Peptide-based non-protein payload delivery

Abstract

Described herein are methods, compositions, kits, and synthetic peptide shuttles involving transduction of protein and / or non-protein cargoes. The methods generally comprise contacting a target eukaryotic cell with a non-protein cargo and a concentration of a synthetic peptide shuttle sufficient to increase the transduction efficiency of the non-protein cargo as compared to the absence of the synthetic peptide shuttle. 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 shuttles active in transducing 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 cargoes for selecting multifunctional synthetic peptide shuttles active in transducing both protein and non-protein cargoes.

Description

[0001] This specification relates to intracellular delivery of non-protein loads. More specifically, this specification relates to the use of synthetic peptide shuttles for intracellular delivery of small molecules and other non-protein loads, and to synthetic peptide shuttles that improve transduction activity for both proteins and small molecules.

[0002] This manual cites numerous references, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Most drugs have traditionally been small-molecule organic compounds, small enough and lipophilic enough to cross cell membranes to bind to intracellular targets. In conventional drug discovery, small-molecule drug candidates are typically selected not only based on their affinity for their biological targets but also on their drug-like physicochemical properties, which in particular control their ability to be delivered intracellularly and reach their targets. Therefore, compounds identified as exhibiting high target-binding affinity and specificity in large-scale screening efforts under conventional drug development thinking may ultimately be abandoned as clinical drug candidates due to their diminished intracellular delivery capabilities. Furthermore, even cell membrane-permeable compounds can benefit from improved intracellular / cytosol delivery, for example, to increase the rate of uptake and / or reduce the concentration required to achieve the desired biological effect. Therefore, there is a need for technologies that facilitate intracellular / cytosol delivery of small-molecule loads to provide greater flexibility in drug design and potentially open the door to novel therapeutic compounds that might otherwise be abandoned based on traditional small-molecule drug design. Summary of the Invention

[0004] Synthetic peptide shuttles represent a recently defined family of peptides previously reported for rapidly and efficiently transducing protein loads into the cytosol and / or nucleus of various target eukaryotic cells. First-generation such peptide shuttles are described in WO / 2016 / 161516, wherein the peptide shuttle comprises an endosome leakage domain (ELD) operably linked to a cell penetration domain (CPD). WO / 2018 / 068135 subsequently describes other synthetic peptide shuttles rationally designed based on a set of fifteen design parameters aimed at improving protein load transduction while reducing the toxicity of first-generation peptide shuttles. This disclosure relates to the finding that such synthetic peptide shuttles, previously reported for transducing large protein loads, also typically possess the ability to rapidly and efficiently transduce smaller non-protein loads (e.g., small molecule organic compounds). The experimental results presented in Example 2 demonstrate that synthetic peptide shuttles comprising representative members of the shuttles described in WO / 2016 / 161516 and WO / 2018 / 068135, as well as other rationally designed shuttles, can transduce the membrane-impermeable fluorescent dye propidium iodide (PI), which can be considered a small molecule organic compound load. Notably, the negative control peptide, which failed to adhere to the key rational design parameters for protein load delivery described in WO / 2018 / 068135, also failed to transduce PI, indicating that the rational design parameters for protein load delivery in WO / 2018 / 068135 can generally also be applied to the design of peptide shuttles for delivering non-protein loads. Example 3 shows that the representative synthetic peptide shuttle can not only deliver a structure-independent small molecule inhibitor of the hedgehog signaling pathway intracellularly into cultured cells, but also that the delivered inhibitor freely binds to its intracellular target and exerts its inhibitory activity. Example 4 demonstrates that the representative synthetic peptide shuttle, upon local application in shaved mice, enables in vivo delivery and activity of a small molecule inhibitor of hedgehog signaling. In Example 5, different representative synthetic peptide shuttles were shown to enable intracellular delivery of a membrane-impermeable small molecule compound (QX-314) as a sodium channel inhibitor, resulting in a correlated reduction in induced current amplitude, as measured by patch clamp. Finally, Examples 6 and 7 show the results of a large-scale screening of over 300 candidate peptide shuttles for PI and GFP-NLS transduction activities, revealing a significant correlation between PI transduction efficiency and GFP-NLS transduction efficiency, indicating that robust PI transduction predicts shuttles with protein loading transduction activity.

[0005] In some respects, this document describes a method for non-protein load transduction, the method comprising contacting target eukaryotic cells with a non-protein load and a certain concentration of a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle is sufficient to increase the transduction efficiency of the non-protein load compared to the absence of the synthetic peptide shuttle.

[0006] In some respects, this document describes a composition for transducing a non-protein load into target eukaryotic cells, the composition comprising a synthetic peptide shuttle formulated with a pharmaceutically suitable excipient, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the non-protein load into the target eukaryotic cells and cytosol and / or nuclear delivery after administration.

[0007] In some respects, this document describes a composition for a therapy comprising the synthetic peptide shuttle formulated together with a non-protein load (e.g., a therapeutic or biologically active non-protein load) to be transduced into target eukaryotic cells by a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the non-protein load into the target eukaryotic cells and cytosol and / or nuclear delivery after administration.

[0008] In some aspects, this document describes synthetic peptide shuttles with transduction activity for both protein and non-protein loads, said shuttles comprising or consisting of an amino acid sequence of any one of SEQ ID NO:1 to 50. In some aspects, this document describes synthetic peptide shuttles with transduction activity for both protein and non-protein loads, said shuttles comprising or consisting of an amino acid sequence that is approximately equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of any one of SEQ ID NO:1 to 50 (e.g., excluding any linker domains, such as linker domains rich in flexible serine / glycine). In some aspects, this document describes synthetic peptide shuttles with transduction activity for both protein and non-protein loads, said shuttles comprising or consisting of an amino acid sequence that is approximately equal to or less than 10 amino acids of any one of SEQ ID NO:1 to 50 (e.g., excluding any linker domains, such as linker domains rich in flexible serine / glycine). In some aspects, this document describes synthetic peptide shuttles with transduction activity for both protein and non-protein loads, said shuttles comprising or consisting of an amino acid sequence that is approximately equal to or less than 10 amino acids of any one of SEQ ID NO:1 to 50. NO: Any one of 1 to 50 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 (e.g., calculated excluding any linker domains, such as linker domains rich in flexible serine / glycine).

[0009] In some respects, this paper describes a synthetic peptide shuttle that exhibits transduction activity against both protein and non-protein loads in target eukaryotic cells, wherein the shuttle is:

[0010] (1) A peptide having a length of at least 17, 18, 19, or 20 amino acids, said peptide comprising

[0011] (2) Amphiphilic α-helical motifs, which have

[0012] (3) Positively charged hydrophilic and hydrophobic outer surfaces.

[0013] Among them, at least five of the following parameters (4) to (15) must be met:

[0014] (4) Based on an open cylindrical representation of an α-helix with 3.6 residues per turn, the hydrophobic outer surface comprises a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W and / or M amino acids, which account for 12% to 50% of the amino acids of the peptide.

