[+] - 1[Φ] - 2[+] -; -2[Φ] - 2[+] - 1[Φ] - 2[ζ] -; -2[Φ] - 2[+] - 1[Φ] - 1[+] - 1[ζ] -; and 2[Φ] - 2[+] - 1[Φ] - 1[ζ] - 1[+] -; [X3] is selected from the following: -4[+] - A -; -3[+] - G - A -; -3[+] - A - A -; -2[+] - 1[Φ] - 1[+] - A -; -2[+] - 1[Φ] - G - A -; -2[+] - 1[Φ] - A - A -; or 2[+] - A - 1[+] - A; -2[+] - A - G - A; -2[+] - A - A - A; -1[Φ] - 3[+] - A -; -1[Φ] - 2[+] - G - A -; -1[Φ] - 2[+] - A - A -; -1[Φ] - 1[+] - 1[Φ] - 1[+] - A; -1[Φ] - 1[+] - 1[Φ] - G - A; -1[Φ] - 1[+] - 1[Φ] - A - A; -1[Φ] - 1[+] - A - 1[+] - A; -1[Φ] - 1[+] - A - G - A; -1[Φ] - 1[+] - A - A - A; -A - 1[+] - A - 1[+] - A; -A - 1[+] - A - G - A; and A - 1[+] - A - A - A; [X4] is selected from the following: -1[ζ]-2A-1[+]-A;-1[ζ]-2A-2[+];-1[+]-2A-1[+]-A;-1[ζ]-2A-1[+]-1[ζ]-A-1[+];-1[ζ]-A-1[ζ]-A-1[+];-2[+]-A-2[+];-2[+]-A-1[+]-A;-2 [+]-A-1[+]-1[ζ]-A-1[+];-2[+]-1[ζ]-A-1[+];-1[+]-1[ζ]-A-1[+]-A;-1[+]-1[ ζ]-A-2[+];-1[+]-1[ζ]-A-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-A-1[+];-1[+]-2[ζ]-2 [+];-1[+]-2[ζ]-1[+]-A;-1[+]-2[ζ]-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-1[ζ]-A-1 [+];-3[ζ]-2[+];-3[ζ]-1[+]-A;-3[ζ]-1[+]-1[ζ]-A-1[+];-1[ζ]-2A-1[+]-A;-1[ ζ]-2A-2[+];-1[ζ]-2A-1[+]-1[ζ]-A-1[+];-2[+]-A-1[+]-A;-2[+]-1[ζ]-1[+]-A;-1[+]-1[ζ]-A-1[+]-A;-1[+]-2A-1[+]-1[ζ]-A-1[+]; and 1[ζ]-A-1[ζ]-A-1[+]; and The linker is selected from the following: -Gn-; -Sn-; -(GnSn)n-; -(GnSn)nGn-; -(GnSn)nSn-; -(GnSn)nGn(GnSn)n-; and (GnSn)nSn(GnSn)n-; In the formula, [Φ] is an amino acid: Leu, Phe, Trp, Ile, Met, Tyr, or Val, preferably Leu, Phe, Trp, or Ile; [+] is an amino acid: Lys or Arg; [ζ] is an amino acid: Gln, Asn, Thr, or Ser; A is an amino acid: Ala; G is an amino acid: Gly; S is an amino acid: Ser; n is an integer between 1 and 20, 1 and 19, 1 and 18, 1 and 17, 1 and 16, 1 and 15, 1 and 14, or 1 and 3.

[0063] In some embodiments, the peptide shuttle agents described herein are any amino acid sequences of SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, or 344, or SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152 disclosed in International Publication No. 2018 / 068135. A peptide that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one amino acid sequence from 169 to 242 and 243 to 10242, or a functional variant thereof. In some embodiments, the peptide shuttle agents described herein may include amino acid sequence motifs of SEQ ID NO: 158 and / or 159 of International Publication No. 2018 / 068135, as found in the peptides FSD5, FSD16, FSD18, FSD19, FSD20, FSD22, and FSD23. In some embodiments, the peptide shuttle agents described herein may include amino acid sequence motifs of SEQ ID NO: 158 of International Publication No. 2018 / 068135, linked to the amino acid sequence motif of SEQ ID NO: 159 of International Publication No. 2018 / 068135 in a functional manner. As used herein, “functional variant” refers to a peptide having cargo transduction activity that differs from the reference peptide by one or more conserved amino acid substitutions. As used herein in the context of a functional variant, “conserved amino acid substitution” means that one amino acid residue is replaced by another amino acid residue having a similar side chain.The family of amino acid residues having similar side chains is well defined in the art and includes basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and possibly proline), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0064] In some embodiments, the peptide shuttle agents described herein do not include one or more of the amino acid sequences of SEQ ID NOs. 57-59, 66-72, or 82-102 of International Publication No. 2018 / 068135. In some embodiments, the peptide shuttle agents described herein do not include one or more of the amino acid sequences of SEQ ID NOs. 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242 disclosed in International Publication No. 2018 / 068135. Rather, in some embodiments, the peptide shuttle agents described herein may relate to variants of such previously described shuttle agent peptides, which are further manipulated for improved dual transduction activity (i.e., more reliably transduction of protein and non-protein cargoes).

[0065] In some embodiments, the peptide shuttle agents described herein may have a minimum threshold for transduction efficiency and / or cargo delivery score for “surrogate” cargo when measured in a eukaryotic cell model system (e.g., immortalized eukaryotic cell lines) or model organism. The term “transduction efficiency” refers to the percentage or ratio of target cell populations to which the cargo of interest is delivered intracellularly, which can be determined, for example, by flow cytometry, immunofluorescence microscopy, and other suitable methods that can be used to evaluate cargo transduction efficiency (e.g., described in International Publication No. 2018 / 068135). In some embodiments, transduction efficiency may be expressed as a percentage of cargo-positive cells. In some embodiments, transduction efficiency may be expressed as a doubling (or doubling) against a suitable negative control evaluated under the same conditions, except in the absence of cargo and shuttle agent ("untreated"; NT) or in the absence of shuttle agent ("cargo alone").

[0066] As used herein, the term “surrogate cargo” refers to any protein or non-protein cargo that can be transduced by a shuttle agent having known cargo transduction activity, and whose intracellular delivery and endosomal escape (i.e., cytoplasmic and / or nuclear delivery) levels can be readily measured and / or tracked (e.g., via fluorescence or functional assays), and the surrogate cargo is intended to evaluate the suitability of a given shuttle agent for transducing a cargo of a different purpose than the surrogate cargo (e.g., a protein or non-protein cargo such as a therapeutically active cargo that binds to an intracellular target). Examples of suitable surrogate cargoes include fluorescent cargoes (e.g., PI or other membrane-impermeable fluorescent DNA insertors, GFP, GFP-NLS or other fluorescent proteins, fluorescent dextran, etc.). Non-protein cargoes such as PI or other membrane-impermeable fluorescent DNA insertors may be particularly advantageous because they are relatively inexpensive and their fluorescence is enhanced only after binding to genomic DNA. This property is particularly suitable for distinguishing endosome-trapped cargo from endosome-escaped cargo (i.e., cargo access to the cytoplasmic / nuclear compartment). Where used herein, any suitable model system (e.g., immortalized cell lines, ex vivo cells, model organisms) can be used to evaluate the transduction activity of shuttle agents against surrogate cargoes. Conveniently, eukaryotic cell line models can be selected as suitable model systems, and the cell line is chosen to be beneficial for evaluating transduction activity in the target eukaryotic cells to which the transduction ultimately occurs. Indeed, several cell cultures and model organisms are commercially available as model systems for studying various diseases.

[0067] In some embodiments, the peptide shuttle agents described herein increase the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA inserts in a suitable eukaryotic cell model system (e.g., HeLa or other suitable immortalized cell lines). In some embodiments, the peptide shuttle agents described herein increase the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA inserts in HeLa cells or other suitable eukaryotic cell line models to evaluate cargo transduction in target eukaryotic cells of interest by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold compared to a corresponding negative control lacking the shuttle agent ("cargo alone"). In some embodiments, the peptide shuttle agent described herein is used in HeLa cells or other suitable eukaryotic cell line models to evaluate cargo transduction in target eukaryotic cells of interest, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 3% This enables transduction efficiencies of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents at 3%, 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% (as determined, for example, by flow cytometry) for propidium iodide or other membrane-impermeable fluorescent DNA insertion agents.

[0068] In some embodiments, the peptide shuttle agents described herein increase the transduction efficiency of GFP-NLS or other suitable protein substitute cargo in a suitable eukaryotic cell model system (e.g., HeLa or other suitable immortalized cell lines). In some embodiments, the peptide shuttle agents described herein increase the transduction efficiency of GFP-NLS or other suitable protein substitute cargo in HeLa cells or other suitable eukaryotic cell line models to evaluate cargo transduction in target eukaryotic cells of interest by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times compared to the corresponding negative control lacking the shuttle agent ("cargo alone"). In some embodiments, the peptide shuttle agent described herein is used in HeLa cells or other suitable eukaryotic cell line models to evaluate cargo transduction in target eukaryotic cells of interest, with concentrations of at least 10%, 11%, 12%, 13%, 14%, 15%, 14%, 16%, 17%, 16%, 17%, 18%, 19%, 20%, 21%, 21%, 22%, 21%, 22%, 23%, 25%, 26%, 26%, and 28%. This enables transduction efficiencies of GFP-NLS or other suitable substitute protein cargoes of %, 29%, 30%, 29%, 32%, 32%, 33%, 33%, 36%, 36%, 39%, 39%, 39%, 39%, 41%, 42%, 44%, 44%, 45%, 47%, 49%, 51%, 52%, 53%, 55%, 55%, 58%, 59%, or 60% (for example, as determined by flow cytometry).

[0069] In some embodiments, the peptide shuttle agents described herein may include or consist of the shuttle agents listed in Figure 6, having an average PI transduction efficiency of at least 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%. In some embodiments, the peptide shuttle agent described herein is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 3 The shuttle agents listed in Figure 6, having a standardized mean PI delivery score of 3, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, or 60, include or consist of the shuttle agents listed in Figure 6.

[0070] In some embodiments, the peptide shuttle agent described herein contains at least 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%, and 36%. , comprising or comprising the shuttle agents listed in Figure 6, having an average GFP-NLS transduction efficiency of 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%. In some embodiments, the peptide shuttle agent described herein contains at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5 ,20,20.5,21,21.5,22,22.5,23,23.5,24,24.5,25,25.5,26,26.5,27,27.5,28,28.5,29,29.5,30,30.5,31,31.5,32,32.5,33,33.5,34,34.5,35,35.5,36,36.5,37,37.5,38,38.5,39,39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 6 The shuttle agents listed in Figure 6, having standardized mean GFP-NLS delivery scores of 0, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200, may include or consist of the shuttle agents listed in Figure 6.

[0071] In some embodiments, the peptide shuttle agents described herein have an average GFP-NLS transduction efficiency of at least 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%, or at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, The shuttle agents listed in Figure 7, having standardized mean GFP-NLS delivery scores of 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, or 30, may include or consist of the shuttle agents listed in Figure 7.

[0072] In some embodiments, the shuttle agents described herein may include shuttle agent variants that differ from the shuttle agents defined herein, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or less. Preferably, linker domains (e.g., flexible serine / glycine-rich linker domains) are excluded from consideration of different amino acids because the length and / or amino acid composition of the linker domain can vary significantly without affecting transduction activity. In some embodiments, the peptide shuttle agents described herein may contain or consist of an amino acid sequence different from any of the shuttle agents described herein by only conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer conservative amino acid substitutions, preferably excluding any linker domain), and the shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold compared to the corresponding negative control lacking the shuttle agent; and / or for evaluating cargo transduction in the target eukaryotic cells. In a suitable eukaryotic cell line model (e.g., HeLa), at least 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%, This enables transduction efficiencies of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents of 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% (as determined, for example, by flow cytometry) for propidium iodide or other membrane-impermeable fluorescent DNA insertion agents. In some embodiments, each conserved amino acid substitution is selected from amino acids within the same amino acid class, which are: aliphatic: G, A, V, L, and I; hydroxyl or sulfur / selenium-containing: S, C, U, T, and M; aromatic: F, Y, and W; basic: H, K, and R; acidic and their amides: D, E, N, and Q.

[0073] Chemically modified and synthetic amino acids In some embodiments, the shuttle agent described herein may comprise an oligomer (e.g., a dimer, trimer, etc.) of the peptide described herein. Such oligomers can be constructed by covalently bonding the same or different types of shuttle agent monomers (e.g., by linking cysteine ​​residues introduced into the monomer sequence using disulfide crosslinks). In some embodiments, the shuttle agent described herein may comprise N-terminal and / or C-terminal cysteine ​​residues.

[0074] In some embodiments, the shuttle agent described herein may include or consist of a cyclic peptide. In some embodiments, the cyclic peptide may be formed via a covalent bond between a first residue positioned toward the N-terminus of the shuttle agent and a second residue positioned toward the C-terminus of the shuttle agent. In some embodiments, the first and second residues are adjacent residues located at the N-terminus and C-terminus of the shuttle agent. In some embodiments, the first and second residues may be linked via an amide bond to form a cyclic peptide. In some embodiments, the cyclic peptide may be formed by a disulfide bond between two cysteine ​​residues in the shuttle agent, with the two cysteine ​​residues positioned toward the N-terminus and C-terminus of the shuttle agent. In some embodiments, the shuttle agent may include, or be manipulated to include, cysteine ​​residues adjacent to the N-terminus and C-terminus, linked via a disulfide bond to form a cyclic peptide. In some embodiments, the cyclic shuttle agents described herein may be more resistant to degradation (e.g., by proteases) and / or may have a longer half-life than the corresponding linear peptide.

[0075] In some embodiments, the shuttle agent described herein may comprise one or more D-amino acids. In some embodiments, the shuttle agent described herein may comprise D-amino acids at the N-terminus and / or C-terminus of the shuttle agent. In some embodiments, the shuttle agent may consist solely of D-amino acids. In some embodiments, the shuttle agent described herein having one or more D-amino acids may be resistant to degradation (e.g., by proteases) and / or may have a longer half-life than the corresponding peptide composed solely of L-amino acids.

[0076] In some embodiments, the shuttle agents described herein may include chemical modifications to one or more amino acids, and the chemical modifications do not destroy the transduction activity of the synthetic peptide shuttle agent. As used herein, the term “destroy” means that the chemical modification irreversibly destroys the cargo transduction activity of the peptide shuttle agent described herein. Chemical modifications that can temporarily inhibit, attenuate, or delay the cargo transduction activity of the peptide shuttle agent described herein may be included in the chemical modifications to the shuttle agents described herein. In some embodiments, the chemical modification to any one of the shuttle agents described herein may be at the N-terminus and / or C-terminus of the shuttle agent. Examples of chemical modifications include the addition of an acetyl group (e.g., an N-terminal acetyl group), a cysteamide group (e.g., a C-terminal cysteamide group), or a fatty acid (e.g., a C4-C16, C6-C14, C6-C12, C6-C8, or C8 fatty acid, preferably at the N-terminus).

[0077] In some embodiments, the shuttle agent described herein comprises a shuttle agent variant having transduction activity to protein cargo and / or non-protein cargo in target eukaryotic cells, the variant being identical to any of the shuttle agents described herein except that at least one amino acid is replaced with a corresponding synthetic amino acid or amino acid analog having a side chain with similar physicochemical properties (e.g., structure, hydrophobicity, or charge) to the amino acid being replaced. In some embodiments, the replacement of the synthetic amino acid is (a) Replace a basic amino acid with any one of the following: α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, 2,6-diamino-4-hexic acid, β-(1-piperazinyl)-alanine, 4,5-dehydroxylysine, δ-hydroxylysine, ω,ω-dimethylarginine, homoarginine, ω,ω'-dimethylarginine, ω-methylarginine, β-(2-quinolyl)-alanine, 4-aminopiperidine-4-carboxylic acid, α-methylhistidine, 2,5-diiodohistidine, 1-methylhistidine, 3-methylhistidine, spinacine, 4-aminophenylalanine, 3-aminotyrosine, β-(2-pyridyl)-alanine, or β-(3-pyridyl)-alanine; (b) Dehydroalanine, β-Fluoroalanine, β-Chloroalanine, β-Iodoalanine, α-Aminobutyric Acid, α-Aminisobutyric Acid, β-Cyclopropylalanine, Azethidine-2-carboxylic Acid, α-Allylglycine, Propargylglycine, tert-Butylalanine, β-(2-Thiazolyl)-Alanine, Thiaproline, 3,4-Dehydroproline, tert-Butylglycine, β-Cyclopentylalanine, β-Cyclohexylalanine, α-Me Tylproline, norvaline, α-methylvaline, penicillamine, β,β-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, pipecolic acid, 4,5-dehydroleucine, alloisoleucine, norleucine, α-methylleucine, cyclohexylglycine, cis-octahydroindole-2-carboxylic acid, β-(2-thienyl)-alanine, phenylglycine, α-methylphenylalanine, homophenylalanine Replace a nonpolar (hydrophobic) amino acid with one of the following: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-(3-benzothienyl)-alanine, 4-nitrophenylalanine, 4-bromophenylalanine, 4-tert-butylphenylalanine, α-methyltryptophan, β-(2-naphthyl)-alanine, β-(1-naphthyl)-alanine, 4-iodophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, 4-methyltryptophan, 4-chlorophenylalanine, 3,4-dichlorophenylalanine, 2,6-difluorophenylalanine, n-in-methyltryptophan, 1,2,3,4-tetrahydronorharmann-3-carboxylic acid, β,β-diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, or 4-benzoylphenylalanine; (c)β-Cyanolalanine, β-Ureidoalanine, Homocysteine, Allothreonine, Pyroglutamic Acid, 2-Oxothiazolidine-4-carboxylic Acid, Citrulline, Thiocitrulline, Homocitrulline, Hydroxyproline, 3,4-Dihydroxyphenylalanine, β-(1,2,4-Triazole-1-yl)alanine, 2-Mercaptohistidine, β-(3,4-Dihydroxyphenyl)-serine, β-(2-Thienyl)-serine, 4-Azidophenylalanine, 4-Cyanofenyl Replace a polar uncharged amino acid with any one of the following: rualanine, 3-hydroxymethyltyrosine, 3-iodotyrosine, 3-nitrotyrosine, 3,5-dinitrotyrosine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 5-hydroxytryptophan, tyronine, β-(7-methoxycoumarin-4-yl)alanine, or 4-(7-hydroxy-4-coumarinyl)-aminobutyric acid; and / or (d) Replace the acidic amino acid with one of the following: γ-hydroxyglutamic acid, γ-methyleneglutamic acid, γ-carboxyglutamic acid, α-aminoadipic acid, 2-aminoheptanedioic acid, α-aminosuberic acid, 4-carboxyphenylalanine, cysteic acid, 4-phosphonophenylalanine, or 4-sulfomethylphenylalanine.