[0015] (5) The peptide has a hydrophobic moment (μ) of 3.5 to 11;

[0016] (6) The peptide has a predicted net charge of at least +4 at physiological pH;

[0017] (7) The peptide has an isoelectric point (pI) of 8 to 13;

[0018] (8) The peptide is composed of 35% to 65% of any combination of the following amino acids: A, C, G, I, L, M, F, P, W, Y and V;

[0019] (9) The peptide is composed of 0% to 30% of any combination of the following amino acids: N, Q, S, and T;

[0020] (10) The peptide is composed of 35% to 85% of any combination of the following amino acids: A, L, K or R;

[0021] (11) The peptide is composed of 15% to 45% of any combination of the following amino acids: A and L, provided that at least 5% L is present in the peptide;

[0022] (12) The peptide is composed of 20% to 45% of any combination of the following amino acids: K and R;

[0023] (13) The peptide is composed of 0% to 10% of any combination of the following amino acids: D and E;

[0024] (14) The difference between the percentage of A and L residues (A+L%) in the peptide and the percentage of K and R residues (K+R) in the peptide is less than or equal to 10%; and

[0025] (15) The peptide is composed of 10% to 45% of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[0026] In a eukaryotic cell line model suitable for evaluating load transduction in the target eukaryotic cells (e.g., HeLa), the shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators 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, and / or achieves at least 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. 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% transduction efficiency (e.g., as determined by flow cytometry).

[0027] In some respects, this article describes a synthetic peptide shuttle that is transducing in target eukaryotic cells for both protein and non-protein loads, wherein the shuttle comprises or consists of the following amino acid sequences: (a) an amino acid sequence of any one of SEQ ID NO: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344; Or (b) differs from (a) only in an amino acid sequence of conserved amino acid substitutions (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, preferably excluding any linker domains, such as linker domains rich in flexible serine / glycine), wherein, in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, the shuttle causes the transduction of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. The conductivity is increased 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; and / or is capable of achieving at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. % , 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% transduction efficiency (e.g., as determined by flow cytometry).

[0028] In some respects, this document describes a synthetic peptide shuttle with protein loading transduction activity in target eukaryotic cells, wherein the shuttle comprises or consists of the following amino acid sequences: (a) an amino acid sequence of any one of SEQ ID NO: 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) only from conserved amino acid substitutions (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conserved amino acid substitutions, preferably excluding any linker domains, such as flexible serine / glycine-rich linker domains), wherein the shuttle is in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating loading transduction in said target eukaryotic cells. In this process, 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 enables the achievement 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% transduction efficiency of GFP-NLS (e.g., as determined by flow cytometry).

[0029] In some respects, this document describes a synthetic peptide shuttle variant that has transduction activity for protein and / or non-protein loads in target eukaryotic cells, said synthetic peptide shuttle variant being identical to any synthetic peptide shuttle as defined herein, except that at least one amino acid is replaced with a corresponding synthetic amino acid containing a side chain having similar physiological and chemical properties (e.g., structure, hydrophobicity, or charge) to the substituted amino acid, wherein said shuttle variant increases the transduction efficiency of said load in target eukaryotic cells compared to the absence of said shuttle variant.

[0030] In some respects, this document describes an in vitro or in vivo method for transduction of protein and / or non-protein loads, the method comprising contacting target eukaryotic cells with the load and a concentration of a synthetic peptide shuttle or a synthetic peptide shuttle variant as defined herein, wherein the concentration of the synthetic peptide shuttle or the synthetic peptide shuttle variant is sufficient to increase the transduction efficiency of the load into the target eukaryotic cells compared to the absence of the synthetic peptide shuttle.

[0031] In some respects, this document describes a composition for therapy comprising a synthetic peptide shuttle or a synthetic peptide shuttle variant as defined herein, formulated together with a protein and / or non-protein load to be transduced into target eukaryotic cells via a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle or synthetic peptide shuttle variant in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency and cytosol delivery of the load to the target eukaryotic cells after administration.

[0032] In some respects, this document describes a kit comprising a synthetic peptide shuttle or a variant thereof as defined herein, and a protein and / or non-protein load to be transduced by said synthetic peptide shuttle or variant thereof.

[0033] In some respects, this document describes a method for generating a candidate synthetic peptide shuttle for transduction of a target load in target eukaryotic cells, the method comprising synthesizing a peptide that is: (1) a peptide of at least 17, 18, 19, or 20 amino acids, the peptide comprising (2) an amphiphilic α-helical motif having (3) a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein at least five of the parameters (4) to (15) defined herein are satisfied, wherein, in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, the shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 compared to a corresponding negative control lacking the shuttle. 7.5, 8, 8.5, 9, 9.5, or 10 times, and / or capable of achieving at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or 31% of a fluorescent DNA intercalator that is impermeable to membranes. %, 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% transduction efficiency (e.g., as determined by flow cytometry).

[0034] In some aspects, this document describes an in vitro or in vivo method for identifying, defining, or selecting synthetic peptide shuttles intended to transduce both protein and non-protein loads in target eukaryotic cells, the method comprising: providing a model eukaryotic cell or model organism suitable for evaluating load transduction in the target eukaryotic cells; providing a candidate synthetic peptide shuttle (e.g., as defined herein); and measuring the transduction activity (e.g., transduction efficiency, such as by flow cytometry) of the candidate synthetic peptide shuttle in transducing propidium iodide or other membrane-impermeable fluorescent DNA intercalators into the model eukaryotic cell or model organism, wherein the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators in the model eukaryotic cell or model organism is increased by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 compared to a corresponding negative control lacking the candidate synthetic peptide shuttle. 8, 8.5, 9, 9.5, or 10 times, and / or 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 When the transduction efficiency is 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., as determined by flow cytometry), the candidate shuttle is expected to have transduction activity against both protein and non-protein loads in the target eukaryotic cells.

[0035] General definition

[0036] Titles and other identifiers (e.g., (a), (b), (i), (ii), etc.) are provided merely for ease of reading the specification and claims. The use of titles or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are provided.

[0037] In the claims and / or description, when used with the term “comprising,” the word “a or an” can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0038] The term "about" is used to refer to a value that includes the standard deviation of the error of the apparatus or method used to determine the value. Generally, the term "about" means a possible variation of up to 10%. Therefore, variations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% of a value are included within the term "about." Unless otherwise specified, when the term "about" precedes a range, it applies to both ends of that range.

[0039] As used in this specification and claims, the terms “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 inclusive or open-ended and do not exclude additional, unlisted elements or method steps.

[0040] As used herein, “protein” or “polypeptide” or “peptide” means any chain of amino acids linked together by a peptide, which may or may not contain any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfation, threonization, isopreneation, ubiquitination, etc.). For further clarity, protein / polypeptide / peptide modifications are contemplated, provided that such modifications do not impair the loading transduction activity of the shuttles described herein. For example, the shuttles described herein may be linear or cyclic, synthesized from one or more D- or L-amino acids, and / or may be conjugated to fatty acids (e.g., at their N-terminus). At least one amino acid of the shuttles described herein may also be substituted with a corresponding synthetic amino acid containing a side chain having similar physiological and chemical properties (e.g., structure, hydrophobicity, or charge) to the substituted amino acid.