[0078] Histidine-rich domain In some embodiments, the peptide shuttle described herein may further comprise one or more histidine-rich domains. In some embodiments, the histidine-rich domain may be a stretch of at least two, at least three, at least four, at least five, or at least six amino acids, comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues. In some embodiments, the histidine-rich domain may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive histidine residues. While not constrained by theory, the histidine-rich domain in the shuttle agent may act as a proton sponge in endosomes by protonating its imidazole group under acidic endosome conditions, providing another mechanism for endosomal membrane destabilization and thus further enhancing the ability of endosome-trapped cargo to reach the cytosol. In some embodiments, the histidine-rich domain may be located at the N and / or C terminus of the peptide shuttle agent or may be oriented toward the N and / or C terminus.

[0079] Linker In some embodiments, the peptide shuttle agents described herein may comprise one or more suitable linkers (e.g., flexible polypeptide linkers). In some embodiments, such linkers may detach two or more amphiphilic alpha-helix motifs (see, for example, shuttle agent FSD18 in Figure 49D of International Publication No. 2018 / 068135). In some embodiments, the linkers may be used to separate two additional domains (CPD, ELD, or histidine-rich domains) from each other. In some embodiments, the linkers may be formed by adding a sequence of small hydrophobic amino acids (e.g., glycine) that do not have rotational ability and polar serine residues that confer stability and mobility. The linkers may be soft and allow the domains of the shuttle agent to move. In some embodiments, proline may be avoided as it may add significant steric rigidity. In some embodiments, the linkers may be serine / glycine-rich linkers (e.g., GS, GGS, GGSGGGS). (Sequence ID 345) , GGSGGGSGGGS (Sequence ID 346) (etc.) In some embodiments, the use of a shuttle agent containing a suitable linker may be advantageous for delivering cargo to suspension cells rather than adherent cells. In some embodiments, the linker includes or consists of -Gn-;-Sn-;-(GnSn)n-;-(GnSn)nGn-;-(GnSn)nSn-;-(GnSn)nGn(GnSn)n-; or (GnSn)nSn(GnSn)n-, where G is the amino acid Gly; S is the amino acid Ser; and n is an integer from 1 to 5.

[0080] Domain-based peptide shuttle agents In some embodiments, the shuttle agent described herein may be the shuttle agent described in International Publication No. 2016 / 161516, and comprises an endosomal leakage domain (ELD) operably linked to a cell membrane permeability domain (CPD).

[0081] Endosome leakage domain (ELD) In some embodiments, the peptide shuttle agents described herein may include an endosomal leakage domain (ELD) to facilitate endosomal escape and access to the cytoplasmic compartment. As used herein, the expression “endosomal leakage domain” refers to a sequence of amino acids that confers the ability of cargo captured by endosomes to reach the cytoplasmic compartment. While not theoretically bound, endosomal leakage domains are short sequences (often derived from viral or bacterial peptides) that are thought to induce destabilization of the endosomal membrane and release of endosomal contents into the cytoplasm. As used herein, the expression “endosomal soluble peptide” refers to this general class of peptides having endosomal membrane destabilizing properties. Accordingly, in some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may include an ELD that is an endosomal soluble peptide. The activity of such peptides can be evaluated, for example, using the calcein endosomal escape assay described in Example 2 of International Publication No. 2016 / 161516.

[0082] In some embodiments, ELDs can be peptides that disrupt membranes at acidic pH, such as pH-dependent membrane-active peptides (PMAPs) or pH-dependent soluble peptides. For example, peptides GALA and INF-7 are amphiphilic peptides that form alpha helices when a decrease in pH modifies the charge of the amino acids they contain. While not bound by theory, more specifically, it is suggested that ELDs such as GALA induce endosomal leakage by forming membrane lipid pores and flip-flops after conformational changes due to pH reduction (Kakudo, Chaki et al., 2004, Li, Nicol et al., 2004). In contrast, it has been suggested that ELDs such as INF-7 induce endosomal leakage by accumulating in the endosomal membrane and destabilizing it (El-Sayed, Futaki et al., 2009). Therefore, a simultaneous decrease in pH during endosomal maturation causes changes in peptide structure, which destabilizes the endosomal membrane and leads to the release of endosomal contents. The same principle is thought to apply to Pseudomonas toxin A (Varkouhi, Scholte et al., 2011). After a decrease in pH, the structure of the toxin's translocation domain changes, allowing its insertion into the pore-forming endosomal membrane (London 1992, O'Keefe 1992). This ultimately leads to endosomal destabilization and the movement of the complex to the outside of the endosome. The above ELD is encompassed within the ELD described herein and other mechanisms of endosomal leakage whose mechanisms of action are not yet fully understood.

[0083] In some embodiments, ELDs may be antimicrobial peptides (AMPs), such as linear cationic alpha-helix antimicrobial peptides (AMPs). These peptides play a crucial role in the innate immune response due to their ability to strongly interact with bacterial membranes. While not theoretically bound, these peptides are thought to exist in a disordered state in aqueous solutions but adopt an alpha-helix secondary structure in hydrophobic environments. The latter structure is thought to contribute to their typical concentration-dependent membrane disruption properties. When accumulated in endosomes at specific concentrations, some antimicrobial peptides can induce endosomal leakage.

[0084] In some embodiments, the ELD may be an antimicrobial peptide (AMP), such as the cecropine-A / melittin hybrid (CM) peptide. Such peptides are considered to be among the smallest and most effective AMP-derived peptides with membrane-disrupting ability. Cecropine is a family of antimicrobial peptides that have membrane-disrupting ability against both Gram-positive and Gram-negative bacteria. Cecropine A (CA), the first identified antimicrobial peptide, consists of 37 amino acids with a linear structure. Melittin (M), a 26-amino acid peptide, is a cell membrane-lytic factor found in bee venom. Cecropine-melittin hybrid peptides have been shown to produce short, efficient antibiotic peptides that are non-cytotoxic to eukaryotic cells (i.e., non-hemolytic), a desirable property for any antimicrobial agent. These chimeric peptides have been constructed from various combinations of the hydrophilic N-terminal domain of cecropine A and the hydrophobic N-terminal domain of melittin and have been tested in bacterial model systems. Two 26-mers, CA(1-13)M(1-13) and CA(1-8)M(1-18) (Boman et al., 1989), have been shown to demonstrate a broader, improved potency of natural secropin A without the cytotoxic effects of melittin.

[0085] In an attempt to produce shorter CM series peptides, Andreu et al., authors of 1992, constructed hybrid peptides such as 26-mer(CA,1-8)M,1-18 and compared them with 20-mer(CA,1-8)M,1-12, 18-mer(CA,1-8)M,1-10, and six 15-mer(CA(1-7)M(1-8), CA(1-7)M(2-9), CA(1-7)M(3-10), CA(1-7)M(4-11), CA(1-7)M(5-12), and CA,1-7)M,6-13. The 20-mer and 18-mer maintained similar activity compared to CA(1-8)M(1-18). Among the 15 meridians of the 6 types, CA(1-7)M(1-8) showed low antibacterial activity, while the other 5 types showed similar antibiotic efficacy compared to the 26 meridians that did not exhibit hemolytic effects. Therefore, in some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may include CM series peptide variants such as those mentioned above, or ELDs derived therefrom.

[0086] In some embodiments, ELD is melittin (YGRKKRRQRRR (Sequence No. 348) Secropin-A (KWKLFKKIGAVLKVLTTG) is fused with residues 2-12 of ) (Sequence ID 347) The CM series peptide CM18, composed of residues 1-7 of [C(1-7)M(2-12)]CM18, when fused with the cell membrane-permeable peptide TAT, independently crosses the cell membrane, destabilizes the endosomal membrane, and allows cargo captured by some endosomes to be released into the cytosol (Salomone et al., 2012). However, the use of the CM18-TAT11 peptide fused with the fluorophore (atto-633) in some of the authors' experiments increased uncertainty about the peptide's contribution to the fluorophore, as the use of the fluorophore itself was found to contribute, for example, to endosomal lysis by photochemical disruption of the endosomal membrane (Erazo-Oliveras et al., 2014).

[0087] In some embodiments, ELD may be a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identity with respect to SEQ ID NO: 1 of International Publication No. 2016 / 16516, and possessing endosomal lytic activity.

[0088] In some embodiments, ELD may be a peptide derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA), which can cause endosomal membrane destabilization when accumulated in endosomes.

[0089] In some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may include ELDs shown in Table I, or ELDs derived therefrom, or variants thereof having endosomal escape activity and / or pH-dependent membrane disruption activity.

[0090] [Table 2]

[0091] In some embodiments, the shuttle agent described herein may comprise one or more ELDs or types of ELDs. More specifically, they may comprise at least two, at least three, at least four, at least five, or more ELDs. In some embodiments, the shuttle agent may comprise 1 to 10 types of ELDs, 1 to 9 types of ELDs, 1 to 8 types of ELDs, 1 to 7 types of ELDs, 1 to 6 types of ELDs, 1 to 5 types of ELDs, 1 to 4 types of ELDs, 1 to 3 types of ELDs, etc.

[0092] In some embodiments, the order or arrangement of ELDs relative to other domains (CPDs, histidine-rich domains) in the shuttle agent described herein may be changed as long as the shuttle agent's reversible transport capability is maintained.

[0093] In some embodiments, the ELD is any one variant or fragment listed in Table I and may have endosomal lytic activity. In some embodiments, the ELD may include, or consist of, any one amino acid sequence of any one of Sequence IDs 1-15, 63, or 64 of International Publication No. 2016 / 16516 or any one of Sequence IDs 1-15, 63, or 64 of International Publication No. 2016 / 16516, and may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to any one of Sequence IDs 1-15, 63, or 64 of International Publication No. 2016 / 16516 and having endosomal lytic activity.

[0094] In some embodiments, the shuttle agents described herein do not contain one or more amino acid sequences from any one of the sequence numbers 1-15, 63, or 64 of International Publication No. 2016 / 16516.

[0095] Cell permeation domain (CPD) In some embodiments, the shuttle agents described herein may include a cell penetration domain (CPD). As used herein, the term “cell penetration domain” means a sequence of amino acids that confers the ability of a CPD-containing polymer (e.g., a peptide or protein) to be transduced into a cell.

[0096] In some embodiments, the CPD may be (or may be derived from) a cell membrane-permeable peptide or a protein transduction domain of a cell membrane-permeable peptide. Cell membrane-permeable peptides can function as carriers for the successful delivery of various cargoes (e.g., polynucleotides, polypeptides, small molecule compounds, or other macromolecules / compounds that are otherwise membrane-impermeable) into cells. Cell membrane-permeable peptides are often rich in basic amino acids and, when fused with (or rather operably linked to) macromolecules, contain short peptides that mediate internal translocation into cells (Shaw, Catchpole et al., 2008). The first cell membrane-permeable peptide was identified by analyzing the cell penetration ability of the transcriptional HIV-1 transactivator (Tat) protein (Green and Loewenstein 1988, Vives, Brodin et al., 1997). This protein contains a short hydrophilic amino acid sequence named "TAT" that facilitates its insertion into the cell membrane and pore formation. Since this discovery, numerous other cell membrane-permeable peptides have been reported. In this regard, in some embodiments, the CPD may be a cell membrane-permeable peptide or a variant thereof having cell membrane-permeable activity, as described in Table II.

[0097] [Table 3]

[0098] While not bound by theory, cell membrane-permeable peptides are thought to interact with the cell membrane and then pass through the membrane via pinocytosis or endocytosis. In the case of TAT peptides, their hydrophilicity and charge are thought to promote insertion into the cell membrane and pore formation (Herce and Garcia 2007). Alpha-helix motifs (e.g., SP) within hydrophobic peptides are also thought to form pores within the cell membrane (Veach, Liu et al., 2004).

[0099] In some embodiments, the shuttle agents described herein may comprise one or more CPDs or types of CPDs. More specifically, they may comprise at least two, at least three, at least four, or at least five or more CPDs. In some embodiments, the shuttle agents may comprise CPDs between 1 and 10, CPDs between 1 and 6, CPDs between 1 and 5, CPDs between 1 and 4, CPDs between 1 and 3, and so on.

[0100] In some embodiments, CPD is a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity with respect to SEQ ID NO: 17 of International Publication No. 2016 / 16516, and having cell membrane permeable activity, or an international publication Penetratin having the amino acid sequence of Sequence ID No. 18 of International Publication No. 2016 / 16516, or a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity with Sequence ID No. 18 of International Publication No. 2016 / 16516 and possessing cell membrane permeable activity.

[0101] In some embodiments, CPD may be PTD4 having the amino acid sequence of SEQ ID NO: 65 of International Publication No. 2016 / 16516, or a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity with SEQ ID NO: 65 of International Publication No. 2016 / 16516.

[0102] In some embodiments, the order or arrangement of CPD relative to other domains (ELD, histidine-rich domain) in the shuttle agent described herein may be changed as long as the reversal transduction ability of the shuttle agent is maintained.

[0103] In some embodiments, the CPD is any one variant or fragment listed in Table II and may have cell membrane permeable activity. In some embodiments, the CPD may include or consist of any amino acid sequence of any one of Sequence IDs 16-27 or 65 of International Publication No. 2016 / 16516 or any one of Sequence IDs 16-27 or 65 of International Publication No. 2016 / 16516 that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to any one of Sequence IDs 16-27 or 65 of International Publication No. 2016 / 16516 and has cell membrane permeable activity.

[0104] In some embodiments, the shuttle agents described herein do not contain one or more amino acid sequences from SEQ ID NOs. 16-27 or 65 of International Publication No. 2016 / 16516.

[0105] Methods, kits, uses, compositions, and cells In some embodiments, this description relates to a method for delivering protein cargo and / or non-protein cargo from the extracellular space of a target eukaryotic cell to the cytoplasm and / or nucleus. The method includes the step of contacting the target eukaryotic cell with the cargo in the presence of a shuttle agent at a concentration sufficient to increase the transduction efficiency of the cargo compared to the absence of the shuttle agent. In some embodiments, contact between the target eukaryotic cell and the cargo in the presence of the shuttle agent results in an increase of at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold in the transduction efficiency of the non-protein cargo compared to the absence of the shuttle agent.

[0106] In some embodiments, this description relates to methods for increasing the transduction efficiency of protein cargo and / or non-protein cargo into the cytoplasm and / or nucleus of target eukaryotic cells. As used herein, the expression “increased transduction efficiency” refers to the ability of the shuttle agent described herein to improve the percentage or ratio of the target cell population to which the cargo of interest (e.g., non-protein cargo) is delivered into the cell. Cargo transduction efficiency can be evaluated using immunofluorescence microscopy, flow cytometry, and other suitable methods. In some embodiments, the shuttle agent described herein can enable transduction efficiencies of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, as measured, for example, by immunofluorescence microscopy, flow cytometry, FACS, and other suitable methods. In some embodiments, the shuttle agents described herein may enable one of the aforementioned transduction efficiencies, along with cell viability of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, as measured, for example, by the assay described in Example 3.3a of International Publication No. 2018 / 068135, or by another suitable assay known in the art.

[0107] In addition to increasing the efficiency of target cell transduction, the shuttle agents described herein may facilitate the delivery of the cargo of interest (e.g., protein cargo and / or non-protein cargo) to the cytoplasm and / or nucleus of target cells. In this regard, efficiently delivering extracellular cargo to the cytoplasm and / or nucleus of target cells using peptides can be difficult. This is because cargo often becomes trapped in intracellular endosomes after crossing the cell membrane, which can limit its intracellular availability and lead to its eventual metabolic degradation. For example, the use of protein transduction domains derived from the HIV-1 Tat protein has been reported to result in the large-scale sequestration of cargo into intracellular vesicles. In some embodiments, the shuttle agents described herein may facilitate the ability of endosome-trapped cargo to escape from the endosome and gain access to the cytoplasmic compartment. In this regard, for example, the expression "to the cytoplasm" in the phrase "increases the transduction efficiency of non-protein cargo into the cytoplasm" is intended to refer to the ability of the shuttle agent described herein to enable the target cargo, once delivered into the cell, to escape from endosomal confinement and gain access to the cytoplasmic and / or nuclear compartments. After the target cargo has gained access to the cytoplasm, it can freely bind to intracellular targets (e.g., the nucleus, nucleolus, mitochondria, peroxisomes). In some embodiments, therefore, the expression "to the cytoplasm" is intended to include not only delivery to the cytoplasm but also delivery to other intracellular compartments where the cargo first needs to gain access to the cytoplasmic compartment.

[0108] In some embodiments, the methods described herein are in vitro methods (e.g., for therapeutic and / or diagnostic purposes). In other embodiments, the methods described herein are in vivo methods (e.g., for therapeutic and / or diagnostic purposes). In some embodiments, the methods described herein involve topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of non-protein cargo and synthetic peptide shuttle agents. In some embodiments, compositions formulated for topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of non-protein cargo and synthetic peptide shuttle agents are described herein.

[0109] In some embodiments, the method described herein may include the step of contacting target eukaryotic cells with a shuttle agent or composition as defined herein, and protein cargo and / or non-protein cargo. In some embodiments, the shuttle agent or composition may be pre-incubated with the cargo to form a mixture before the target eukaryotic cells are exposed to the mixture. In some embodiments, the type of shuttle agent may be selected based on the identity and / or physicochemical properties of the cargo to be delivered into the cells. In other embodiments, the type of shuttle agent may be selected to take into account the identity and / or physicochemical properties of the cargo to be delivered into the cells, the type of cell, the type of tissue, etc.

[0110] In some embodiments, the method may involve multiple treatments of target cells with the shuttle agent or composition (e.g., one, two, three, four or more times per day and / or according to a predetermined schedule). In such cases, lower concentrations of the shuttle agent or composition may be desirable (e.g., to reduce toxicity). In some embodiments, the cells may be suspension cells or adherent cells. In some embodiments, those skilled in the art may adapt the teachings described herein to suit specific needs for delivering protein cargo and / or non-protein cargo to specific cells with desired viability, using different combinations of shuttles, domains, uses, and methods.

[0111] In some embodiments, the methods described herein can be applied to methods for delivering protein cargo and / or non-protein cargo into cells in vivo. Such methods can be achieved by parenteral administration or direct injection into tissues, organs, or systems.

[0112] In some embodiments, the synthetic peptide shuttle agents described herein can be used in vitro or in vivo methods to increase the transduction efficiency of protein cargoes and / or non-protein cargoes (e.g., therapeutically or biologically active protein cargoes and / or non-protein cargoes) into target eukaryotic cells, and the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant may be used or formulated for use at concentrations sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the cargoes into target eukaryotic cells compared to the absence of the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant.

[0113] In some embodiments, the synthetic peptide shuttle agents described herein can be used therapeutically, and the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant transduces therapeutically or biologically active protein cargo and / or non-protein cargo into the cytoplasm and / or nucleus of target eukaryotic cells, and the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is used (or formulated for use) at a concentration sufficient to increase the transduction efficiency of cargo into target eukaryotic cells compared to the absence of the synthetic peptide shuttle agent.

[0114] In some embodiments, this specification describes compositions for use in transducing protein cargoes and / or non-protein cargoes into target eukaryotic cells, the compositions comprising a synthetic peptide shuttle formulation with pharmaceutically appropriate excipients, the concentration of the synthetic peptide shuttle in the composition being sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the cargoes to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle. In some embodiments, the compositions further comprise cargoes. In some embodiments, the compositions may be mixed with the cargoes before administration or therapeutic use.

[0115] In some embodiments, this specification describes compositions for therapeutic use, comprising a synthetic peptide shuttle agent formulated with protein cargo and / or non-protein cargo to be transduced into target eukaryotic cells by the synthetic peptide shuttle agent, wherein the concentration of the synthetic peptide shuttle agent in the composition is sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the cargo to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle agent.

[0116] In some embodiments, the shuttle agent or composition, as well as the protein cargo and / or non-protein cargo, may be exposed to target cells in or out of the presence of serum. In some embodiments, the method may be suitable for clinical or therapeutic use.

[0117] In some embodiments, this description relates to a kit for delivering protein cargo and / or non-protein cargo from the extracellular space of a target eukaryotic cell to the cytoplasm and / or nucleus. In some embodiments, this description relates to a kit for increasing the transduction efficiency of protein cargo and / or non-protein cargo into the cytoplasm of a target eukaryotic cell. The kit may comprise a shuttle agent, or composition as defined herein, and a suitable container.

[0118] In some embodiments, the target eukaryotic cells may be animal cells, mammalian cells, or human cells. In some embodiments, the target eukaryotic cells may be stem cells (e.g., embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, peripheral blood stem cells), primary cells (e.g., myoblasts, fibroblasts), immune cells (e.g., NK cells, T cells, dendritic cells, antigen-presenting cells), epithelial cells, skin cells, gastrointestinal cells, mucosal cells, or lung cells. In some embodiments, the target cells may include those having a cellular apparatus for endocytosis (i.e., producing endosomes).

[0119] In some embodiments, this description relates to isolated cells containing a synthetic peptide shuttle agent as defined herein. In some embodiments, the cells may be protein-inducible pluripotent stem cells. It is understood that cells that are often resistant to or unsuitable for DNA transfection may be interesting candidates for the synthetic peptide shuttle agents described herein.

[0120] In some embodiments, this description relates to a method for producing a synthetic peptide shuttle agent for delivering protein and / or nonprotein cargo from the extracellular space of a target eukaryotic cell to the cytoplasm and / or nucleus, the method comprising the step of synthesizing a peptide which (1) is at least 17, 18, 19, or 20 amino acids long, (2) is an amphiphilic alpha-helix motif, and (3) comprises an amphiphilic alpha-helix motif having a positively charged hydrophilic outer surface and a hydrophobic outer surface, and satisfies at least five of the parameters (4) to (15) defined herein.

[0121] In some embodiments, this description relates to a method for identifying or selecting a shuttle agent for delivering protein and / or non-protein cargo from the extracellular space of a target eukaryotic cell to the cytoplasm and / or nucleus, the method comprising: (a) synthesizing a peptide which is a peptide as defined herein; (b) contacting a target eukaryotic cell with the cargo in the presence of the peptide; (c) measuring the transduction efficiency of the cargo in the target eukaryote; and (d) identifying or selecting a peptide which is a shuttle agent for transducing the cargo if an increase in the transduction activity (e.g., transduction efficiency) of the cargo is observed in the target eukaryotic cell.

[0122] In some embodiments, this description relates to a composition for use in transducing protein cargoes and / or non-protein cargoes into target eukaryotic cells, the composition comprising a synthetic peptide shuttle formulation with pharmaceutically suitable excipients, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the cargoes to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle. In some embodiments, the composition further comprises cargoes.

[0123] In some embodiments, this description relates to the shuttle agents and cargoes described herein, including oral formulations such as enteric-coated oral administration forms.

[0124] In some embodiments, the shuttle agents described herein may be used in food, agriculture, and / or agricultural industries. In some embodiments, the shuttle agents described herein may be formulated as feed additives to aid in weight gain and / or nutrient absorption. In some embodiments, the shuttle agents described herein may be formulated as feed additives to aid in weight gain and / or nutrient absorption.

[0125] In some embodiments, this specification describes a method for producing candidate synthetic peptide shuttles that are expected to have transduction activity to target protein cargoes and / or non-protein cargoes in target eukaryotic cells, the method comprising the step of synthesizing a peptide that (1) is at least 17, 18, 19, or 20 amino acid length, (2) is an amphiphilic alpha-helix motif, and (3) comprises an amphiphilic alpha-helix motif having a positively charged hydrophilic outer surface and a hydrophobic outer surface, and satisfies at least five of the parameters (4) to (15) defined herein, wherein the shuttle is at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times more potent than the corresponding negative control lacking the shuttle, and contains propidopropyl iodide. In a eukaryotic cell line model (e.g., HeLa) suitable for increasing the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertors and / or evaluating cargo transduction in the target eukaryotic cells, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 2% of propidium iodide or other membrane-impermeable fluorescent DNA insertors are used. This enables transduction efficiencies of 5%, 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% (for example, as determined by flow cytometry).

[0126] In some embodiments, this specification describes in vitro or in vivo methods for identifying or selecting synthetic peptide shuttles expected to have transduction activity for protein cargo and / or non-protein cargo in target eukaryotic cells, the methods comprising: providing a model eukaryotic cell or model organism suitable for evaluating cargo transduction in target eukaryotic cells; providing a candidate synthetic peptide shuttle (e.g., any of the shuttles defined herein); and measuring the transduction activity (e.g., cargo transduction efficiency, e.g., by flow cytometry) of the candidate synthetic peptide shuttle for transducing propidium iodide or other membrane-impermeable fluorescent DNA insertors into a eukaryotic cell line model, wherein the transduction activity (e.g., transduction efficiency) of the candidate shuttle for propidium iodide or other membrane-impermeable fluorescent DNA insertors is compared to a corresponding negative control lacking the candidate synthetic peptide shuttle. If the amount increases by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times, and / or if the amount of propidium iodide or other membrane-impermeable fluorescent DNA insertion agent increases by at least 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%, 3 If transduction efficiencies of 7%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% (e.g., measured by flow cytometry) occur in model eukaryotic cells or model organisms, it is expected that the substance will have transduction activity for both protein and non-protein cargoes in target eukaryotic cells.

[0127] Item I In some embodiments, this may include one or more of the following items: 1. A method for transduction of a non-protein cargo, comprising the step of contacting a target eukaryotic cell with a synthetic peptide shuttle agent at a concentration sufficient to increase the transduction efficiency of the non-protein cargo compared to the absence of the non-protein cargo and the synthetic peptide shuttle agent. 2. The method according to item 1, wherein the non-protein cargo is (a) an organic compound; (b) has a molecular weight of less than 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000 or 1000 Da, or between 50 and 5000, 50 and 4000, 50 and 3000, 50 and 2000 or 50 and 1000 Da; (c) is a small molecule such as a small molecule drug that binds to an intracellular biological or therapeutic target; (d) is not a biomolecule such as a polynucleotide or polysaccharide; (e) is not covalently bonded to a synthetic peptide shuttle agent during transduction; or (f) any combination of (a) to (e). 3. The method according to item 1 or 2, wherein the non-protein cargo is a drug for treating cancer (e.g., skin cancer, basal cell carcinoma, nevus basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye disease, atrophic or exudative age-related macular degeneration, finger ulcers, actinic keratosis, idiopathic pulmonary fibrosis), pain (e.g., chronic or acute), or diseases affecting the lungs (e.g., cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), or idiopathic pulmonary fibrosis). 4. The method according to any one of items 1 to 3, wherein the non-protein cargo is a pain inhibitor such as a hedgehog inhibitor (e.g., itraconazole, posaconazole, arsenic trioxide (ATO), Gant61, PF-4708671, HPI-1, HPI-4), a voltage-gated sodium (Nav) channel inhibitor (e.g., QX-314), and / or an inflammation inhibitor (e.g., an inhibitor of inflammatory cytokine production, or an NF-Kappa B pathway inhibitor), or includes such an inhibitor. 5. The shuttle agent is a peptide comprising an amphiphilic alpha-helix motif that (1) has a length of at least 20 amino acids, (2) is an amphiphilic alpha-helix motif, and (3) has a positively charged hydrophilic outer surface and a hydrophobic outer surface, and has the following parameters (4)~(15): (4) The hydrophobic outer surface contains a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12~50% of the amino acids of the peptide, based on the open cylinder representation of the alpha-helix having 3.6 residues per turn; (5) The peptide has a hydrophobic moment (μ) of 3.5~11; (6) The peptide has a predicted net charge of at least +4 at physiological pH; (7) The peptide has an isoelectric point (pI) of 8~13; (8) The peptide consists of 35~65% amino acids: any combination of A, C, G, I, L, M, F, P, W, Y, and V; (9) The peptide consists of 0%~30% amino acids (10) The peptide consists of any combination of acids: N, Q, S, and T; (11) The peptide consists of any combination of amino acids: A, L, K, or R, provided that at least 5% L is present in the peptide; (12) The peptide consists of any combination of amino acids: A and L, provided that at least 5% L is present in the peptide; (13) The peptide consists of any combination of amino acids: K and R, providing at least 20% to 45% L; The method according to any one of items 1 to 4, wherein at least five of the following conditions are met: (14) the peptide is composed of any combination of amino acids: D and E; (2) the difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R) is 10% or less; and (3) the peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H. 6. (a) The shuttle agent satisfies at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or all parameters (4) to (15); (b) The shuttle agent is a peptide having a minimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 110, 120, 130, 140, or 150 amino acids Yes; (c) The amphiphilic alpha helix motif has lower limits of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, and (d) The amphiphilic alpha-helix motif includes a positively charged hydrophilic outer surface, which includes (i) at least two, three, or four adjacent positively charged K and / or R residues on a helical wheel projection; and / or (ii) a segment of six adjacent residues on a helical wheel projection, based on an alpha-helix with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn; and (e) the amphiphilic The alpha-helix motif includes a hydrophobic outer surface, which includes (i) at least two adjacent L residues on a helical wheel projection; and / or (ii) a segment of 10 adjacent residues on a helical wheel projection, based on an alpha-helix with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn, including at least five hydrophobic residues selected from L, I, F, V, W, and M; and (f) the hydrophobic outer surface includes 12.5%, 13%, 13.5%, 14%, and 14% of the amino acids of the shuttle agent.(g) The shuttle agent comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% to 25%, 30%, 35%, 40%, or 45%; (g) the shuttle agent comprises 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6 The method according to item 5, wherein the shuttle agent has a hydrophobic moment (μ) between a lower limit of 0.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5, and (h) the shuttle agent has a predicted effective charge of +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, or +15; (i) the shuttle agent has a predicted pI of 10 to 13; or (j) any combination of (a) to (i). 7. The method according to any one of items 1 to 6, wherein the shuttle agent satisfies at least one, at least two, at least three, at least four, at least five, at least six, or all of the following parameters: (8) The shuttle agent comprises 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60% of amino acids: any combination of A, C, G, I, L, M, F, P, W, Y, and V (9) The shuttle agent consists of any combination of amino acids: N, Q, S, and T in amounts of 1%-29%, 2%-28%, 3%-27%, 4%-26%, 5%-25%, 6%-24%, 7%-23%, 8%-22%, 9%-21%, or 10%-20%; (10) The shuttle agent consists of 36%-80%, 37%-75%, 38%-70%, 39%-65%, or 40%-60% of amino acids: A, L, K, or R (11) The shuttle agent consists of any combination of amino acids A and L in amounts of 15%-40%, 20%-40%, 20%-35%, or 20%-30%; (12) The shuttle agent consists of any combination of amino acids K and R in amounts of 20%-40%, 20%-35%, or 20%-30%; (13) The shuttle agent consists of any combination of amino acids D and E in amounts of 5%-10% (14) The difference between the percentage of A and L residues in the shuttle agent (A+L%) and the percentage of K and R residues in the shuttle agent (K+R) is 9%, 8%, 7%, 6%, or 5% or less; and (15) the shuttle agent consists of 15-40%, 20-35%, or 20-30% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H. 8. The method according to any one of items 1 to 7, wherein the shuttle agent comprises a histidine-rich domain, and optionally the histidine-rich domain is (i) positioned toward the N-terminus and / or C-terminus of the shuttle agent; (ii) a stretch of at least 3, at least 4, at least 5, at least 6 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues, and / or comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive histidine residues; or (iii) both (i) and (ii). 9. The method according to any one of items 1 to 8, wherein the shuttle agent comprises a flexible linker domain concentrated with serine and / or glycine residues. 10. The method according to any one of items 1 to 9, wherein the shuttle agent contains or consists of the following amino acid sequences: (a) [X1]-[X2]-[linker]-[X3]-[X4](formula 1); (b) [X1]-[X2]-[linker]-[X4]-[X3](formula 2); (c) [X2]-[X1]-[linker]-[X3]-[X4](formula 3); (d) [X2]-[X1]-[linker]-[X4]-[X3](formula 4); (e) [X3]-[X4]-[linker]-[X1]-[X2](formula 5); (f) [X3]-[X4]-[linker]-[X 2]-[X1](Equation 6);(g)[X4]-[X3]-[Linker]-[X1]-[X2](Equation 7);or(h)[X4]-[X3]-[Linker]-[X2]-[X1](Equation 8);wherein [X1] is selected from the following: 2[Φ]-1[+]-2[Φ]-1[ζ]-1[+]-;2[Φ]-1[+]-2[Φ]-2[+]-;1[+]-1[Φ]-1[+]-2[Φ]-1[ζ]-1[+]-;and 1[+]-1[Φ]-1[+]-2[Φ]-2[+]-;[X2] is selected from the following:-2[Φ]-1[+]-2[Φ]-2[ζ]-;-2 [Φ]-1[+]-2[Φ]-2[+]-;-2[Φ]-1[+]-2[Φ]-1[+]-1[ζ]-;-2[Φ]-1[+]-2[Φ ]-1[ζ]-1[+]-;-2[Φ]-2[+]-1[Φ]-2[+]-;-2[Φ]-2[+]-1[Φ]-2[ζ]-;-2[Φ ]-2[+]-1[Φ]-1[+]-1[ζ]-; and -2[Φ]-2[+]-1[Φ]-1[ζ]-1[+]-;[X3] from the following Selected:-4[+]-A-;-3[+]-GA-;-3[+]-AA-;-2[+]-1[Φ]-1[+]-A-;-2[+]-1[ Φ]-GA-;-2[+]-1[Φ]-AA-; or -2[+]-A-1[+]-A;-2[+]-AGA;-2[+]-AAA-;- 1[Φ]-3[+]-A-;-1[Φ]-2[+]-GA-;-1[Φ]-2[+]-AA-;-1[Φ]-1[+]-1[Φ]-1[ +]-A;-1[Φ]-1[+]-1[Φ]-GA;-1[Φ]-1[+]-1[Φ]-AA;-1[Φ]-1[+]-A-1[+]- A;-1[Φ]-1[+]-AGA;-1[Φ]-1[+]-AAA;-A-1[+]-A-1[+]-A;-A-1[+]-AGA;And -A-1[+]-AAA;[X4] is selected from the following: -1[ζ]-2A-1[+]-A;-1[ζ]-2A-2[+];-1[+]-2A-1[+]-A;-1[ζ]-2A-1[+]-1[ζ]-A-1[+];-1[ζ]-A-1[ζ]-A-1[+];-2[+]-A-2[+];-2[+]-A-1[+]-A;-2[+]-A-1[+]-1[ζ]-A-1[+];-2[+]-1[ζ]-A-1[+];-1[+]-1[ζ]-A-1[+]-A;-1[+]-1[ζ]-A-2[+];-1 [+]-1[ζ]-A-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-A-1[+];-1[+]-2[ζ]-2 [+];-1[+]-2[ζ]-1[+]-A;-1[+]-2[ζ]-1[+]-1[ζ]-A-1[+];-1[+]-2[ ζ]-1[ζ]-A-1[+];-3[ζ]-2[+];-3[ζ]-1[+]-A;-3[ζ]-1[+]-1[ζ]-A-1 [+];-1[ζ]-2A-1[+]-A;-1[ζ]-2A-2[+];-1[ζ]-2A-1[+]-1[ζ]-A-1[+ ];-2[+]-A-1[+]-A;-2[+]-1[ζ]-1[+]-A;-1[+]-1[ζ]-A-1[+]-A;-1[+]-2A-1[+]-1[ζ]-A-1[+]; and -1[ζ]-A-1[ζ]-A-1[+]; and [linker] is selected from the following:-Gn-;-Sn-;-(GnSn)n-;-(GnSn)nGn-;-(GnSn)nSn-;-(GnSn)nGn(GnSn)n-; and -(GnSn)nSn(GnSn)n-;[Φ] is an amino acid: Leu, Phe, Trp, Ile, Met, Tyr, or Val, preferably Leu, Phe, Trp, or Ile; [+] is the amino acid Lys or Arg; [ζ] is the amino acid Gln, Asn, Thr, or Ser; A is the amino acid Ala; G is the amino acid Gly; S is the amino acid Ser; n is an integer between 1 and 20, 1 and 19, 1 and 18, 1 and 17, 1 and 16, 1 and 15, 1 and 14, 1 and 13, 1 and 12, 1 and 11, 1 and 10, 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, or 1 and 3. 11. The method according to any one of items 1 to 10, wherein the shuttle agent comprises or consists of a peptide which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of any one of sequence numbers 19 to 50. 12. The method according to any one of items 1 to 11, wherein the shuttle agent comprises an endosomal leakage domain (ELD) and / or a cell membrane permeability domain (CPD). 13. (i) The ELDs are endosomal soluble peptides; antimicrobial peptides (AMP); linear cationic alpha-helix antimicrobial peptides; secropine-A / melittin hybrid (CM) peptides; pH-dependent membrane-active peptides (PAMP); amphiphilic peptides; peptides derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1; VSVG; pseudomonas toxin; melittin; KALA; JS (ii) The method according to any one of items 1 to 12, wherein the CPD is T-1;C(LLKK)3C;G(LLKK)3G or any combination thereof or derived therefrom; or (iii) the method according to any one of items 1 to 12, wherein the CPD is a cell membrane permeable peptide or a protein transduction domain derived from a cell membrane permeable peptide;TAT;PTD4;Penetratin;pVEC;M918;Pep-1;Pep-2;Xcentry;Arginine stretch;Transportan;SynB1;SynB3; or any combination thereof or derived therefrom; or (iii)(i) and (ii). 14. The method according to any one of items 1 to 13, wherein the shuttle agent is a cyclic peptide and / or comprises one or more D-amino acids. 15. An in vitro method for therapeutic and / or diagnostic purposes, etc., as described in any one of items 1 to 14. 16. An in vivo method for therapeutic and / or diagnostic purposes, etc., as described in any one of items 1 to 14. 17. The method according to item 16, comprising topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of non-protein cargo and synthetic peptide shuttle agents. 18. A composition for use in transducing a non-protein cargo into target eukaryotic cells, comprising a synthetic peptide shuttle formulation with pharmaceutically appropriate excipients, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the non-protein cargo to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle. 19. The composition according to item 17, further comprising non-protein cargo. 20. A composition according to item 18 or 19, wherein (a) the synthetic peptide shuttle is as defined in any one of items 1 or 5-14; (b) the non-protein cargo is as defined in any one of items 2-4; (c) the composition is for use in an in vitro or in vivo method as defined in any one of items 15-17; or (d) any combination of (a)-(c). 21. A kit for use in the method described in any one of items 1 to 17, comprising a synthetic peptide shuttle agent as defined in item 1 or any one of items 5 to 14, and a non-protein cargo as defined in any one of items 2 to 4. 22. The method according to any one of items 1 to 17, the composition according to any one of items 18 to 20, or the kit according to item 21, wherein the target eukaryotic cell is an animal cell, mammalian cell, human cell, stem cell, primary cell, immune cell, T cell, NK cell, dendritic cell, epithelial cell, skin cell, or gastrointestinal cell. 23. A synthetic peptide shuttle agent having transduction activity for both protein cargo and non-protein cargo, wherein the shuttle agent contains an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs. 19 to 50. 24. A synthetic peptide shuttle agent as defined in any one of items 5-13, as described in item 23.