[0041] As used herein, a "domain" or "protein domain" generally refers to a portion of a protein that has a specific function or purpose. Some domains retain their function when separated from the rest of the protein and can therefore be used in a modular manner. The modularity of many protein domains can provide flexibility in their location within the shuttle as described in this specification. However, some domains may perform better when engineered at certain locations within the shuttle (e.g., in the N-terminal or C-terminal regions, or in between). The location of a domain within its endogenous protein sometimes indicates where within the shuttle the domain should be engineered and what type / length of adapter should be used. In light of this disclosure, those skilled in the art can use standard recombinant DNA techniques to manipulate the location and / or number of domains within the shuttle as described in this specification. Furthermore, the assays disclosed herein, as well as others known in the art, can be used to assess the functionality of each domain in the context of the shuttle (e.g., its ability to facilitate cell penetration across the plasma membrane, endosome escape, and / or entry into the cytosol). Standard methods can also be used to assess whether the domains of a shuttle affect the activity of the load to be delivered intracellularly. In this regard, the expression "operably connected" as used herein refers to the ability of the domains to perform one or more of their intended functions (e.g., cell penetration, endosome escape, and / or subcellular targeting) in the context of the shuttle described in this specification. For clarity, the expression "operably connected" is intended to define a functional connection between two or more domains, and is not limited to a specific order or distance between them.

[0042] As used herein, the term "synthetic" in expressions such as "synthetic peptide," "synthetic peptide shuttle," or "synthetic polypeptide" is intended to refer to a non-naturally occurring molecule that can be produced in vitro (e.g., chemically synthesized and / or produced using recombinant DNA technology). The purity of various synthetic formulations can be assessed, for example, by high-performance liquid chromatography and mass spectrometry. Chemical synthesis methods may be superior to cell expression systems (e.g., yeast or bacterial protein expression systems) because they eliminate the need for extensive recombinant protein purification steps (e.g., required for clinical use). In contrast, producing longer synthetic polypeptides via chemical synthesis methods can be more complex and / or more costly, and cell expression systems are more advantageous for producing such polypeptides. In some embodiments, the peptides or shuttles of this specification may be synthesized by chemical methods (e.g., solid-phase or liquid-phase peptide synthesis) in contrast to expression by recombinant host cells. In some embodiments, the peptides or shuttles of this specification may lack an N-terminal methionine residue. Those skilled in the art can modify the synthetic peptides or shuttles described herein by using one or more modified amino acids (e.g., non-naturally occurring amino acids) or by chemically modifying them to suit specific stability requirements or other needs.

[0043] As used herein, the term "independent" is generally intended to refer to molecules or agents that are not covalently bound to each other. For example, the expression "independent load" is intended to refer to a load that is not covalently bound (e.g., not fused) to the shuttle of this specification and is intended for intracellular delivery (transduction). In some respects, a shuttle independent of (not fused to) the load may be advantageous because it provides increased shuttle versatility—for example, the ability to readily change the shuttle-to-load ratio (in contrast to a fixed ratio limited by covalent bonds between the shuttle and the load).

[0044] As used herein, the expression “is or is from” or “from” includes functional variants of a given protein domain (e.g., CPD or ELD), such as conserved amino acid substitutions, deletions, modifications, and variants or functional derivatives that do not eliminate the activity of the protein domain.

[0045] Other objects, advantages and features of this specification will become more apparent upon reading the following non-limiting description of specific embodiments thereof, which are given by way of example only with reference to the accompanying drawings. Attached Figure Description

[0046] In the attached diagram:

[0047] Figures 1A-1D show the delivery and viability results of HeLa cells co-incubated for 1 minute with different classes of synthetic peptide shuttles along with a non-protein load (propidium iodide, PI; Figures 1A and 1B) or a protein load (GFP-NLS protein; Figures 1C and 1D). Results were obtained by flow cytometry two hours after load delivery and are expressed as a percentage of fluorescent cells (PI+cells% or GFP+cells%). Peptide classes shown (from left to right): synthetic peptide shuttles containing an endosome leakage domain (ELD) operatively linked to a cell penetration domain (CPD) as described in WO / 2016 / 161516; rationally designed synthetic peptide shuttles as described in WO / 2018 / 068135; other rationally designed synthetic peptide shuttles as described herein; cyclic peptides as described herein; and negative control peptides that failed to comply with several rational design parameters listed in WO / 2018 / 068135. In Figure 1A, “FS then PI” indicates that PI is added 1 hour after treatment with the synthetic peptide shuttle, thus ensuring that the positive PI signal is not due to cell death. “Negative control” is cells incubated with the load only (“PI” in Figures 1A and 1B or “GFP-NLS” in Figures 1C and 1D) or untreated cells not exposed to the load or peptide shuttle (“NT”, Figures 1A-1D).

[0048] Figure 2 is a table summarizing the results from Figures 1A-1D.

[0049] Figure 3 shows the activity of small molecule inhibitors of hedgehog signaling (Gant61, HPI-4, itraconazole, or ATO) transduced into NIH3T3 Gli-luciferase reporter cells via the peptide shuttle FSD250D. Successful small molecule transduction in the presence of the peptide shuttle ("+FSD250D"; SEQ ID NO:36) resulted in decreased luminescence intensity in NIH3T3 Gli-luciferase reporter cells stimulated with recombinant mouse sound hedgehog factor protein (+mShh) compared to the absence of the peptide shuttle ("-FSD250D").

[0050] Figure 4 shows the successful in vivo transduction of small molecule hedgehog signaling inhibitors (Gant61 and itraconazole) in skin cells of shaved mice using the peptide shuttle FSD250D. Hair removal in mouse skin induces hair growth strongly associated with the hedgehog pathway. The experiment consisted of activating the hedgehog pathway in mice through hair removal, followed by measuring the delay in hair regrowth induced by delivering small molecule hedgehog pathway inhibitors (Gant61 or itraconazole) that bind to intracellular targets in skin cells. The results showed that mice treated with the small molecule hedgehog inhibitor Gant61 or itraconazole ("FSD250D+Gant61100μM" and "FSD250D+itraconazole100μM") in the presence of FSD250D, compared with mice in the absence of FSD250D ("Gant61 100μM" and "FSD250D+itraconazole100μM") or mice in the presence of shuttle peptide alone ("FSD250D"), exhibited delayed hair regrowth 10 days after treatment (*).

[0051] Figures 5A-5C show representative patch-clamp electrophysiological whole-cell current traces of HEK293 cells stably expressing the sodium channel Nav1.7 after exposure to the membrane-impermeable sodium channel inhibitor QX-314 with or without FSD194. A decrease in current amplitude was observed when cells were temporarily 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), consistent with the presence of intracellular QX-314 (Figure 5C). This same decrease in current amplitude was not observed in the absence of QX-314 (i.e., 15 μM GFP-NLS + 5 μM FSD194; Figure 5A) or 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 QX-314+GFP-NLS+FSD194 and FSD194+GFP-NLS conditions, but no GFP-NLS-positive cells were identified under QX-314+GFP-NLS conditions, indicating that GFP-NLS was indeed co-transduced with the peptide shuttle along with QX-314.

[0052] Figures 6 and 7 show the results of a large-scale screening of over 300 candidate peptide shuttles for PI and GFP-NLS transduction activities. Figure 6 shows the results of all screened candidate peptide shuttles with an average PI transduction efficiency of 10% or higher, ranked according to their average PI transduction efficiency level. Figure 7 shows the results of 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%, ranked according to their average GFP-NLS transduction efficiency level.

[0053] sequence list

[0054] This application contains a sequence list in a computer-readable form, created on April 15, 2020, which is approximately 122 kb in size. The computer-readable form is incorporated herein by reference.

[0055]

[0056]

[0057]

[0058] *The peptide name is a variation of the name used in CA 3,040,645. Detailed Implementation

[0059] In some respects, this document describes methods for transduction of non-protein and / or protein loads. These methods typically involve contacting target eukaryotic cells with a non-protein and / or protein load and a concentration of a synthetic peptide shuttle sufficient to increase the transduction efficiency of the load compared to the absence of the synthetic peptide shuttle. This document also describes the use of multifunctional synthetic peptide shuttles with dual transduction activity for both protein and non-protein loads, and the use of PI or other membrane-impermeable fluorescent DNA intercalators as “alternative” loads for selecting synthetic peptide shuttles with such dual transduction activity.

[0060] Non-protein load

[0061] In some embodiments, the non-protein loading may be a compound (e.g., an organic compound) with a molecular weight less than 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 Da. In some embodiments, the non-protein loading may be a compound (e.g., an organic compound) with a molecular weight between 50 and 5,000, 50 and 4,000, 50 and 3,000, 50 and 2,000, or 50 and 1,000 Da. In some embodiments, the non-protein loading may be a small molecule, such as a small molecule drug that binds to an intracellular biological or therapeutic target. In some embodiments, the non-protein loading is not a biopolymer, such as a polynucleotide or polysaccharide, particularly a biopolymer with a uniform negative charge, such as a polynucleotide with a length greater than 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides. In some embodiments, the non-protein loading may have a net cationic charge in aqueous solution. In some embodiments, the non-protein load is not covalently bound to the synthetic peptide shuttle (i.e., independent of the synthetic peptide shuttle) (e.g., during transduction).

[0062] In some embodiments, the non-protein load may be a load that is impermeable to the cell membrane or has low membrane permeability (e.g., prevents its free diffusion across the cell membrane due to the physicochemical properties of the load), wherein the peptide shuttle described herein promotes or increases its intracellular delivery and / or entry into the cytosol. In some embodiments, the non-protein load may be a load that is permeable to the cell membrane, wherein the peptide shuttle described herein still increases its intracellular delivery and / or entry into the cytosol. In some embodiments, the peptide shuttle described herein may reduce the amount or concentration of load required to achieve its intended biological effect compared to applying a load alone.

[0063] In some embodiments, the non-protein load to be transduced may be a drug for treating any disease or condition with an intracellular biological or therapeutic target. In some embodiments, the non-protein load may be a drug for treating cancer (e.g., skin cancer, basal cell carcinoma, nevus-like basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye disease, dry or wet 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).

[0064] In certain embodiments, the non-protein load to be transduced may be or contains hedgehog inhibitors (e.g., itraconazole, posaconazole, arsenic trioxide (ATO), Gant61, PF-4708671, HPI-1, HPI-4). In certain embodiments, the non-protein load to be transduced may be or contains pain inhibitors, such as voltage-gated sodium (Nav) channel inhibitors (e.g., QX-314). In certain embodiments, the non-protein load to be transduced may be or contains inflammation inhibitors, such as inhibitors of pathways leading to the production of inflammatory cytokines (e.g., NF-κB pathway inhibitors).

[0065] In some implementations, the shuttle described herein may have the ability to transduce both non-protein and protein loads to the cytosol of target eukaryotic cells.

[0066] Reasonable design parameters and peptide shuttle

[0067] In some aspects, the shuttle agent described herein may be a peptide that has transduction activity in target eukaryotic cells for protein loads, non-protein loads, or both protein and non-protein loads. In some embodiments, the shuttle agent described herein preferably satisfies one or more of the following fifteen rational design parameters.

[0068] (1) In some embodiments, the shuttle is a peptide with a length of at least 17, 18, 19, or 20 amino acids. For example, the peptide may contain 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 implementations, 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, which may be more suitable for clinical use (as a control for recombinant proteins, which must be purified from cell expression systems). While numbers and ranges in this specification are generally listed as multiples of 5, this specification should not be limited thereto. For example, the maximum length described in this specification should be understood to also include lengths of 56, 57, 58…61, 62, etc., and the non-enumeration herein is merely for the sake of brevity. The same reasoning applies to the percentages of identity listed herein.

[0069] (2) In some embodiments, the peptide shuttle comprises an amphiphilic α-helical motif. As used herein, unless otherwise stated, the expressions “α-helical motif” or “α-helix” refer to a right-handed coiled or helical conformation (helix) with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn. As used herein, the expressions “comprising an α-helical motif” or “amphiphilic α-helical motif” refer to a three-dimensional conformation that is predicted to be adopted by the peptide (a segment of the peptide) in this specification based on the primary amino acid sequence of the peptide in a biological environment, regardless of whether the peptide actually adopts that conformation when used as a shuttle in a cell. Furthermore, the peptides in this specification may contain one or more α-helical motifs at different positions on the peptide. For example, the shuttle FSD5 predicted in WO / 2018 / 068135 employs an α-helix along its entire length (see Figure 49C of WO / 2018 / 068135), while the shuttle FSD18 predicted in WO / 2018 / 068135 contains two separated α-helices toward the N and C terminal regions of the peptide (see Figure 49D of WO / 2018 / 068135). In some embodiments, the shuttles predicted in this specification do not contain β-sheet motifs, as shown, for example, in Figures 49E and 49F of WO / 2018 / 068135. Methods for predicting the presence of α-helices and β-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 de novo peptide structures (http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD / ) (Lamiable et al., 2016; Shen et al., 2014; Thévenet et al., 2012). Other methods for predicting the presence of α-helices in peptides and proteins are known and readily available to those skilled in the art.

[0070] As used herein, the term "amphiphilic" refers to a peptide having both hydrophobic and hydrophilic elements (e.g., based on the side chains of the amino acids comprising the peptide). For example, the terms "amphiphilic α-helix" or "amphiphilic α-helix motif" refer to peptides predicted to employ an α-helix motif having both a nonpolar hydrophobic surface and a polar hydrophilic surface, based on the properties of the amino acid side chains that form the helix.