[0128] Item II In some embodiments, this may include one or more of the following items: 1. A method for transduction of a non-protein cargo, comprising the step of contacting a target eukaryotic cell with a synthetic peptide shuttle agent at a concentration sufficient to increase the transduction efficiency of the non-protein cargo compared to the absence of the non-protein cargo and the synthetic peptide shuttle agent. 2. The method according to item 1, wherein the non-protein cargo is (a) an organic compound; (b) has a molecular weight of less than 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000 or 1000 Da, or between 50 and 5000, 50 and 4000, 50 and 3000, 50 and 2000 or 50 and 1000 Da; (c) is a small molecule such as a small molecule drug that binds to an intracellular biological or therapeutic target; (d) is not a biomolecule such as a polynucleotide or polysaccharide; (e) is not covalently bonded to a synthetic peptide shuttle agent during transduction; or (f) any combination of (a) to (e). 3. The method according to item 1 or 2, wherein the non-protein cargo is a drug for treating cancer (e.g., skin cancer, basal cell carcinoma, nevus basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye disease, atrophic or exudative age-related macular degeneration, finger ulcers, actinic keratosis, idiopathic pulmonary fibrosis), pain (e.g., chronic or acute), or diseases affecting the lungs (e.g., cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), or idiopathic pulmonary fibrosis). 4. The method according to any one of items 1 to 3, wherein the non-protein cargo is a pain inhibitor such as a hedgehog inhibitor (e.g., itraconazole, posaconazole, arsenic trioxide (ATO), Gant61, PF-4708671, HPI-1, HPI-4), a voltage-gated sodium (Nav) channel inhibitor (e.g., QX-314), and / or an inflammation inhibitor (e.g., an inhibitor of inflammatory cytokine production, or an NF-Kappa B pathway inhibitor), or includes such an inhibitor. 5. The shuttle agent is a peptide comprising an amphiphilic alpha-helix motif that (1) has a length of at least 20 amino acids, (2) is an amphiphilic alpha-helix motif, and (3) has a positively charged hydrophilic outer surface and a hydrophobic outer surface, and has the following parameters (4)~(15): (4) The hydrophobic outer surface contains a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12~50% of the amino acids of the peptide, based on the open cylinder representation of the alpha-helix having 3.6 residues per turn; (5) The peptide has a hydrophobic moment (μ) of 3.5~11; (6) The peptide has a predicted net charge of at least +4 at physiological pH; (7) The peptide has an isoelectric point (pI) of 8~13; (8) The peptide consists of 35~65% amino acids: any combination of A, C, G, I, L, M, F, P, W, Y, and V; (9) The peptide consists of 0%~30% amino acids (10) The peptide consists of any combination of acids: N, Q, S, and T; (11) The peptide consists of any combination of amino acids: A, L, K, or R, provided that at least 5% L is present in the peptide; (12) The peptide consists of any combination of amino acids: A and L, provided that at least 5% L is present in the peptide; (13) The peptide consists of any combination of amino acids: K and R, providing at least 20% to 45% L; The method according to any one of items 1 to 4, wherein at least five of the following conditions are met: (14) the peptide is composed of any combination of amino acids: D and E; (2) the difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R) is 10% or less; and (3) the peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H. 6. (a) The shuttle agent satisfies at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or all parameters (4) to (15); (b) The shuttle agent is a peptide having a minimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 110, 120, 130, 140, or 150 amino acids Yes; (c) The amphiphilic alpha helix motif has lower limits of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, and (d) The amphiphilic alpha-helix motif includes a positively charged hydrophilic outer surface, which includes (i) at least two, three, or four adjacent positively charged K and / or R residues on a helical wheel projection; and / or (ii) a segment of six adjacent residues on a helical wheel projection, based on an alpha-helix with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn; and (e) the amphiphilic The alpha-helix motif includes a hydrophobic outer surface, which includes (i) at least two adjacent L residues on a helical wheel projection; and / or (ii) a segment of 10 adjacent residues on a helical wheel projection, based on an alpha-helix with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn, including at least five hydrophobic residues selected from L, I, F, V, W, and M; and (f) the hydrophobic outer surface includes 12.5%, 13%, 13.5%, 14%, and 14% of the amino acids of the shuttle agent.(g) The shuttle agent comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% to 25%, 30%, 35%, 40%, or 45%; (g) the shuttle agent comprises 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6 The method according to item 5, wherein the shuttle agent has a hydrophobic moment (μ) between a lower limit of 0.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5, and (h) the shuttle agent has a predicted effective charge of +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, or +15; (i) the shuttle agent has a predicted pI of 10 to 13; or (j) any combination of (a) to (i). 7. The method according to any one of items 1 to 6, wherein the shuttle agent satisfies at least one, at least two, at least three, at least four, at least five, at least six, or all of the following parameters: (8) The shuttle agent comprises 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60% of amino acids: any combination of A, C, G, I, L, M, F, P, W, Y, and V (9) The shuttle agent consists of any combination of amino acids: N, Q, S, and T in amounts of 1%-29%, 2%-28%, 3%-27%, 4%-26%, 5%-25%, 6%-24%, 7%-23%, 8%-22%, 9%-21%, or 10%-20%; (10) The shuttle agent consists of 36%-80%, 37%-75%, 38%-70%, 39%-65%, or 40%-60% of amino acids: A, L, K, or R (11) The shuttle agent consists of any combination of amino acids A and L in amounts of 15%-40%, 20%-40%, 20%-35%, or 20%-30%; (12) The shuttle agent consists of any combination of amino acids K and R in amounts of 20%-40%, 20%-35%, or 20%-30%; (13) The shuttle agent consists of any combination of amino acids D and E in amounts of 5%-10% (14) The difference between the percentage of A and L residues in the shuttle agent (A+L%) and the percentage of K and R residues in the shuttle agent (K+R) is 9%, 8%, 7%, 6%, or 5% or less; and (15) the shuttle agent consists of 15-40%, 20-35%, or 20-30% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H. 8. The method according to any one of items 1 to 7, wherein the shuttle agent comprises a histidine-rich domain, and optionally the histidine-rich domain is (i) positioned toward the N-terminus and / or C-terminus of the shuttle agent; (ii) a stretch of at least 3, at least 4, at least 5, at least 6 amino acids comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues, and / or comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 consecutive histidine residues; or (iii) both (i) and (ii). 9. The method according to any one of items 1 to 8, wherein the shuttle agent comprises a flexible linker domain concentrated with serine and / or glycine residues. 10. The method according to any one of items 1 to 9, wherein the shuttle agent contains or consists of the following amino acid sequences: (a) [X1]-[X2]-[linker]-[X3]-[X4](formula 1); (b) [X1]-[X2]-[linker]-[X4]-[X3](formula 2); (c) [X2]-[X1]-[linker]-[X3]-[X4](formula 3); (d) [X2]-[X1]-[linker]-[X4]-[X3](formula 4); (e) [X3]-[X4]-[linker]-[X1]-[X2](formula 5); (f) [X3]-[X4]-[linker]-[X 2]-[X1](Equation 6);(g)[X4]-[X3]-[Linker]-[X1]-[X2](Equation 7);or(h)[X4]-[X3]-[Linker]-[X2]-[X1](Equation 8);wherein [X1] is selected from the following: 2[Φ]-1[+]-2[Φ]-1[ζ]-1[+]-;2[Φ]-1[+]-2[Φ]-2[+]-;1[+]-1[Φ]-1[+]-2[Φ]-1[ζ]-1[+]-;and 1[+]-1[Φ]-1[+]-2[Φ]-2[+]-;[X2] is selected from the following:-2[Φ]-1[+]-2[Φ]-2[ζ]-;-2 [Φ]-1[+]-2[Φ]-2[+]-;-2[Φ]-1[+]-2[Φ]-1[+]-1[ζ]-;-2[Φ]-1[+]-2[Φ ]-1[ζ]-1[+]-;-2[Φ]-2[+]-1[Φ]-2[+]-;-2[Φ]-2[+]-1[Φ]-2[ζ]-;-2[Φ ]-2[+]-1[Φ]-1[+]-1[ζ]-; and -2[Φ]-2[+]-1[Φ]-1[ζ]-1[+]-;[X3] from the following Selected:-4[+]-A-;-3[+]-GA-;-3[+]-AA-;-2[+]-1[Φ]-1[+]-A-;-2[+]-1[ Φ]-GA-;-2[+]-1[Φ]-AA-; or -2[+]-A-1[+]-A;-2[+]-AGA;-2[+]-AAA-;- 1[Φ]-3[+]-A-;-1[Φ]-2[+]-GA-;-1[Φ]-2[+]-AA-;-1[Φ]-1[+]-1[Φ]-1[ +]-A;-1[Φ]-1[+]-1[Φ]-GA;-1[Φ]-1[+]-1[Φ]-AA;-1[Φ]-1[+]-A-1[+]- A;-1[Φ]-1[+]-AGA;-1[Φ]-1[+]-AAA;-A-1[+]-A-1[+]-A;-A-1[+]-AGA;And -A-1[+]-AAA;[X4] is selected from the following: -1[ζ]-2A-1[+]-A;-1[ζ]-2A-2[+];-1[+]-2A-1[+]-A;-1[ζ]-2A-1[+]-1[ζ]-A-1[+];-1[ζ]-A-1[ζ]-A-1[+];-2[+]-A-2[+];-2[+]-A-1[+]-A;-2[+]-A-1[+]-1[ζ]-A-1[+];-2[+]-1[ζ]-A-1[+];-1[+]-1[ζ]-A-1[+]-A;-1[+]-1[ζ]-A-2[+];-1 [+]-1[ζ]-A-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-A-1[+];-1[+]-2[ζ]-2 [+];-1[+]-2[ζ]-1[+]-A;-1[+]-2[ζ]-1[+]-1[ζ]-A-1[+];-1[+]-2[ ζ]-1[ζ]-A-1[+];-3[ζ]-2[+];-3[ζ]-1[+]-A;-3[ζ]-1[+]-1[ζ]-A-1 [+];-1[ζ]-2A-1[+]-A;-1[ζ]-2A-2[+];-1[ζ]-2A-1[+]-1[ζ]-A-1[+ ];-2[+]-A-1[+]-A;-2[+]-1[ζ]-1[+]-A;-1[+]-1[ζ]-A-1[+]-A;-1[+]-2A-1[+]-1[ζ]-A-1[+]; and -1[ζ]-A-1[ζ]-A-1[+]; and [linker] is selected from the following:-Gn-;-Sn-;-(GnSn)n-;-(GnSn)nGn-;-(GnSn)nSn-;-(GnSn)nGn(GnSn)n-; and -(GnSn)nSn(GnSn)n-;[Φ] is an amino acid: Leu, Phe, Trp, Ile, Met, Tyr, or Val, preferably Leu, Phe, Trp, or Ile; [+] is the amino acid Lys or Arg; [ζ] is the amino acid Gln, Asn, Thr, or Ser; A is the amino acid Ala; G is the amino acid Gly; S is the amino acid Ser; n is an integer between 1 and 20, 1 and 19, 1 and 18, 1 and 17, 1 and 16, 1 and 15, 1 and 14, 1 and 13, 1 and 12, 1 and 11, 1 and 10, 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, or 1 and 3. 11. The shuttle agent is any amino acid sequence of any one of SEQ ID NOs: 1-50; any amino acid sequence of any one of SEQ ID NOs: 1-50 differing by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or fewer amino acids (e.g., excluding any linker domain); or any amino acid sequence of any one of SEQ ID NOs: 1-50 differing by at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, The method according to any one of items 1 to 10, comprising or consisting of amino acid sequences that are 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (calculated, for example, excluding any linker domain). 12. The shuttle agent is one amino acid sequence of any of the following: SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, or 344; SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141 An amino acid sequence that differs from any one of the following: ~146, 149~161, 163~169, 171, 174~234, 236~240, 242~260, 262~285, 287~294, 296~300, 302~308, 310, 311, 313~324, 326~332, 338~342, or 344 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acids (for example, excluding any linker domain); or SEQ ID NOs: 1~50, 58~78, 80 ~107, 109~139, 141~146, 149~161, 163~169, 171, 174~234, 236~240, 242~260, 262~285, 287~294, 296~300, 302~308, 310, 311, 313~324, 326~332, 338~342, or 344, and at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65 The method according to any one of items 1 to 10, comprising or consisting of amino acid sequences that are identical by %, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (calculated, for example, excluding any linker domain). 13. The method according to any one of items 1 to 12, wherein the shuttle agent comprises an endosomal leakage domain (ELD) and / or a cell membrane permeability domain (CPD). 14. (i) The ELDs are endosomal soluble peptides; antimicrobial peptides (AMP); linear cationic alpha-helix antimicrobial peptides; secropine-A / melittin hybrid (CM) peptides; pH-dependent membrane-active peptides (PAMP); amphiphilic peptides; peptides derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1; VSVG; pseudomonas toxin; melittin; KALA; JS (ii) The method according to any one of items 1 to 13, wherein the CPD is a cell membrane permeable peptide or a protein transduction domain derived from a cell membrane permeable peptide; TAT; PTD4; penetratin; pVEC; M918; Pep-1; Pep-2; xentory; arginine stretch; transportan; SynB1; SynB3; or any combination thereof; or (iii) both (i) and (ii). 15. The method according to any one of items 1 to 14, wherein the shuttle agent is a cyclic peptide and / or comprises one or more D-amino acids. 16. In a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in target eukaryotic cells, the shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to a corresponding negative control lacking the shuttle agent, and / or at least 10%, 11%, 12%, 13%, 14%, 15%, 1% of propidium iodide or other membrane-impermeable fluorescent DNA insertion agent. The method described in any one of items 1 to 15, enabling transduction efficiencies of 6%, 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% (for example, as determined by flow cytometry). 17. The shuttle agent increases the transduction efficiency of GFP-NLS by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to the corresponding negative control lacking the shuttle agent, and / or in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in the target eukaryotic cells, at least 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% of GFP-NLS. The method according to any one of items 1 to 16, enabling transduction efficiencies of 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% (for example, as determined by flow cytometry). 18. The method according to any one of items 1 to 17, wherein the shuttle agent further comprises chemical modifications to one or more amino acids, the chemical modifications not destroying the transduction activity of the synthetic peptide shuttle agent. 19. The method according to item 18, wherein the chemical modification is located at the N-terminus and / or C-terminus of the shuttle agent. 20. The method according to item 18 or 19, wherein the chemical modification is the addition of an acetyl group (e.g., an N-terminal acetyl group), a cysteamide group (e.g., a C-terminal cysteamide group), or a fatty acid (e.g., a C4-C16 fatty acid, preferably N-terminal). 21. An in vitro method for therapeutic and / or diagnostic purposes, etc., as described in any one of items 1 to 20. 22. An in vivo method for therapeutic and / or diagnostic purposes, etc., as described in any one of items 1 to 20. 23. The method according to item 22, comprising topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of non-protein cargo and synthetic peptide shuttle agents. 24. A composition for use in transducing a non-protein cargo into target eukaryotic cells, comprising a synthetic peptide shuttle formulation with pharmaceutically appropriate excipients, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the non-protein cargo to the target eukaryotic cells at administration compared to the absence of the synthetic peptide shuttle. 25. The composition according to item 24, further comprising non-protein cargo. 26. A composition for therapeutic use, comprising a synthetic peptide shuttle formulation together with a non-protein cargo to be transduced into target eukaryotic cells by the synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the non-protein cargo to the target eukaryotic cells at administration compared to the absence of the synthetic peptide shuttle. 27. (a) The synthetic peptide shuttle is as defined in any one of items 1 or 5-20; (b) The non-protein cargo is as defined in any one of items 2-4; (c) The composition is for use in an in vitro or in vivo method as defined in any one of items 21-23; or (d) Any combination of (a)-(c) as described in any one of items 24-26. 28. A kit for use in the method described in any one of items 1 to 23, comprising a synthetic peptide shuttle agent as defined in item 1 or any one of items 5 to 20, wherein the non-protein cargo is as defined in any one of items 2 to 4. 29. The method according to any one of items 1 to 23, the composition according to any one of items 24 to 27, or the kit according to item 28, wherein the target eukaryotic cell is an animal cell, mammalian cell, human cell, stem cell, primary cell, immune cell, T cell, NK cell, dendritic cell, epithelial cell, skin cell, or gastrointestinal cell. 30. A synthetic peptide shuttle agent having transduction activity for both protein cargo and non-protein cargo, wherein the shuttle agent is an amino acid sequence that is any one of the SEQ ID NOs: 19-50; an amino acid sequence that is different from any one of the SEQ ID NOs: 19-50 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or fewer amino acids (e.g., excluding any linker domain); or an amino acid sequence that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 5% of any one of the SEQ ID NOs: 19-50 A synthetic peptide shuttle containing or comprising amino acid sequences that are 9%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (calculated, for example, excluding any linker domain). 31. A synthetic peptide shuttle agent as defined in any one of items 5-20, as described in item 30.32. A synthetic peptide shuttle having transduction activity for both protein and non-protein cargo in target eukaryotic cells, wherein (1) it is at least 17, 18, 19, or 20 amino acid lengths, (2) it is an amphiphilic alpha-helix motif, and (3) it is a peptide comprising an amphiphilic alpha-helix motif having a positively charged hydrophilic outer surface and a hydrophobic outer surface, and the following parameters (4)~(15): (4) the hydrophobic outer surface is an open alpha-helix having 3.6 residues per turn (5) The peptide contains a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12-50% of the peptide's amino acids, based on Linder notation; (6) The peptide has a hydrophobic moment (μ) of 3.5-11; (7) The peptide has a predicted net charge of at least +4 at physiological pH; (8) The peptide has an isoelectric point (pI) of 8-13; (9) The peptide is composed of 35-65% of any combination of amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (10) The peptide is (10) The peptide consists of any combination of amino acids: N, Q, S, and T, from 0% to 30%; (11) The peptide consists of any combination of amino acids: A, L, K, or R, provided that at least 5% of L is present in the peptide; (12) The peptide consists of any combination of amino acids: A and L, from 20% to 45%; (13) The peptide consists of any combination of amino acids: K and R, from 0% to 10%; (14) The difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R) is 10% or less; and (15) The peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H. At least five of these conditions are met, and the peptide is at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9 compared to the corresponding negative control lacking the shuttle agent.5 or 10 times, in a eukaryotic cell line model (e.g., HeLa) suitable for increasing the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertors and / or evaluating cargo transduction in the target eukaryotic cells, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% of propidium iodide or other membrane-impermeable fluorescent DNA insertors A synthetic peptide shuttle agent that enables transduction efficiencies of 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% (as determined, for example, by flow cytometry). 33. The shuttle agent increases the transduction efficiency of GFP-NLS by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to the corresponding negative control lacking the shuttle agent, and / or in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in the target eukaryotic cells, at least 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% of GFP-NLS. A synthetic peptide shuttle agent as described in item 32, enabling transduction efficiencies of 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% (as determined, for example, by flow cytometry). 