[0071] (3) In some embodiments, the peptide shuttle of this specification comprises an amphiphilic α-helical motif having a positively charged hydrophilic outer surface, such as an outer surface rich in R and / or K residues. As used herein, the expression “positively charged hydrophilic outer surface” means, based on an α-helical wheel projection, the presence of at least three lysine (K) and / or arginine (R) residues clustered on one side of the amphiphilic α-helical motif (see, for example, Figure 49A, left inset, WO / 2018 / 068135). Such a wheel projection can be prepared using various programs, such as the online wheel projection tool available at http: / / rzlab.ucr.edu / scripts / wheel / wheel.cgi. In some embodiments, the amphiphilic α-helical motif may comprise a positively charged hydrophilic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, wherein the positively charged hydrophilic outer surface comprises: (a) at least two, three, or four adjacent positively charged K and / or R residues when projected onto the helical wheel; and / or (b) a segment of six adjacent residues comprising three to five K and / or R residues when projected onto the helical wheel.

[0072] In some embodiments, the peptide shuttle of this specification comprises an amphiphilic α-helical motif comprising a hydrophobic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, said hydrophobic outer surface comprising: (a) at least two adjacent L residues in the helical wheel projection; and / or (b) a segment in the helical wheel projection comprising ten adjacent residues containing at least five hydrophobic residues selected from L, I, F, V, W and M.

[0073] (4) In some embodiments, the peptide shuttle of this specification comprises an amphiphilic α-helical 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, based on an open cylindrical representation of an α-helix with 3.6 residues per turn, the highly hydrophobic core may consist of spatially adjacent L, I, F, V, W, and / or M amino acids, calculated to constitute 12% to 50% of the amino acids in the peptide, excluding any histidine-rich domains (see below), as shown in the right-hand inset of Figure 49A in WO / 2018 / 068135. In some embodiments, the highly hydrophobic core may consist of spatially adjacent L, I, F, V, W, and / or M amino acids, representing 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 in the peptide. More specifically, the highly hydrophobic core parameter can be calculated by first representing the amino acids of the peptide in an open cylindrical shape, and then depicting a region of consecutive highly hydrophobic residues (L, I, F, V, W, M), as shown in the right-hand inset of Figure 49A in WO / 2018 / 068135. The number of highly hydrophobic residues in the depicted highly hydrophobic core is then divided by the total amino acid length of the peptide, excluding any histidine-rich domains (e.g., domains rich in N- and / or C-terminal histidines). For example, for the peptide shown in Figure 49A of WO / 2018 / 068135, there are 8 residues in the depicted highly hydrophobic core, and the peptide has a total of 25 residues (excluding the terminal 12 histidines). Therefore, the highly hydrophobic core is 32% (8 / 25).

[0074] (5) Hydrophobic moment is a measure of the amphiphilicity of a helix, peptide, or part thereof, calculated by the vector sum of the hydrophobicity of the amino acid side chains (Eisenberg et al., 1982). Online tools for calculating the hydrophobic moment of peptides are available at: http: / / rzlab.ucr.edu / scripts / wheel / wheel.cgi. High hydrophobic moments indicate strong amphiphilicity, while low hydrophobic moments indicate weak amphiphilicity. In some embodiments, the peptide shuttle of this specification may comprise or consist of a peptide or α-helical domain with a hydrophobic moment (μ) of 3.5 to 11. In some embodiments, the shuttle agent may be a peptide comprising an amphiphilic α-helical motif having 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. The hydrophobic torque between the lower limits of 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 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 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, any histidine-rich domains that may be present in the peptide are excluded when calculating the hydrophobic moment.

[0075] (6) In some embodiments, the peptide shuttle of this specification may have a predicted net charge of at least +4 at physiological pH, calculated from the side chains of K, R, D, and E residues. For example, the net charge of the peptide at physiological pH 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. These positive charges are typically conferred by the increased presence of positively charged lysine and / or arginine residues, in contrast to negatively charged aspartic acid and / or glutamic acid residues.

[0076] (7) In some embodiments, the peptide shuttle of this specification may have a predicted isoelectric point (pI) of 8 to 13, preferably 10 to 13. Procedures and methods for calculating and / or measuring the isoelectric point of peptides or proteins are known in the art. For example, pI can be calculated using Prot Param software available at: http: / / web.expasy.org / protparam /

[0077] (8) In some embodiments, the peptide shuttle of this specification may consist of 35% to 65% of hydrophobic residues (A, C, G, I, L, M, F, P, W, Y, V). In a particular embodiment, the peptide shuttle may consist of any combination of the following amino acids: A, C, G, I, L, M, F, P, W, Y, and V, at 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60%.

[0078] (9) In some embodiments, the peptide shuttle of this specification may consist of 0 to 30% neutral hydrophilic residues (N, Q, S, T). In specific embodiments, the peptide shuttle may consist of any combination of the following amino acids: N, Q, S, and T, 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%.

[0079] (10) In some embodiments, the peptide shuttle of this specification may consist of 35% to 85% of the following amino acids: A, L, K and / or R. In specific embodiments, the peptide shuttle may consist of any combination of the following amino acids: A, L, K or R, at 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60%.

[0080] (11) In some embodiments, the peptide shuttle of this specification may consist of 15% to 45% of amino acids A and / or L, provided that at least 5% L is present in the peptide. In a particular embodiment, the peptide shuttle may consist of any combination of the following amino acids: A and L, at 15% to 40%, 20% to 40%, 20% to 35%, or 20% to 30%, provided that at least 5% L is present in the peptide.

[0081] (12) In some embodiments, the peptide shuttle of this specification may consist of 20% to 45% of amino acids K and / or R. In a particular embodiment, the peptide shuttle may consist of any combination of 20% to 40%, 20% to 35%, or 20% to 30% of the following amino acids: K and R.

[0082] (13) In some embodiments, the peptide shuttle of this specification may consist of 0 to 10% of amino acids D and / or E. In a particular embodiment, the peptide shuttle may consist of 5% to 10% of any combination of the following amino acids: D and E.

[0083] (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 less than or equal to 10%. In specific 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 less than or equal to 9%, 8%, 7%, 6%, or 5%.

[0084] (15) In some embodiments, the peptide shuttle of this specification may consist of 10% to 45% of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, or H (i.e., not A, L, K, or R). In a particular embodiment, the peptide shuttle may consist of 15% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[0085] In some embodiments, the peptide shuttle of this specification conforms to 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 nine, at least ten, at least eleven, at least twelve, thirteen, at least fourteen, or all of the parameters (1) to (15) described herein. In certain embodiments, the peptide shuttle of this specification conforms to all of the parameters (1) to (3) and 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.

[0086] In some embodiments, when the peptide shuttle of this specification contains only one histidine-rich domain, the residues of said histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein. In some embodiments, when the peptide shuttle of this specification contains more than one histidine-rich domain, the residues of only one histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein. For example, when the peptide shuttle of this specification contains two histidine-rich domains: a first histidine-rich domain toward the N-terminus and a second histidine-rich domain toward the C-terminus, only the first histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein.

[0087] In some implementations, machine learning or computer-aided design methods can be implemented to generate peptides that conform to one or more of the parameters (1) to (15) described herein. Some parameters, such as parameters (1) and (5)-(15), may be better suited to computer-aided design methods, while structural parameters, such as parameters (2), (3), and (4), may be better suited to manual design methods. Therefore, in some implementations, peptides that conform to one or more of the parameters (1) to (15) can be generated by combining computer-aided and manual design methods. For example, multiple sequence alignment analysis of various peptides (and other peptides) that act as effective shuttles as shown herein revealed the presence of several common sequences—namely, the commonly found alternating patterns of hydrophobic, cationic, hydrophilic, alanine, and glycine amino acids. The presence of these common sequences may lead to conformation to structural parameters (2), (3), and (4) (i.e., amphiphilic α-helix formation, positively charged faces, and a highly hydrophobic core of 12%–50%). Therefore, these and other common sequences can be used in machine learning and / or computer-aided design methods to generate peptides that conform to one or more of the parameters (1)-(15).

[0088] Therefore, in some embodiments, the peptide shuttle described herein may comprise or consist of the following amino acid sequence:

[0089] (a)[X1]-[X2]-[connector]-[X3]-[X4](Equation 1);

[0090] (b)[X1]-[X2]-[Connector]-[X4]-[X3](Equation 2);

[0091] (c)[X2]-[X1]-[Connector]-[X3]-[X4](Equation 3);

[0092] (d)[X2]-[X1]-[Connector]-[X4]-[X3](Equation 4);

[0093] (e)[X3]-[X4]-[Connector]-[X1]-[X2](Equation 5);

[0094] (f)[X3]-[X4]-[Connector]-[X2]-[X1](Formula 6);

[0095] (g)[X4]-[X3]-[Connector]-[X1]-[X2](Formula 7); or

[0096] (h)[X4]-[X3]-[connector]-[X2]-[X1](Equation 8),

[0097] in:

[0098] [X1]Selected from: 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[+]-;

[0099] [X2]Selected from:-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

[0100] [X3]Select from:-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[+]-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[+]-A;-1[Φ]-1[+]-A;-1[Φ]-1[+]-A;-A-1[+]-A;-A-1[+]-A;and-A-1[+]-AAA;

[0101] [X4] Selected from: -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

[0102] [Connector] is selected from: -Gn-; -Sn-; -(GnSn)n-; -(GnSn)nGn-; -(GnSn)nSn-; -(GnSn)nGn(GnSn)n-; and -(GnSn)nSn(GnSn)n-;

[0103] Wherein: [Φ] is an amino acid, which is: Leu, Phe, Trp, Ile, Met, Tyr or Val, preferably Leu, Phe, Trp or Ile; [+] is an amino acid, which is: Lys or Arg; [ζ] is an amino acid, which is: Gln, Asn, Thr or Ser; A is amino acid Ala; G is amino acid Gly; S is amino acid Ser; and n is an integer from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 1 to 4 or 1 to 3.

[0104] In some embodiments, the peptide shuttle of the present invention may comprise or consist of the following peptides or functional variants thereof, wherein the peptide is an amino acid sequence of any one of SEQ ID NO: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344, or SEQ ID NO: as disclosed in WO / 2018 / 068135. NO:104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242 and 243-10 The amino acid sequence of any one of 242 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. In some embodiments, the peptide shuttle of the present invention may comprise the amino acid sequence motif of SEQ ID NO:158 and / or 159 of WO / 2018 / 068135, which is found in each of peptides FSD5, FSD16, FSD18, FSD19, FSD20, FSD22, and FSD23. In some embodiments, the peptide shuttle of the present invention may comprise the amino acid sequence motif of SEQ ID NO:159 of WO / 2018 / 068135, which is operatively linked to the amino acid sequence motif of SEQ ID NO:158 of WO / 2018 / 068135. As used herein, a “functional variant” refers to a peptide having transduction-loaded activity that differs from a reference peptide by one or more conserved amino acid substitutions. As used herein in the context of a functional variant, a “conserved amino acid substitution” is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain.Families of amino acid residues with similar side chains have been clearly defined in this art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and optional proline), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0105] In some embodiments, the peptide shuttle of this specification does not contain one or more of the amino acid sequences of any one of SEQ ID NO: 57-59, 66-72, or 82-102 of WO / 2018 / 068135. In some embodiments, the peptide shuttle of the present invention does not contain one or more of the amino acid sequences of any one of SEQ ID NO: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242 of WO / 2018 / 068135. More specifically, in some embodiments, the peptide shuttle of this specification may relate to variants of such previously described shuttle peptides, wherein said variants are further engineered to improve dual transduction activity (i.e., the ability to more robustly transduce protein and non-protein loads).

[0106] In some embodiments, such as in a eukaryotic cell model system (e.g., an immortalized eukaryotic cell line) or in a model organism, the peptide shuttle of this specification may have a minimum threshold for transduction efficiency and / or load delivery score for “alternative” loading. The term “transduction efficiency” refers to the percentage or proportion of the target cell population to which the target load is delivered intracellularly, which can be determined by, for example, flow cytometry, immunofluorescence microscopy, and other suitable methods that can be used to assess load transduction efficiency (e.g., as described in WO / 2018 / 068135). In some embodiments, transduction efficiency may be expressed as the percentage of load-positive cells. In some embodiments, transduction efficiency may be expressed as a fold increase (or fold decrease) compared to a suitable negative control, assessed under identical conditions other than the absence of load and shuttle (“no treatment”; NT) or the absence of shuttle (“load only”).

[0107] As used herein, the term "alternative load" refers to any protein or non-protein load that can be transduced by a shuttle with known load transduction activity, the levels of its intracellular delivery and endosome escape (i.e., cytosol and / or nuclear delivery) of which can be readily measured and / or tracked (e.g., via fluorescence or functional assays), wherein the alternative load is intended to assess the suitability of a given shuttle for transducing a target load (e.g., a protein or non-protein load, such as a therapeutically active load that binds to an intracellular target) that differs from the alternative load. Examples of suitable alternative loads may include fluorescent loads (e.g., PI or other membrane-impermeable fluorescent DNA intercalators, GFP, GFP-NLS or other fluorescent proteins, fluorescent dextran, etc.). Non-protein loads (such as PI or other membrane-impermeable fluorescent DNA intercalators) may be particularly advantageous because they are relatively inexpensive and exhibit enhanced fluorescence only upon binding to genomic DNA—a characteristic that makes them particularly suitable for distinguishing between endosome-captured loads and endosome-escaped loads (i.e., loads that enter the cytosol / nuclear compartment). As used herein, any suitable model system (e.g., immortalized cell lines, ex vivo cells, model laboratory organisms) can be used to assess shuttle transduction activity of alternative loadings. Conveniently, eukaryotic cell line models can be selected as suitable model systems, where the cell line is chosen to provide information for assessing transduction activity in the target eukaryotic cells to be transduced. In fact, a variety of cell cultures and model organisms are commercially available as model systems for studying a wide range of diseases.