34. (a) The shuttle agent satisfies at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or all parameters (4) to (15); (b) The shuttle agent has a minimum length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 110, 120, 130, 140, or 150 amino acids. (c) A peptide having length; (c) The amphiphilic alpha-helix motif has lower limits of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 1 (d) The hydrophobic moment (μ) is between the upper limits of 0.8, 10.9, or 11.0, and the amphiphilic alpha-helix motif includes a positively charged hydrophilic outer surface, which includes (i) at least two, three, or four adjacent positively charged K and / or R residues on a helical wheel projection; and / or (ii) a segment of six adjacent residues on a helical wheel projection, based on an alpha-helix with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn, including three to five K and / or R residues. (e) The amphiphilic alpha-helix motif includes a hydrophobic outer surface, which includes (i) at least two adjacent L residues on a helical wheel projection; and / or (ii) a segment of 10 adjacent residues on a helical wheel projection, based on an alpha-helix with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn, including at least five hydrophobic residues selected from L, I, F, V, W, and M; (f) the hydrophobic outer surface includes 12.5%, 13%, and 13% of the amino acids of the shuttle agent.(g) A highly hydrophobic core comprising spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% to 25%, 30%, 35%, 40%, or 45%; (g) a shuttle agent comprising 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3 A synthetic peptide shuttle agent as described in item 32 or 33, having a hydrophobic moment (μ) between a lower limit of 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5, wherein (h) the shuttle agent has a predicted effective charge of +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, or +15; (i) the shuttle agent has a predicted pI of 10 to 13; or (j) any combination of (a) to (i). 35. The synthetic peptide shuttle agent according to any one of items 32-34, wherein the shuttle agent satisfies at least one, at least two, at least three, at least four, at least five, at least six, or all of the following parameters: (8) The shuttle agent comprises 36%-64%, 37%-63%, 38%-62%, 39%-61%, or 40%-60% of amino acids: A, C, G, I, L, M, F, P, W, Y, and V (9) The shuttle agent consists of any combination of amino acids: N, Q, S, and T in amounts of 1%-29%, 2%-28%, 3%-27%, 4%-26%, 5%-25%, 6%-24%, 7%-23%, 8%-22%, 9%-21%, or 10%-20%; (10) The shuttle agent consists of 36%-80%, 37%-75%, 38%-70%, 39%-65%, or 40%-60% of amino acids: A, L (11) The shuttle agent consists of any combination of amino acids A and L in amounts of 15%-40%, 20%-40%, 20%-35%, or 20%-30%; (12) The shuttle agent consists of any combination of amino acids K and R in amounts of 20%-40%, 20%-35%, or 20%-30%; (13) The shuttle agent consists of any combination of amino acids D and E in amounts of 5%-10% (14) The difference between the percentage of A and L residues in the shuttle agent (A+L%) and the percentage of K and R residues in the shuttle agent (K+R) is 9%, 8%, 7%, 6%, or 5% or less; and (15) the shuttle agent consists of 15-40%, 20-35%, or 20-30% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H. 36. A synthetic peptide shuttle according to any one of items 32 to 35, wherein the shuttle agent comprises (i) a histidine-rich domain as defined in item 8; (ii) a flexible linker domain as defined in item 9; (iii) a shuttle agent as defined in any one of items 10 to 14; and (iv) any combination of (i) to (iii). 37. A synthetic peptide shuttle according to any one of items 32 to 36, further comprising chemical modifications to one or more amino acids, wherein the chemical modifications do not impair the transduction activity of the synthetic peptide shuttle. 38. A synthetic peptide shuttle agent as described in item 37, wherein the chemical modification is located at the N-terminus and / or C-terminus of the shuttle agent. 39. A synthetic peptide shuttle according to item 37 or 38, wherein the chemical modification is the addition of an acetyl group (e.g., an N-terminal acetyl group), a cysteamide group (e.g., a C-terminal cysteamide group), or a fatty acid (e.g., a C4-C16 fatty acid, preferably N-terminal). 40. The shuttle agent is one amino acid sequence of any of the following: SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, or 344; SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141-1 46, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, or 344, and an amino acid sequence that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acids (for example, excluding any linker domain); or SEQ ID NOs: 1-50, 58-78, 80-107, 109~139, 141~146, 149~161, 163~169, 171, 174~234, 236~240, 242~260, 262~285, 287~294, 296~300, 302~308, 310, 311, 313~324, 326~332, 338~342, or 344, and at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 6 A synthetic peptide shuttle according to any one of items 32 to 39, comprising or consisting of amino acid sequences (calculated, for example, excluding any linker domain) that are 7%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. 41. A synthetic peptide shuttle having transduction activity for both protein cargo and non-protein cargo in target eukaryotic cells, comprising (a) any one amino acid sequence of SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, or 344; or (b) SEQ ID NOs. 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, or 344 differs from any one of these by only conservative amino acid substitutions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer conservative amino acid substitutions, preferably excluding any linker domain). In a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in target eukaryotic cells, the propidium iodide or other membrane-impermeable fluorescent DNA inserter contains or comprises an amino acid sequence; and increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA inserter by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to a corresponding negative control lacking the shuttle agent, and / or contains at least 10%, 11%, 12%, 13% of propidium iodide or other membrane-impermeable fluorescent DNA inserter. A synthetic peptide shuttle agent that enables transduction efficiencies of 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% (for example, as determined by flow cytometry). 42. A synthetic peptide shuttle agent as defined in any one of items 32-39, as described in item 41. 43. A synthetic peptide shuttle having protein cargo transduction activity in target eukaryotic cells, comprising: (a) any one amino acid sequence of SEQ ID NOs. 52, 57, 79, 108, 140, 147, 148, 173, 241, 261, 286, 295, 301, 309, 312, 325, 333-337, or 343; or (b) an amino acid sequence that differs from any one of SEQ ID NOs. 52, 57, 79, 108, 140, 147, 148, 173, 241, 261, 286, 295, 301, 309, 312, 325, 333-337, or 343 by only conservative amino acid substitutions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer conservative amino acid substitutions, preferably excluding any linker domains). A synthetic peptide shuttle comprising or comprising; a synthetic peptide shuttle that increases the transduction efficiency of GFP-NLS by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to a corresponding negative control lacking the shuttle, and / or enables a transduction efficiency of at least 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% (as determined, for example, by flow cytometry) of GFP-NLS in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating cargo transduction in the target eukaryotic cells. 44. A synthetic peptide shuttle according to any one of items 41-43, wherein each conservative amino acid substitution is selected from amino acids within the same amino acid class, where the amino acid class is aliphatic: G, A, V, L, and I; hydroxyl or sulfur / selenium-containing: S, C, U, T, and M; aromatic: F, Y, and W; basic: H, K, and R; acidic and their amides: D, E, N, and Q. 45. A synthetic peptide shuttle variant having transduction activity for protein cargo and / or non-protein cargo in target eukaryotic cells, which is identical to a synthetic peptide shuttle as defined in any one of items 32-44, except that at least one amino acid is replaced by a corresponding synthetic amino acid having a side chain with similar physicochemical properties (e.g., structure, hydrophobicity, or charge) to the amino acid being replaced, and which increases the transduction efficiency of the cargo in target eukaryotic cells compared to the absence of the shuttle variant. 46. ​​The substitution of synthetic amino acids involves replacing a basic amino acid with one of the following: (a) α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, 2,6-diamino-4-hexic acid, β-(1-piperazinyl)-alanine, 4,5-dehydrolysine, δ-hydroxylysine, ω,ω-dimethylarginine, homoarginine, ω,ω'-dimethylarginine, ω-methylarginine, β-(2-quinolyl)-alanine, 4-aminopiperidine-4-carboxylic acid, α-methylhistidine, 2,5-diiodohistidine, 1-methylhistidine, 3-methylhistidine, spinacine, 4-aminophenylalanine, 3-aminotyrosine, β-(2-pyridyl)-alanine, or β-(3-pyridyl)-alanine;(b) Dehydroalanine, β-fluoroalanine, β-chloroalanine, β-rhodoalanine, α-aminobutyric acid, α-aminoisobutyric acid, β-cyclopropylalanine, azetidine-2-carboxylic acid, α-allylglycine, propargylglycine, tert-butylalanine, β-(2-thiazolyl)-alanine, thiaproline, 3,4-dehydroproline, tert-butylglycine, β-cyclopentylalanine, β-cyclohexylalanine, α-meth Luproline, norvaline, α-methylvaline, penicillamine, β, β-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, pipecolic acid, 4,5-dehydroleucine, alloisoleucine, norleucine, α-methylleucine, cyclohexylglycine, cis-octahydroindole-2-carboxylic acid, β-(2-thienyl)-alanine, phenylglycine, α-methylphenylalanine, homophenylalanine, Replace a nonpolar (hydrophobic) amino acid with one of the following: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-(3-benzothienyl)-alanine, 4-nitrophenylalanine, 4-bromophenylalanine, 4-tert-butylphenylalanine, α-methyltryptophan, β-(2-naphthyl)-alanine, β-(1-naphthyl)-alanine, 4-iodophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, 4-methyltryptophan, 4-chlorophenylalanine, 3,4-dichlorophenylalanine, 2,6-difluorophenylalanine, n-in-methyltryptophan, 1,2,3,4-tetrahydronorharmann-3-carboxylic acid, β,β-diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, or 4-benzoylphenylalanine;(c)β-Cyanolalanine, β-Ureidoalanine, Homocysteine, Allosreonine, Pyroglutamic acid, 2-Oxothiazolidine-4-carboxylic acid, Citrulline, Thiocitrulline, Homocitrulline, Hydroxyproline, 3,4-Dihydroxyphenylalanine, β-(1,2,4-Triazole-1-yl)alanine, 2-Mercaptohistidine, β-(3,4-Dihydroxyphenyl)-serine, β-(2-Thienyl)-serine, 4-Azidophenylalanine, 4-Cyanolalanine, 3-Hydroxymethyltyrosine, 3-Iodotyrosine, 3-Nitrotyrosine, 3,5-Dinitrotyrosine, 3,5-Dibromotyrosine, 3,5-Diiodotyrosine, 7-Hydroxy-1,2 A synthetic peptide shuttle variant as described in item 45, in which a polar uncharged amino acid is replaced with one of the following: 3,4-tetrahydroisoquinoline-3-carboxylic acid, 5-hydroxytryptophan, thyronine, β-(7-methoxycoumarin-4-yl)-alanine, or 4-(7-hydroxy-4-coumarinyl)-aminobutyric acid; and / or (d) an acidic amino acid is replaced with one of the following: γ-hydroxyglutamic acid, γ-methyleneglutamic acid, γ-carboxyglutamic acid, α-aminoadipic acid, 2-aminoheptanedioic acid, α-aminosuberic acid, 4-carboxyphenylalanine, cysteic acid, 4-phosphonophenylalanine, or 4-sulfomethylphenylalanine. 47. A synthetic peptide shuttle or synthetic peptide shuttle variant as defined in any one of items 32 to 46, for use in an in vitro or in vivo method to increase the transduction efficiency of protein cargo and / or non-protein cargo (e.g., therapeutically active protein cargo and / or non-protein cargo) to target eukaryotic cells, used at a concentration sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the cargo to target eukaryotic cells compared to the absence of the synthetic peptide shuttle or synthetic peptide shuttle variant. 48. A synthetic peptide shuttle or synthetic peptide shuttle variant as defined in any one of items 32-47, for therapeutic use, which transduces therapeutically active protein cargo and / or non-protein cargo into the cytoplasm and / or nucleus of a target eukaryotic cell, and which is used at a concentration sufficient to increase the transduction efficiency of the cargo into the target eukaryotic cell compared to the absence of the synthetic peptide shuttle. 49. An in vitro or in vivo method for transduction of a protein cargo and / or a non-protein cargo, comprising the step of contacting a target eukaryotic cell with a synthetic peptide shuttle or a synthetic peptide shuttle variant as defined in any one of items 32 to 46, at a concentration sufficient to increase the transduction efficiency of the cargo into the target eukaryotic cell compared to the absence of the cargo and the synthetic peptide shuttle. 50. An in vitro or in vivo method described in item 49, which is for therapeutic and / or diagnostic purposes. 51. A composition for therapeutic use comprising a synthetic peptide shuttle or synthetic peptide shuttle variant as defined in any one of items 32 to 46, formulated with a protein cargo and / or non-protein cargo to be transduced into target eukaryotic cells by a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle or synthetic peptide shuttle variant in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the cargo to the target eukaryotic cells at administration compared to the absence of the synthetic peptide shuttle. 52. The composition described in item 51, formulated for topical, enteral / gastrointestinal (e.g., oral), or parenteral administration. 53. A kit comprising a synthetic peptide shuttle agent or a synthetic peptide shuttle agent variant as defined in any one of items 32-46, and a protein cargo and / or non-protein cargo transduced by the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant. 54. A synthetic peptide shuttle agent or synthetic peptide shuttle agent variant as described in any one of items 32-48, an in vitro or in vivo method as described in item 49 or 50, a composition as described in item 51 or 52, or a kit as described in item 53, wherein the target eukaryotic cells are animal cells, mammalian cells, human cells, stem cells, primary cells, immune cells, T cells, NK cells, dendritic cells, epithelial cells, skin cells, or gastrointestinal cells. 55. A synthetic peptide shuttle agent or synthetic peptide shuttle agent variant as described in any one of items 32-48 or 54, wherein the non-protein cargo is as defined in any one of items 2-4, an in vitro or in vivo method of item 49 or 50 or 54, a composition of item 51, 52 or 54, or a kit of item 53 or 54. 56. A method for producing a candidate synthetic peptide shuttle agent that is expected to have transduction activity to a target cargo in target eukaryotic cells, comprising: (1) a peptide having a length of at least 17, 18, 19, or 20 amino acids; (2) an amphiphilic alpha-helix motif; and (3) a peptide comprising an amphiphilic alpha-helix motif having a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein the following parameters (4)~(15): (4) the hydrophobic outer surface is an open syringe of an alpha-helix having 3.6 residues per turn. (1) Based on the labeling, the peptide contains a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12-50% of the peptide's amino acids; (2) The peptide has a hydrophobic moment (μ) of 3.5-11; (3) The peptide has a predicted net charge of at least +4 at physiological pH; (4) The peptide has an isoelectric point (pI) of 8-13; (5) The peptide consists of 35-65% of any combination of amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (6) The peptide contains 0-30% of amino acids (10) The peptide consists of any combination of amino acids: N, Q, S, and T; (11) The peptide consists of any combination of amino acids: A, L, K, or R, provided that at least 5% of L is present in the peptide; (12) The peptide consists of any combination of amino acids: A and L, provided that at least 5% of L is present in the peptide; (13) The peptide consists of any combination of amino acids: K and R, providing that at least 5% of L is present in the peptide; (14) The peptide consists of any combination of amino acids: K and R, providing that at least 5% of L is present in the peptide; (15) The peptide consists of any combination of amino acids: D and E, providing that at least 0% of L is present in the peptide; (16) The peptide consists of any combination of amino acids: K and R, providing that at least 0% of L is present in the peptide; (17) The peptide consists of any combination of amino acids: D and E, providing that at least 0% of L is present in the peptide; (18) The peptide The difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R) is 10% or less; and (15) the peptide comprises the step of synthesizing a peptide that satisfies at least five of the following conditions: the peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H, wherein the shuttle agent is at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.In a eukaryotic cell line model (e.g., HeLa) suitable for increasing the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertors by 5 or 10 times, and / or evaluating cargo transduction in the target eukaryotic cells, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23% of propidium iodide or other membrane-impermeable fluorescent DNA insertors are used. A method enabling transduction efficiencies of %, 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% (for example, as determined by flow cytometry). 57. The method according to item 56, wherein the candidate synthetic peptide shuttle agent is a synthetic peptide shuttle agent or synthetic peptide shuttle agent variant as defined in any one of items 32 to 46. 58. An in vitro or in vivo method for identifying, selecting, or qualifying a synthetic peptide shuttle agent expected to have transduction activity for both protein and non-protein cargoes in target eukaryotic cells, comprising the steps of: providing a model eukaryotic cell or model organism suitable for evaluating cargo transduction in target eukaryotic cells; providing a candidate synthetic peptide shuttle agent (e.g., as defined in any one of items 5-20 or 32-46); and measuring the transduction activity (e.g., cargo transduction efficiency, e.g., by flow cytometry) of the candidate synthetic peptide shuttle agent for transducing propidium iodide or other membrane-impermeable fluorescent DNA insertors into model eukaryotic cells or model organisms, wherein the candidate shuttle agent exhibits transduction activity (e.g., transduction efficiency) of propidium iodide or other membrane-impermeable fluorescent DNA insertors at least 3,3. If the amount increases by 5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times, and / or if the amount of propidium iodide or other membrane-impermeable fluorescent DNA insertion agent is increased by at least 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%, An in vitro or in vivo method that, if a transduction efficiency (e.g., measured by flow cytometry) of 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% occurs in a model eukaryotic cell or model organism, is expected to have transduction activity for both protein and non-protein cargo in target eukaryotic cells. [Examples]

[0129] (Example 1) Materials and methods All materials and methods not described or specified herein were generally those as demonstrated in International Publication No. 2016 / 161516 and / or International Publication No. 2018 / 068135.

[0130] [Table 4]

[0131] [Table 5]

[0132] 1.4 Propidium Iodide Transduction Protocol HeLa cells were seeded in 96-well dishes (20,000 cells / well) the day before the experiment. Each delivery mixture containing synthetic peptide shuttle (10 μM) and propidium iodide (PI) (10 μg / mL) or GFP-NLS (10 μM) was prepared and diluted to 50 μL with phosphate-buffered saline (PBS). The cells were washed once with PBS and the shuttle / PI or shuttle / GFP-NLS was added to the cells for 1 minute. Next, 100 μL of DMEM containing 10% FBS was added to the mixture and removed. The cells were washed once with PBS and incubated in DMEM containing 10% FBS. After 2 hours of incubation by flow cytometry, the cells were analyzed. For the condition "FS, then PI", only synthetic peptide shuttle (10 μM) was added to HeLa cells for 1 minute, and after 1 hour, PI (10 μg / mL) was added for 1 minute after the same washing step. Cells were analyzed one hour after treatment with PI or GFP-NLS.

[0133] 1.5 Transduction protocol for Hedgehog pathway inhibitors in Gli reporter NIH3T3 cells Cargo stock solutions were prepared as follows: Gant61 stock (20 mM in DMSO); HPI4 stock (40 mM in DMSO); itraconazole stock (4.8 mM (4 mg / mL) in DMSO); and arsenic trioxide stock (ATO) stock (40 mM in H2O). Peptide shuttle agent (5 μM) and hedgehog pathway inhibitor (100 μM) were mixed, and the volume was increased to 50 μL in PBS.

[0134] Hedgehog signaling pathway Gli Reporter NIH3T3 cells were cultured in DMEM containing 10% bovine serum. The cells were trypsinized, centrifuged, and resuspended in PBS at a concentration of 10 million cells / mL. 50 μL of cells (500,000 cells / well) were distributed into a round-bottom, untreated 96-well plate. The suspended cells were mixed with a delivery mixture containing a peptide shuttle agent (5 μM) and a Hedgehog pathway inhibitor (100 μM). The cells were incubated with the delivery mixture at room temperature for 90 seconds, 200 μL of DMEM containing 10% calf serum (200 μL) was added to each well, the cells were centrifuged (400 g, 4 minutes), and washed with 200 μL of PBS. The cells were then resuspended in 200 μL of DMEM and transferred to the wells of a 6-well plate containing 1 mL of DMEM with 10% calf serum, and incubated at 37°C for 2 hours. The culture medium was gently removed, and 1 ml of control medium (Opti-MEM®) or activated medium (Opti-MEM, 5 μg / mL mShh) was added to each well. The cells were incubated at 37°C for 24–30 hours.

[0135] For analysis, cells were trypsinized, resuspended in 200 μL of Opti-MEM® in each well, and then equally divided into two wells of a round-bottom 96-well plate. Viability was assessed using flow cytometry, and luminescence was measured using a one-step luciferase assay according to the manufacturer's instructions.

[0136] 1.6 In vivo transduction protocol for Hedgehog pathway inhibitors Cargo was suspended as recommended: 20 mM Gant61 stock in DMSO; 4.8 mM (4 mg / mL) itraconazole stock in DMSO. Female C57BL6 mice 6-7 weeks old were shaved and depilated using the depilation product (Nair®). Five days after depilation, 30 μL of a mixture containing PBS, synthetic peptide shuttle FSD250D (SEQ ID NO: 36), and / or cargo was placed 3 cm from the depilated skin. 2 The solution was applied to the mice. Mice were imaged 3, 10, and 17 days after treatment.

[0137] 1.7 Simultaneous transduction of QX-314 and GFP-NLS and patch-clamp technology Cell culture. HEK293 cells stably expressing Nav1.7 were grown in Dulbecco's Minimum Essential Medium (DMEM, Gibco BRL Life Technologies) supplemented with fetal bovine serum (FBS, 10%), L-glutamine (2 mM), penicillin (100 U / mL), and streptomycin (10 mg / mL). The cells were incubated at 37°C in a 5% CO2 humidified atmosphere.

[0138] Delivery. Cells were seeded in 24-well plates 24 hours prior to the experiment. Cells were washed twice with PBS. Solutions containing 1 mM QX-314, 5 μM FSD194, and 15 μM GFP-NLS protein were applied to the cells for 90 seconds and removed by aspiration. Controls were performed in the presence of GFP-NLS using 5 μM FSD194 or 2.5 mM QX-314. Cells were washed with 800 μL of DMEM containing 10% FBS, transferred to recording solution, and electrophysiology was performed. GFP-positive cells were measured under a microscope and then selected for patch-clamp analysis.

[0139] Electrophysiology. A frequency protocol using QX-314 was recorded within 30 seconds after the entire cell configuration was formed. The frequency protocol consisted of a 10 ms pulse at -20 mV, starting from a holding potential of -140 mV at 10 Hz. The total cellular Na+ current in HEK293 cells was recorded at room temperature using an Axopatch 200B with the entire cell configuration in patch-clamp technique (Molecular Devices). pClamp v10.0 was used for pulse stimulation and recording (Molecular Devices). The current was filtered at 5 kHz, digitized at 100 kHz using a Digidata 1550 AD converter (Molecular Devices), and stored on a computer for further analysis. Series resistance was corrected by 70-80%. Linear leakage current artifacts were removed using online leakage subtraction as needed. Fire-polished low-resistance electrodes (2 MΩ) were drawn from 8161 glass (Corning).

[0140] Recording solutions. Bath solution: 35 mM NaCl, 115 mM NMDG, 2 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 10 mM glucose, 10 mM HEPES. pH adjusted to 7.3 with 1 M NaOH. Pipette solution: 35 mM NaCl, 105 mM CsF, 10 mM EGTA, and 10 mM HEPES. pH adjusted to 7.4 with 1 M CsOH.

[0141] (Example 2) Synthetic peptide shuttle agents enable intracellular delivery of propidium iodide. Propidium iodide (PI) is a fluorescent DNA insertion dye frequently used as a nuclear stain in fluorescence microscopy and flow cytometry applications. Binding of PI to DNA results in a 20-30x fluorescence enhancement and a shift in its maximum excitation / emission spectrum. Because PI typically cannot penetrate the cell membrane of living cells, it is routinely used to detect dead cells in cell populations. Surprisingly, it has been found herein that synthetic peptide shuttles, including the shuttle peptides described in International Publications 2016 / 161516 and 2018 / 068135, for the transduction of protein cargoes, can transduce PI and other non-protein cargoes.

[0142] HeLa cells were cultured as described in Example 1.3 and subjected to the PI transduction protocol as described in Example 1.4, and the protein cargo GFP-NLS was transduced separately as a control in several experiments. The results were obtained by flow cytometry 2 hours after delivery and are shown in Figures 1A-1D and summarized in the table shown in Figure 2, expressed as the percentage of fluorescent cells (%PI+ cells or %GFP+ cells).

[0143] Figures 1 and 2 show the delivery and viability results of HeLa cells co-incubated for 1 minute with either a non-protein cargo PI (Figures 1A and 1B) or a protein cargo GFP-NLS (Figures 1C and 1D) and a synthetic peptide shuttle or control peptide. Multiple members from different families of peptide shuttles or control peptides were tested. The first group of synthetic peptide shuttles tested contained an endosomal leakage domain (ELD) operably linked to a cell membrane permeable domain (CPD), as already described in International Publication 2016 / 161516 for its ability to transduce protein cargoes. The second and third groups of synthetic peptide shuttles tested correspond to those reasonably designed and optimized for protein cargo delivery, with the second group being peptides already described in International Publication 2018 / 068135. The fourth group of synthetic peptide shuttles tested corresponds to cyclic peptides having either an amide bond between its C-terminus and N-terminus (e.g., "FSD268 cyclic amide"; SEQ ID NO: 49) or a disulfide bridge between two adjacent cysteines added at the N and C-terminal positions (e.g., "FSD268 cyclic disulfide"; SEQ ID NO: 50). The fifth group of peptides consists of negative control peptides that do not satisfy some of the synthetic peptide shuttle rational design parameters described in International Publication No. 2018 / 068135 (e.g., FSN3, FSN4, and FSN8; SEQ ID NOs. 54, 55, and 57, respectively). These negative control peptides also include the "FSD10 Scramble" (SEQ ID NO: 51), "FSD268 Scramble" (SEQ ID NO: 52), and "FSD174 Scramble" (SEQ ID NO: 53) peptides, which have the same amino acid composition as the peptide shuttles FSD10, FSD268, and FSD174 (SEQ ID NOs: 13, 43, and 32, respectively), but with altered amino acid order (i.e., primary amino acid sequence) to deviate from some of the rational design parameters described in International Publication No. 2018 / 068135. In Figures 1A and 1B, "FS, then PI" indicates that PI was added 1 hour after treatment with the synthetic peptide shuttle to ensure that the PI-positive signal was not attributable to cell death.Finally, the rightmost bar in Figures 1A-1D corresponds to negative controls ("PI" in Figures 1A and 1B, or "GFP-NLS" in Figures 1C and 1D) where cells were incubated with cargo only, or untreated cells ("NT" in Figures 1A-1D) where cells were not exposed to cargo or shuttle peptides.

[0144] In summary, the results reveal that members of the family of synthetic peptide shuttles, including ELDs operably linked to CPDs (described in International Publication No. 2016 / 161516), as well as those rationally designed for protein cargo transduction (described in International Publication No. 2018 / 068135), can increase the transduction efficiency of relatively low molecular weight non-protein cargoes such as PIs (in addition to their protein transduction activity). Notably, several negative control peptides that did not meet the rational design parameters described in International Publication No. 2018 / 068135 for protein cargo delivery also did not transduce PIs. This suggests that the rational design parameters of International Publication No. 2018 / 068135 are also applicable to the design of peptide shuttles for non-protein cargo delivery.