[0108] In some embodiments, the peptide shuttle of this specification increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators in suitable eukaryotic cell model systems (e.g., HeLa or other suitable immortalized cell lines). In some embodiments, in HeLa cells or other suitable eukaryotic cell line models used to evaluate load transduction in target eukaryotic cells, the peptide shuttle of this specification increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators 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 said shuttle (“load only”). In some embodiments, the peptide shuttle of this specification is capable of achieving at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, or 32% loading in HeLa cells or other suitable eukaryotic cell line models for evaluating target eukaryotic cell loading. The transduction efficiency of 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% of propidium iodide or other membrane-impermeable fluorescent DNA intercalators (e.g., as determined by flow cytometry).

[0109] In some embodiments, the peptide shuttle of this specification increases the transduction efficiency of GFP-NLS or other suitable protein alternative loading in a suitable eukaryotic cell model system (e.g., in HeLa or other suitable immortalized cell lines). In some embodiments, in HeLa cells or other suitable eukaryotic cell line models used to evaluate loading transduction in target eukaryotic cells, the peptide shuttle of this specification increases the transduction efficiency of GFP-NLS or other suitable protein alternative loading 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-fold compared to a corresponding negative control lacking said shuttle (“loading only”). In some embodiments, the peptide shuttle of this specification is capable of achieving at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, or 32% loading transduction in HeLa cells or other suitable eukaryotic cell line models for evaluating target eukaryotic cells. The transduction efficiency of GFP-NLS or other suitable protein replacement loads of 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., as determined by flow cytometry).

[0110] In some embodiments, the peptide shuttle of this specification may comprise or consist of a shuttle having the following average PI transduction efficiency as listed in Figure 6: 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 of this specification may comprise or consist of a shuttle having a normalized average PI delivery score as listed in Figure 6, having the following: 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, 2 8.5, 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 or 60.

[0111] In some embodiments, the peptide shuttle of this specification may comprise or consist of a shuttle having, as listed in Figure 6, 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%, 2 7%, 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 of this specification may comprise or consist of a shuttle having the following normalized average GFP-NLS delivery score as listed in Figure 6: 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, 5 4, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 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.

[0112] In some embodiments, the peptide shuttle of this specification may comprise the peptides listed in Figure 7, having 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%, or 30%. Shuttles or components thereof with normalized average GFP-NLS delivery scores of 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, or 30.

[0113] In some embodiments, the shuttle of this specification may comprise shuttle variants that are approximately equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids as defined herein. Preferably, the linker domain (e.g., a linker domain rich in flexible serine / glycine) is excluded from consideration of different amino acids, because the length and / or amino acid composition of the linker domain can vary greatly without affecting transduction activity. In some embodiments, the peptide shuttle of this specification may comprise or consist of an amino acid sequence differing only from any shuttle described herein by conserved amino acid substitutions (e.g., by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, preferably excluding any adapter domains), wherein, in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, the shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators 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 is capable of achieving 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., as determined by flow cytometry) of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. In some embodiments, each conserved amino acid substitute is an amino acid selected from the same amino acid class, which 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 its amides: D, E, N and Q.

[0114] Chemical modification and synthesis of amino acids

[0115] In some embodiments, the shuttle of this specification may comprise an oligomer (e.g., a dimer, trimer, etc.) of the peptide described herein. Such oligomers may be constructed by covalently binding the same or different types of shuttle monomers (e.g., using disulfide bridges to link cysteine ​​residues introduced into the monomer sequence). In some embodiments, the shuttle of this specification may comprise N-terminal and / or C-terminal cysteine ​​residues.

[0116] In some embodiments, the shuttle of this specification may comprise or consist of cyclic peptides. In some embodiments, the cyclic peptide may be formed via a covalent link between a first residue positioned toward the N-terminus of the shuttle and a second residue positioned toward the C-terminus of the shuttle. In some embodiments, the first and second residues are side-attached residues located at the N and C-termini of the shuttle. In some embodiments, the first and second residues may be linked via an amide bond to form the cyclic peptide. In some embodiments, the cyclic peptide may be formed by a disulfide bond between two cysteine ​​residues within the shuttle, wherein the two cysteine ​​residues are positioned toward the N and C-termini of the shuttle. In some embodiments, the shuttle may comprise or be designed to comprise side-attached cysteine ​​residues at the N and C-termini, which are linked via disulfide bonds to form the cyclic peptide. In some embodiments, the cyclic shuttles described herein may be more resistant to degradation (e.g., by protease degradation) and / or may have a longer half-life compared to corresponding linear peptides.

[0117] In some embodiments, the shuttle of this specification may comprise one or more D-amino acids. In some embodiments, the shuttle of this specification may comprise a D-amino acid at the N and / or C-terminus of the shuttle. In some embodiments, the shuttle may consist entirely of D-amino acids. In some embodiments, the shuttle having one or more D-amino acids described herein may be more resistant to degradation (e.g., degradation by proteases) and / or may have a longer half-life compared to the corresponding peptide consisting only of L-amino acids.

[0118] In some embodiments, the shuttles of this specification may comprise chemical modifications to one or more amino acids, wherein said chemical modifications do not impair the transduction activity of the synthetic peptide shuttle. As used herein, the term “impairment” means that the chemical modification irreversibly eliminates the loading transduction activity of the peptide shuttle described herein. Chemical modifications that may temporarily inhibit, attenuate, or delay the loading transduction activity of the peptide shuttle described herein may be included in the chemical modifications of the shuttles of this specification. In some embodiments, the chemical modification of any of the shuttles described herein may be at the N and / or C-terminus of the shuttle. Examples of chemical modifications include the addition of an acetyl group (e.g., an N-terminal acetyl group), a cysteine ​​group (e.g., a C-terminal cysteine ​​group), or a fatty acid (e.g., a C4-C16, C6-C14, C6-C12, C6-C8, or C8 fatty acid, preferably N-terminal).

[0119] In some embodiments, the shuttle agent of this specification comprises a shuttle agent variant having transduction activity against protein and / or non-protein loads in target eukaryotic cells, said variant being identical to any shuttle agent of this specification, except that at least one amino acid is replaced with a corresponding synthetic amino acid or amino acid analog containing a side chain having similar physiological and chemical properties (e.g., structure, hydrophobicity, or charge) to the substituted amino acid. In some embodiments, the synthetic amino acid replacement is as follows:

[0120] (a) Replace the basic amino acid with any of the following: α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, 2,6-diamino-4-hexynic acid, β-(1-piperazinyl)-alanine, 4,5-dehydro-lysine, δ-hydroxylysine, ω,ω-dimethylarginine, homoarginine, ω,ω'-dimethylarginine, ω-methylarginine, β-(2-quinolinyl)-alanine, 4-aminopiperidin-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.