[0145] Furthermore, the same synthetic peptide shuttle in linear form (FSD268; SEQ ID NO: 43), cyclic form using an amide bond (FSD268 cyclic amide; SEQ ID NO: 49), or cyclic form using a disulfide bond (FSD268 cyclic disulfide; SEQ ID NO: 50) increased PI delivery, confirming that the synthetic shuttle peptide does not need to be functional.

[0146] (Example 3) Synthetic peptide shuttle agents enable intracellular delivery of small molecule inhibitors of the Hedgehog signaling pathway. FSD250D (SEQ ID NO: 36), a rationally designed peptide shuttle agent with efficient transduction activity for protein cargoes, was evaluated for its ability to transduce small molecule inhibitors of the Hedgehog signaling pathway in cultured cells, as described in Example 1.5. The FSD250D peptide has the same amino acid sequence as FSD250 (SEQ ID NO: 35), except that all amino acids in FSD250D are D-amino acids. The results are shown in Figure 3 and Table 1.

[0147] [Table 6]

[0148] In short, the NIH3T3 Gli-luciferase reporter cell line was designed to monitor the activity of the Hedgehog signaling pathway and contains the firefly luciferase gene under the control of a Gli response element stably integrated into NIH3T3 cells. As shown in Figure 3 and Table 1, as a positive control, exposure of recombinant mouse sonic HedgeHog protein NIH3T3 Gli-luciferase reporter cells ("Ctrl+mShh") results in an increase in luminescence intensity not observed in negative control cells ("Ctrl-mSh") that were not exposed to mShh. The presence of the peptide shuttle agent FSD250D did not affect the cellular luminescence intensity after mShh stimulation (data not shown). This was expected given that the receptor for mShh (patched) is located on the cell surface (not intracellular). However, exposure of reporter cells to structurally different small molecule inhibitors of the Hedgehog signaling pathway that bind to intracellular targets (Gant61, HPI-4, itraconazole, or ATO) significantly reduced cell fluorescence intensity in the presence of FSD250D compared to the absence of FSD250D, suggesting successful small molecule transduction by the peptide shuttle. Similar results were observed using peptide FSD19 (data not shown).

[0149] (Example 4) Synthetic peptide shuttle agents enable intracellular delivery of small molecule inhibitors of the Hedgehog signaling pathway. FSD250D (SEQ ID NO: 36), a rationally designed peptide shuttle agent with efficient transduction activity for protein cargoes, was evaluated for its ability to transduce small molecule inhibitors of the Hedgehog signaling pathway in a mouse model of alopecia, as described in Example 1.6.

[0150] In short, alopecia in mouse skin induces hair growth associated with potent induction of the Hedgehog pathway and increased Gli1 expression. This experiment involved activating the Hedgehog pathway in mice by alopecia, and then measuring the delay in hair regrowth by delivering small molecule Hedgehog pathway inhibitors (Gant61 or itraconazole) that bind to intracellular targets to skin cells. The results in Figure 4 show that mice treated with the small molecule Hedgehog inhibitor Gant61 or itraconazole (100 μM) in the presence of FSD250D showed delayed hair regrowth 10 days after treatment (*) compared to mice in the absence of FSD250D.

[0151] (Example 5) The synthetic peptide shuttle enables co-intracellular delivery of small molecule sodium channel inhibitor (QX-314) and GFP-NLS in HEK293 cells. The small molecule compound QX-314 (lidocaine N-ethyl bromide) is a quaternary derivative of lidocaine. QX-314 is not membrane-permeable. When QX-314 is delivered to the cytoplasm, it undergoes rapid Na₂ + Voltage-dependent action potential and voltage-dependent deinactivated sodium +Blocking both conductances (Ilfeld and Yaksh, 2009). To evaluate the simultaneous transduction of small molecules and protein cargoes by peptide shuttles, HEK293 cells stably expressing sodium channel Nav1.7 were exposed to a mixture of QX-314 and GFP-NLS in or without the peptide shuttle FSD194 (SEQ ID NO: 33). As a control, cells were also treated with GFP-NLS and peptide shuttle FSD194 in the absence of QX-314. Results were evaluated using the patch-clamp technique described in Example 1.7, and representative total cellular Na of treated HEK293 cells. + The currents are shown in Figures 5A to 5C. Currents were induced by a 10 ms depolarization pulse at 10 Hz. When cells were incubated for 90 seconds with QX-314 and GFP-NLS (i.e., 1 mM QX-314 + 15 μM GFP-NLS + 5 μM FSD194) in the presence of the peptide shuttle FSD194, a reduction in current amplitude was observed, which was consistent with the presence of QX-314 in the cells (Figure 5C). In contrast, the same reduction in current amplitude was not observed when cells were incubated without QX-314 (i.e., 15 μM GFP-NLS + 5 μM FSD194 +; Figure 5A) or with QX-314, but not in the absence of FSD194 (i.e., 2.5 mM QX-314 + 15 μM GFP-NLS; Figure 5B). Furthermore, GFP-NLS-positive cells were identified under the QX-314+GFP-NLS+FSD194 and FSD194+GFP-NLS conditions, but not under the QX-314+GFP-NLS condition. This indicates that GFP-NLS was indeed co-transduced with QX-314 by the peptide shuttle agent.

[0152] (Example 6) Reliable PI transduction predicts the presence of shuttle agents with protein cargo transduction activity. High-throughput screening attempts to identify, select, and / or qualify novel peptide shuttles with protein transduction activity can quickly become prohibitively expensive, especially for complex proteins such as recombinant immunoglobulins, due to the high cost of manufacturing and purifying large quantities of recombinant proteins as cargo. Using GFP or GFP-NLS as protein cargo is advantageous for enabling rapid screening by flow cytometry and for evaluating intracellular delivery. However, the use of GFP-NLS requires microscopic validation of each peptide shuttle in parallel with flow cytometry measurements to ensure that candidate shuttles allow GFP-NLS cargoes to access the cytoplasm / nucleus, which is a resource and time-consuming process and avoids endosomal confinement. Therefore, a more cost-effective "surrogate" cargo that can reliably predict protein transduction activity and endosomal escape is highly desirable.

[0153] The results of Example 2 demonstrate that synthetic peptide shuttles with validated transduction activity for GFP (and other protein cargoes) can also transduce small molecules such as PI. This raises the intriguing possibility that the reverse is true: whether PI can be used as a reliable "surrogate" cargo to screen, identify / select / qualify novel shuttles with reliable transduction activity for protein cargoes. Commercially, PI is widely available and relatively inexpensive. Furthermore, PI exhibits 20-30 times enhanced fluorescence and a detectable shift in the maximum excitation / emission spectrum only after binding to genomic DNA. This property is particularly suitable for distinguishing cargo trapped in endosomes from cargo released by endosomes that access the cytoplasmic / nuclear compartment. Therefore, since no PI remaining trapped in endosomes reaches the nucleus and does not exhibit enhanced fluorescence or spectral shift, both intracellular delivery and endosomal escape are measurable by flow cytometry.

[0154] To evaluate the suitability of PI as a "surrogate" cargo for novel shuttle agents, a proprietary library of over 300 candidate peptide shuttle agents was screened in parallel for both PI and GFP-NLS transduction activity in HeLa cells using flow cytometry, as generally described in Example 1.4. The transduction protocols were identical except for the cargo concentration (i.e., 10 μg / mL of PI versus 10 μM of GFP-NLS).

[0155] For the numerous peptides screened, negative controls were performed in parallel for each experimental batch, including “untreated” (NT) controls in which cells were not exposed to the shuttle peptide or cargo, and “cargo alone” controls in which cells were exposed to the cargo in the absence of the shuttle agent. The results are shown in Figures 6 and 7, where “transduction efficiency” refers to the percentage of overall viable cells that are positive for the cargo (PI or GFP-NLS). “Mean delivery score” provides a further indicator of the total amount of cargo delivered per cell among all cargo-positive cells. The mean PI or GFP-NLS delivery score was calculated by multiplying the mean fluorescence intensity measured for viable PI+ or GFP+ cells (at least for duplicate samples) by the mean percentage of viable PI+ or GFP+ cells, and dividing by 100,000 for GFP delivery, or by 10,000 for PI delivery. Next, the average delivery scores for PI and GFP-NLS for each candidate shuttle agent were standardized by dividing them by the average delivery score for the "cargo alone" negative control, which was performed in parallel for each experimental batch. Therefore, the "standardized average delivery score" in Figures 6 and 7 shows that the average delivery score doubled compared to the "cargo only" negative control.

[0156] The batch-to-batch variability observed in the negative control was relatively small with GFP-NLS, but considerably higher with PI as the cargo. For example, the variability in transduction efficiency of the "cargo alone" negative control ranged from 0.4% to 1.3% with GFP-NLS and from 0.9% to 6.3% with PI. Furthermore, the transduction efficiency of several negative control peptides (i.e., peptides known to have low or no GFP transduction activity) tested in parallel (e.g., FSD174 scramble; data not shown) sometimes showed that PI (but not GFP-NLS) had a transduction efficiency that was sometimes as low as 5% lower than the "cargo alone" negative control, likely due to nonspecific interactions between PI and the peptide. This phenomenon was not observed in the GFP-NLS transduction experiments. The above suggests that the transduction efficiency of the shuttle agent, at least for PI, may be more appropriate compared to that of the negative control peptide rather than under the "cargo alone" condition.

[0157] Screening of over 300 candidate peptide shuttles for PI and GFP-NLS transduction activity revealed that shuttles exhibiting reliable transduction efficiency for PI generally correlated with reliable transduction efficiency for GFP-NLS. Surprisingly, progressively higher PI transduction efficiency was generally associated with progressively higher GFP-NLS transduction efficiency. This is demonstrated by grouping all screened candidate shuttles into incremental windows according to their PI transduction efficiency, as shown in the table below, and then calculating the average GFP transduction efficiency for all shuttles within that %PI window.

[0158] [Table 7]

[0159] Figure 6 shows the results for all screened candidate peptide shuttles with an average PI transduction efficiency of 10% or higher, selected based on the level of average PI transduction efficiency. Surprisingly, of the 306 candidate peptide shuttles with an average PI transduction efficiency of at least 10%, 96% showed a GFP transduction efficiency of 10% or higher. The thresholds for PI transduction efficiencies of at least 15% and 20% correspond to values ​​at least 2.5 and 3 times higher than the best PI transduction efficiencies observed for the "cargo alone" negative control (approximately 6%) in all experimental batches. Of the 273 candidate peptide shuttles listed in Figure 6 with an average PI transduction efficiency of at least 15%, 97% showed a GFP transduction efficiency of 15% or higher. Furthermore, of the 256 candidate peptide shuttles listed in Figure 6 that had an average PI transduction efficiency of at least 20%, 99.6% of the candidate peptide shuttles showed a GFP transduction efficiency of 10% or more, and 96% of the candidate peptide shuttles showed a GFP transduction efficiency of 20% or more.

[0160] These results strongly suggest that reliable PI delivery predicts peptide shuttle agents with reliable protein cargo transduction activity, and therefore PI can be practically used as a "surrogate" cargo for screening and identifying / selecting / qualifying novel peptide shuttle agents with dual cargo transduction activity (i.e., small molecules and proteins).

[0161] Among the candidate peptide shuttles in Figure 6 that had an average PI transduction efficiency of at least 20%, peptides with lengths of less than 20 residues were included: FSD390 (17aa), FSD367 (19aa), and FSD366 (18aa). Furthermore, among the candidate peptide shuttle agents in Figure 6, which had an average PI transduction efficiency of at least 20%, were peptides containing either non-physiological amino acid analogs (e.g., FSD435, corresponding to FSD395 except for the lysine residue (K) replaced by an L-2,4-diaminobutyric acid residue) or chemical modifications (e.g., FSD438, corresponding to FSD10 except for the N-terminal octanoic acid modification; FSD436, corresponding to FSD222 except for the phenylalanine residue (F) replaced by a (2-naphthyl)-L-alanine residue; and FSD171, corresponding to FSD168 except for having an N-terminal acetyl group and a C-terminal cysteamide group). These results confirm the reliability of the peptide shuttle agent platform technology to withstand the use of non-physiological amino acids or their analogs instead of physiological amino acids and / or chemical modifications.

[0162] (Example 7) Low levels of PI delivery result in poor predictiveness for peptide shuttle agents with protein cargo transduction activity. Figure 7 shows the results for over 300 candidate peptide shuttle agents screened in Example 6, which had an average PI transduction efficiency of less than 10% but an average GFP-NLS transduction efficiency of at least 7%, and at this point were sorted according to their average GFP transduction efficiency levels.

[0163] For candidate peptides with PI transduction efficiencies of less than 10%, the large-scale nature of the screening approach used herein may prevent any firm conclusions regarding their potential lack of cargo transduction activity. Indeed, International Publications 2016 / 161516 and 2018 / 068135 disclose that shuttle peptides function in a concentration-dependent manner, and that multiple factors such as shuttle concentration, cargo concentration, exposure time, and cell type can affect shuttle performance in transduction assays. The large-scale screening of candidate peptide shuttles described herein imposed a single "blanket" shuttle concentration, a single cargo concentration, and a single exposure time / protocol for each of the peptides tested. Thus, it is difficult to draw firm conclusions regarding the cargo transduction activity of non-proteins based solely on the low PI transduction efficiencies observed in this large-scale screening.

[0164] [References] TIFF0007883371000010.tif47170TIFF0007883371000011.tif187170TIFF0007883371000012.tif192170TIFF0007883371000013.tif117170

Claims

1. A synthetic peptide shuttle agent having transduction activity for both protein cargo and non-protein cargo in target eukaryotic cells, wherein the synthetic peptide shuttle agent is (3) (a) on a helical wheel projection, at least two adjacent positively charged K and / or R residues; and (b) on a helical wheel projection, a positively charged hydrophilic outer surface including a segment of six adjacent residues containing three to five K and / or R residues, based on an alpha helix with a rotation angle of 100 degrees between consecutive amino acids and / or an alpha helix with 3.6 residues per turn, and (4) A hydrophobic outer surface comprising a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12-50% of the amino acids of the peptide, based on an open cylinder representation of an alpha helix having 3.6 residues per turn, wherein the hydrophobic outer surface comprises (a) at least two adjacent L residues on a helical wheel projection; and / or (b) a hydrophobic outer surface comprising a segment of 10 adjacent residues, based on an alpha helix having a rotation angle of 100 degrees between consecutive amino acids and / or an alpha helix having 3.6 residues per turn, comprising at least five hydrophobic residues selected from L, I, F, V, W, and M. Having, (2) Amphipathic alpha helix motif including, (1) Peptides with a length of at least 20 amino acids And, The following parameters (5) to (15): (5) The peptide has a hydrophobic moment (μ) of 3.5 to 11; (6) The peptide has a predicted net charge of at least +4 at physiological pH; (7) The peptide has an isoelectric point (pI) of 8 to 13; (8) The peptide is composed of 35% to 65% of any combination of amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (9) The peptide is composed of any combination of amino acids N, Q, S, and T, from 0% to 30%; (10) The peptide is composed of 35% to 85% of any combination of amino acids: A, L, K, or R; (11) The peptide is composed of any combination of amino acids A and L, with at least 5% of L present in the peptide; (12) The peptide is composed of any combination of amino acids K and R, from 20% to 45%; (13) The peptide is composed of any combination of amino acids D and E, from 0% to 10%; (14) The difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R%) is 10% or less; and (15) The peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H At least eight of the following conditions must be met: In a eukaryotic cell line model suitable for evaluating cargo transduction in target eukaryotic cells, the synthetic peptide shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to a corresponding negative control lacking the synthetic peptide shuttle, and / or the synthetic peptide shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by at least 10 times compared to the synthetic peptide shuttle. Enabling transduction efficiencies of %, 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%, and / or The synthetic peptide shuttle increases the transduction efficiency of GFP-NLS by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to the corresponding negative control lacking the synthetic peptide shuttle, and / or in eukaryotic cell line models suitable for evaluating cargo transduction in target eukaryotic cells, at least 7%, 8, 9, 10%, 11%, 12%, 13%, 1% of GFP-NLS. It enables transduction efficiencies of 4%, 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%, and The aforementioned synthetic peptide shuttle agent (a) Amino acid sequence of Sequence ID No. 35; (b) One, two, or three or fewer amino acids, excluding the GGSGGGS linker domain of SEQ ID NO: 35, that have an amino acid sequence different from the amino acid sequence of SEQ ID NO: 35; or (c) An amino acid sequence that, after calculation excluding the GGSGGGS linker domain of SEQ ID NO: 35, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35; Includes or consists of Synthetic peptide shuttle agent.

2. (a) The synthetic peptide shuttle agent satisfies at least nine, at least ten, or all of parameters (5) to (15); (b) The synthetic peptide shuttle is a peptide having a minimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids; (c) The amphiphilic alpha helix motif is 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, It has a hydrophobic moment (μ) between the lower limit of 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and the upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.

0. (d) The amphiphilic alpha-helix motif includes a positively charged hydrophilic outer surface, the outer surface including at least three or four adjacent positively charged K and / or R residues on the helical wheel projection; (e) The amphiphilic alpha-helix motif includes a hydrophobic outer surface, the outer surface including (i) at least two adjacent L residues on a helical wheel projection; and (ii) a segment of 10 adjacent residues on a helical wheel projection, based on an alpha-helix having a rotation angle of 100 degrees between consecutive amino acids and / or an alpha-helix having 3.6 residues per turn, including at least five hydrophobic residues selected from L, I, F, V, W, and M; (f) The hydrophobic outer surface includes a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% to 25%, 30%, 35%, 40%, or 45% of the amino acids of the synthetic peptide shuttle; (g) The synthetic peptide shuttle agent has a hydrophobic moment (μ) between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5, (h) The synthetic peptide shuttle agent has a predicted net charge between +4, +5, +6, +7, +8, +9 and +10, +11, +12, +13, +14, or +15; (i) The synthetic peptide shuttle agent has a predicted pI of 10 to 13; or (j)(a) to (i) any combination of the synthetic peptide shuttle agent according to claim 1.

3. The synthetic peptide shuttle agent according to claim 1 or 2, wherein the synthetic peptide shuttle agent satisfies at least one, at least two, at least three, at least four, at least five, at least six, or all of the following parameters: (8) The synthetic peptide shuttle agent is composed of any combination of amino acids A, C, G, I, L, M, F, P, W, Y, and V in amounts of 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60%; (9) The synthetic peptide shuttle agent consists of any combination of amino acids:N,Q,S, andT in amounts of 1% to 29%, 2% to 28%, 3% to 27%, 4% to 26%, 5% to 25%, 6% to 24%, 7% to 23%, 8% to 22%, 9% to 21%, or 10% to 20%; (10) The synthetic peptide shuttle consists of any combination of amino acids A, L, K, or R in amounts of 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60%; (11) The synthetic peptide shuttle agent is composed of any combination of amino acids A and L in amounts of 15% to 40%, 20% to 40%, 20% to 35%, or 20% to 30%; (12) The synthetic peptide shuttle agent consists of any combination of amino acids K and R in amounts of 20% to 40%, 20% to 35%, or 20% to 30%; (13) The synthetic peptide shuttle agent is composed of any combination of amino acids D and E in an amount of 5% to 10%; (14) The difference between the percentage of A and L residues in the synthetic peptide shuttle (A+L%) and the percentage of K and R residues in the synthetic peptide shuttle (K+R%) is 9%, 8%, 7%, 6%, or 5% or less; and (15) The synthetic peptide shuttle agent is composed of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H in amounts of 15-40%, 20-35%, or 20-30%.

4. The synthetic peptide shuttle according to any one of claims 1 to 3, wherein the synthetic peptide shuttle comprises a histidine-rich domain.

5. The synthetic peptide shuttle according to any one of claims 1 to 4, wherein the synthetic peptide is a cyclic peptide; comprises one or more D-amino acids; and / or further comprises chemical modifications to one or more amino acids, the chemical modifications not destroying the transduction activity of the synthetic peptide shuttle.

6. The synthetic peptide shuttle according to claim 5, wherein the chemical modification is located at the N-terminus and / or C-terminus of the synthetic peptide shuttle, and / or the chemical modification is the addition of an acetyl group, a cysteamide group, or a fatty acid.

7. A synthetic peptide shuttle agent having transduction activity for both protein cargo and non-protein cargo in target eukaryotic cells, wherein the synthetic peptide shuttle agent is - The amino acid sequence defined in claim 1(a); or, - An amino acid sequence that differs from the amino acid sequence defined in claim 1(a) from the amino acid sequence of SEQ ID NO: 35, except for the GGSGGGS linker domain, by only conserved amino acid substitutions, wherein each conserved amino acid substitution is selected from amino acids within the same amino acid class, and the amino acid class is aliphatic: G, A, V, L, and I; hydroxyl or sulfur / selenium-containing: S, C, U, T, and M; aromatic: F, Y, and W; basic: H, K, and R; acidic and their amides: D, E, N, and Q, and the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35, calculated excluding the GGSGGGS linker domain of SEQ ID NO:

35. It includes or consists of; In a eukaryotic cell line model suitable for evaluating cargo transduction in target eukaryotic cells, the synthetic peptide shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to a corresponding negative control lacking the synthetic peptide shuttle, and / or the addition of at least 1 propidium iodide or other membrane-impermeable fluorescent DNA insertion agent. Enabling transduction efficiencies of 0%, 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%. Synthetic peptide shuttle agent.

8. A synthetic peptide shuttle variant having transduction activity for protein cargo and / or non-protein cargo in target eukaryotic cells, wherein the synthetic peptide shuttle variant is identical to the synthetic peptide shuttle according to any one of claims 1 to 7, except that at least one amino acid is replaced by a corresponding synthetic amino acid having a side chain with similar physiological and chemical properties to the amino acid being replaced, wherein the synthetic peptide shuttle variant comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35, calculated excluding the GGSGGGS linker domain of SEQ ID NO: 35, and the synthetic peptide shuttle variant increases the transduction efficiency of the cargo in the target eukaryotic cells compared to the absence of the synthetic peptide shuttle variant, and the replacement of the synthetic amino acid is (a) Replace a basic amino acid with any one of the following: α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, 2,6-diamino-4-hexic acid, β-(1-piperazinyl)-alanine, 4,5-dehydrolysine, δ-hydroxylysine, ω,ω-dimethylarginine, homoarginine, ω,ω'-dimethylarginine, ω-methylarginine, β-(2-quinolyl)-alanine, 4-aminopiperidine-4-carboxylic acid, α-methylhistidine, 2,5-diiodohistidine, 1-methylhistidine, 3-methylhistidine, spinacine, 4-aminophenylalanine, 3-aminotyrosine, β-(2-pyridyl)-alanine, or β-(3-pyridyl)-alanine; (b) Dehydroalanine, β-fluoroalanine, β-chloroalanine, β-rhodoalanine, α-aminobutyric acid, α-aminoisobutyric acid, β-cyclopropylalanine, azetidine-2-carboxylic acid, α-allylglycine, propargylglycine, tert-butylalanine, β-(2-thiazolyl)-alanine, thiaproline, 3,4-dehydroproline, tert-butylglycine, β-cyclopentylalanine, β-cyclohexylalanine, α-meth Luproline, norvaline, α-methylvaline, penicillamine, β,β-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, pipecolic acid, 4,5-dehydroleucine, alloisoleucine, norleucine, α-methylleucine, cyclohexylglycine, cis-octahydroindole-2-carboxylic acid, β-(2-thienyl)-alanine, phenylglycine, α-methylphenylalanine, homophenylalanine, Replace a nonpolar (hydrophobic) amino acid with one of the following: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-(3-benzothienyl)-alanine, 4-nitrophenylalanine, 4-bromophenylalanine, 4-tert-butylphenylalanine, α-methyltryptophan, β-(2-naphthyl)-alanine, β-(1-naphthyl)-alanine, 4-iodophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, 4-methyltryptophan, 4-chlorophenylalanine, 3,4-dichlorophenylalanine, 2,6-difluorophenylalanine, n-in-methyltryptophan, 1,2,3,4-tetrahydronorharmann-3-carboxylic acid, β,β-diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, or 4-benzoylphenylalanine; (c)β-Cyanolalanine, β-Ureidoalanine, Homocysteine, Allosreonine, Pyroglutamic acid, 2-Oxothiazolidine-4-carboxylic acid, Citrulline, Thiocitrulline, Homocitrulline, Hydroxyproline, 3,4-Dihydroxyphenylalanine, β-(1,2,4-Triazole-1-yl)alanine, 2-Mercaptohistidine, β-(3,4-Dihydroxyphenyl)-serine, β-(2-Thienyl)-serine, 4-Azidophenylalanine, 4-Cyanofenyl Replace any polar uncharged amino acid with one of the following: rualanine, 3-hydroxymethyltyrosine, 3-iodotyrosine, 3-nitrotyrosine, 3,5-dinitrotyrosine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 5-hydroxytryptophan, tyronine, β-(7-methoxycoumarin-4-yl)alanine, or 4-(7-hydroxy-4-coumarinyl)-aminobutyric acid; and / or (d) Replace the acidic amino acid with one of the following: γ-hydroxyglutamic acid, γ-methyleneglutamic acid, γ-carboxyglutamic acid, α-aminoadipic acid, 2-aminoheptanedioic acid, α-aminosuberic acid, 4-carboxyphenylalanine, cysteic acid, 4-phosphonophenylalanine, or 4-sulfomethylphenylalanine. A variant of a synthetic peptide shuttle agent.

9. - For use in in vitro or ex vivo methods to increase the transduction efficiency of protein cargo and / or non-protein cargo to target eukaryotic cells, wherein the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is used at a concentration sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the cargo to the target eukaryotic cells compared to the absence of the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant; or - For therapeutic use, the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant transduces therapeutically active protein cargo and / or non-protein cargo into the cytoplasm and / or nucleus of target eukaryotic cells, and the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is used at a concentration sufficient to increase the transduction efficiency of the cargo into the target eukaryotic cells compared to the absence of the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant. A synthetic peptide shuttle agent or a synthetic peptide shuttle agent variant according to any one of claims 1 to 8.

10. A composition comprising a synthetic peptide shuttle agent or synthetic peptide shuttle agent variant according to any one of claims 1 to 8 for use in an in vitro or ex vivo method for transduction of protein cargo and / or non-protein cargo, wherein the method comprises the step of contacting target eukaryotic cells with the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant according to any one of claims 1 to 8 in a concentration sufficient to increase the transduction efficiency of the cargo into the target eukaryotic cells compared to the absence of the cargo and the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant.

11. A composition for therapeutic use, comprising a synthetic peptide shuttle agent or synthetic peptide shuttle agent variant according to any one of claims 1 to 8, wherein the composition is formulated together with protein cargo and / or non-protein cargo to be transduced into target eukaryotic cells by the synthetic peptide shuttle agent, the concentration of the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the cargo to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant.

12. A composition for treating a disease that can be treated by intracellular delivery of therapeutically active protein cargo and / or non-protein cargo, wherein the composition comprises a synthetic peptide shuttle or synthetic peptide shuttle variant according to any one of claims 1 to 8, formulated together with the therapeutically active protein cargo and / or non-protein cargo to be transduced into target eukaryotic cells by the synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle or synthetic peptide shuttle variant in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the cargo to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle or synthetic peptide shuttle variant.

13. The composition for use according to any one of claims 10 to 12, further comprising a non-protein cargo that is transduced into a target eukaryotic cell by the synthetic peptide shuttle agent or a synthetic peptide shuttle agent variant.

14. A method for producing a candidate synthetic peptide shuttle agent that is expected to have transduction activity to a target cargo in target eukaryotic cells, wherein the method comprises a step of synthesizing a peptide, and the peptide is (3) (a) on a helical wheel projection, at least two adjacent positively charged K and / or R residues; and (b) on a helical wheel projection, a positively charged hydrophilic outer surface including a segment of six adjacent residues containing three to five K and / or R residues, based on an alpha helix with a rotation angle of 100 degrees between consecutive amino acids and / or an alpha helix with 3.6 residues per turn, and (4) A hydrophobic outer surface comprising a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12-50% of the amino acids of the peptide, based on an open cylinder representation of an alpha helix having 3.6 residues per turn, wherein the hydrophobic outer surface comprises (a) at least two adjacent L residues on a helical wheel projection; and / or (b) a hydrophobic outer surface comprising a segment of 10 adjacent residues, based on an alpha helix having a rotation angle of 100 degrees between consecutive amino acids and / or an alpha helix having 3.6 residues per turn, comprising at least five hydrophobic residues selected from L, I, F, V, W, and M. Having, (2) Amphipathic alpha helix motif including, (1) Peptides with a length of at least 20 amino acids And, The following parameters (5) to (15): (5) The peptide has a hydrophobic moment (μ) of 3.5 to 11; (6) The peptide has a predicted net charge of at least +4 at physiological pH; (7) The peptide has an isoelectric point (pI) of 8 to 13; (8) The peptide is composed of 35% to 65% of any combination of amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (9) The peptide is composed of any combination of amino acids N, Q, S, and T, from 0% to 30%; (10) The peptide is composed of 35% to 85% of any combination of amino acids: A, L, K, or R; (11) The peptide is composed of any combination of amino acids A and L, with at least 5% of L present in the peptide; (12) The peptide is composed of any combination of amino acids K and R, from 20% to 45%; (13) The peptide is composed of any combination of amino acids D and E, from 0% to 10%; (14) The difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R%) is 10% or less; and (15) The peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H; At least eight of the following conditions must be met: In a eukaryotic cell line model suitable for evaluating cargo transduction in target eukaryotic cells, the synthetic peptide shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to a corresponding negative control lacking the synthetic peptide shuttle, and / or the synthetic peptide shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA insertion agents by at least 10 times compared to the synthetic peptide shuttle. Enabling transduction efficiencies of %, 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%, and / or The synthetic peptide shuttle increases the transduction efficiency of GFP-NLS by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times compared to the corresponding negative control lacking the synthetic peptide shuttle, and / or in eukaryotic cell line models suitable for evaluating cargo transduction in target eukaryotic cells, at least 7%, 8, 9, 10%, 11%, 12%, 13%, 1% of GFP-NLS. It enables transduction efficiencies of 4%, 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%, and The aforementioned synthetic peptide shuttle agent (a) Amino acid sequence of Sequence ID No. 35; (b) One, two, or three or fewer amino acids, excluding the GGSGGGS linker domain of SEQ ID NO: 35, that have an amino acid sequence different from the amino acid sequence of SEQ ID NO: 35; or, (c) An amino acid sequence that, after calculation excluding the GGSGGGS linker domain of SEQ ID NO: 35, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

35. Includes or consists of method.

15. A composition for use in an in vivo or ex vivo method for transduction of a non-protein cargo, the method comprising the step of contacting target eukaryotic cells with a concentration of the synthetic peptide shuttle or synthetic peptide shuttle variant sufficient to increase the transduction efficiency of the non-protein cargo compared to the absence of the non-protein cargo and the synthetic peptide shuttle or synthetic peptide shuttle variant, wherein the non-protein cargo (a) It is an organic compound; (b) Molecular weight less than 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000 or 1000 Da, or between 50 and 5000, 50 and 4000, 50 and 3000, 50 and 2000 or 50 and 1000 Da; (c) It is a small molecule; (d) Not polynucleotides or polysaccharides; (e) Not covalently bonded with the synthetic peptide shuttle agent during transduction; or (f) Any combination of (a) to (e), The synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is as defined in any one of claims 1 to 8. composition.

16. A composition comprising a synthetic peptide shuttle agent or a synthetic peptide shuttle agent variant, - For use in in vitro or ex vivo methods to increase the transduction efficiency of non-protein cargo into target eukaryotic cells, provided that the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is used at a concentration sufficient to increase the transduction efficiency and cytoplasmic and / or nuclear delivery of the cargo into the target eukaryotic cells compared to the absence of the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant; or - For therapeutic use, the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is used at a concentration sufficient to transduce a therapeutically active non-protein cargo into the cytoplasm and / or nucleus of target eukaryotic cells, and the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is used at a concentration sufficient to increase the transduction efficiency of the cargo into the target eukaryotic cells compared to the absence of the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant. The aforementioned non-protein cargo, (a) It is an organic compound; (b) Molecular weight less than 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000 or 1000 Da, or between 50 and 5000, 50 and 4000, 50 and 3000, 50 and 2000 or 50 and 1000 Da; (c) It is a small molecule; (d) Not polynucleotides or polysaccharides; (e) Not covalently bonded with the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant during transduction; or (f) Any combination of (a) to (e), The synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is as defined in any one of claims 1 to 8. composition.

17. A therapeutic composition comprising a synthetic peptide shuttle or synthetic peptide shuttle variant formulated with a therapeutically active non-protein cargo that is transduced into target eukaryotic cells by the synthetic peptide shuttle or synthetic peptide shuttle variant, wherein the concentration of the synthetic peptide shuttle or synthetic peptide shuttle variant in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the cargo to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle or synthetic peptide shuttle variant, and the non-protein cargo is (a) It is an organic compound; (b) Molecular weight less than 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000 or 1000 Da, or between 50 and 5000, 50 and 4000, 50 and 3000, 50 and 2000 or 50 and 1000 Da; (c) It is a small molecule; (d) Not polynucleotides or polysaccharides; (e) Not covalently bonded with the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant during transduction; or (f) Any combination of (a) to (e), The synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is as defined in any one of claims 1 to 8. composition.

18. A composition for treating a disease treatable by intracellular delivery of a therapeutically active non-protein cargo, wherein the composition comprises a synthetic peptide shuttle or synthetic peptide shuttle variant formulated with the therapeutically active non-protein cargo, which is transduced into target eukaryotic cells by the synthetic peptide shuttle or synthetic peptide shuttle variant, wherein the concentration of the synthetic peptide shuttle or synthetic peptide shuttle variant in the composition is sufficient to increase the transduction efficiency and cytoplasmic delivery of the cargo to the target eukaryotic cells upon administration compared to the absence of the synthetic peptide shuttle or synthetic peptide shuttle variant, and the non-protein cargo is (a) It is an organic compound; (b) Molecular weight less than 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000 or 1000 Da, or between 50 and 5000, 50 and 4000, 50 and 3000, 50 and 2000 or 50 and 1000 Da; (c) It is a small molecule; (d) Not polynucleotides or polysaccharides; (e) Not covalently bonded with the synthetic peptide shuttle agent or synthetic peptide shuttle agent variant during transduction; or (f) Any combination of (a) to (e), The synthetic peptide shuttle agent or synthetic peptide shuttle agent variant is as defined in any one of claims 1 to 8. composition.

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