[0121] (b) Replace the nonpolar (hydrophobic) amino acid with any of the following: dehydroalanine, β-fluoroalanine, β-chloroalanine, β-iodoalanine, α-aminobutyric acid, α-aminoisobutyric acid, β-cyclopropylalanine, aziridine-2-carboxylic acid, α-allylglycine, propargylglycine, tert-butylalanine, β-(2-thiazolyl)-alanine, thioproline, 3,4-dehydroproline, tert-butylalanine. Amino acids, β-cyclopentylalanine, β-cyclohexylalanine, α-methylproline, pentylalanine, α-methylvaline, penicillamine, β,β-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, pipercoic acid, 4,5-dehydroleucine, alloleucine, ortholeucine, α-methylleucine, cyclohexylglycine, cis-octahydroindole-β-2-carboxylic acid, β-(2-thienyl)-alanine Phenylenetetrine, α-methylphenylalanine, homophenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-(3-benzothiophene)-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-tetrahydronorharman-3-carboxylic acid, β,β-diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine or 4-benzoylphenylalanine;

[0122] (c) Replace the polar, uncharged amino acid with any of the following: β-cyanoalanine, β-ureidoalanine, homocysteine, alletine, pyroglutamic acid, 2-oxothiazolidin-4-carboxylic acid, citrulline, thiocitrulline, homocitrulline, hydroxyproline, 3,4-dihydroxyphenylalanine, β-(1,2,4-triazol-1-yl)-alanine, 2-mercaptohistidine, β-(3,4-dihydroxyphenyl)-serine, β-(2-thienyl)-serine 4-Azide-phenylalanine, 4-cyanophenylalanine, 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, thyroxine, β-(7-methoxycoumarin-4-yl)-alanine or 4-(7-hydroxy-4-coumarinyl)-aminobutyric acid; and / or

[0123] (d) Replace the acidic amino acid with any of the following: γ-hydroxyglutamic acid, γ-methyleneglutamic acid, γ-carboxyglutamic acid, α-aminoadipic acid, 2-aminopimelic acid, α-aminooctanoic acid, 4-carboxyphenylalanine, sulfoalanine, 4-phosphonophenylalanine or 4-sulfomethylphenylalanine.

[0124] Histidine-rich domains

[0125] In some embodiments, the peptide shuttle of this specification may further comprise one or more histidine-rich domains. In some embodiments, the histidine-rich domain may be an extension comprising at least 2, 3, 4, 5, or 6 amino acids comprising at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of histidine residues. In some embodiments, the histidine-rich domain may comprise at least 2, 3, 4, 5, 6, 7, 8, or 9 consecutive histidine residues. Without being bound by theory, the histidine-rich domain in the shuttle may act as a proton sponge in endosomes by protonating its imidazole group under acidic conditions, providing another mechanism for endosome membrane instability, thereby further enhancing the ability of endosome-captured loads to enter the cytosol. In some embodiments, the histidine-rich domain may be located at or toward the N and / or C ends of the peptide shuttle.

[0126] connector

[0127] In some embodiments, the peptide shuttle of this specification may comprise one or more suitable linkers (e.g., flexible peptide linkers). In some embodiments, such linkers may separate two or more amphiphilic α-helical motifs (e.g., see shuttle FSD18 in Figure 49D of WO / 2018 / 068135). In some embodiments, the linker may be used to separate two or more domains (CPD, ELD, or histidine-rich domains) from one another. In some embodiments, the linker may be formed by adding a small hydrophobic amino acid sequence with no rotational potential (e.g., glycine) and a polar serine residue that imparts stability and flexibility. The linker may be flexible and allow for regional movement of the shuttle. In some embodiments, proline may be avoided as it can increase significant conformational rigidity. In some embodiments, the linker may be a serine / glycine-rich linker (e.g., GS, GGS, GGSGGGS (SEQ ID NO:345), GGSGGGSGGGS (SEQ ID NO:346), etc.). In some embodiments, the use of a shuttle containing a suitable adapter can facilitate the delivery of loads to suspended cells rather than adherent cells. In some embodiments, the adapter may comprise or consist of the following: -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.

[0128] Domain-based peptide shuttles

[0129] In some respects, the shuttles described herein may be shuttles as described in WO / 2016 / 161516, which include an endosomal leakage domain (ELD) operatively linked to a cell penetration domain (CPD).

[0130] Internal leakage domain (ELD)

[0131] In some aspects, the peptide shuttles of this specification may comprise an endosome leak-through domain (ELD) for facilitating endosome escape and entry into cytoplasmic compartments. As used herein, the expression "endosome leak-through domain" refers to an amino acid sequence that confers the ability of endosome-captured loads into cytoplasmic compartments. Not bound by theory, endosome leak-through domains are short sequences (typically derived from viral or bacterial peptides) that are thought to induce instability of the endosome membrane and release of endosome contents into the cytoplasm. As used herein, the expression "endosome-dissolving peptide" is intended to refer to peptides of this general class that possess endosome membrane instability properties. Therefore, in some embodiments, the synthetic peptides or peptide-based shuttles of the present invention may comprise an ELD as an endosome-dissolving peptide. The activity of such a peptide can be assessed, for example, using the calcein endosome escape assay described in Example 2 of WO / 2016 / 161516.

[0132] In some implementations, the ELD can be a membrane-disrupting peptide at acidic pH, such as a pH-dependent membrane-active peptide (PMAP) or a pH-dependent cleavage peptide. For example, peptides GALA and INF-7 are amphiphilic peptides that form α-helices when their charge changes due to a decrease in pH. More specifically, without being bound by theory, it has been shown that ELDs such as GALA induce endosome leakage by forming pores and flipping membrane lipids after a conformational change due to a decrease in pH (Kakudo, Chaki et al., 2004; Li, Nicol et al., 2004). In contrast, it has been shown that ELDs such as INF-7 induce endosome leakage by accumulating on and destabilizing the endosome membrane (El-Sayed, Futaki et al., 2009). Thus, during endosome maturation, the accompanying pH decrease causes a conformational change in the peptide and destabilizes the endosome membrane, leading to the release of endosome contents. The same principle is thought to apply to Pseudomonas toxin A (Varkouhi, Scholte et al., 2011). Upon pH decrease, the toxin translocation domain undergoes a conformational change, allowing it to insert into the endosome membrane at pore formation sites (London 1992, O'Keefe 1992). This ultimately facilitates endosome instability and translocation of the complex to the endosome exterior. The aforementioned ELD encompasses the ELDs described in this specification and other endosome leakage mechanisms whose mechanisms of action are not fully defined.

[0133] In some implementations, the ELD can be an antimicrobial peptide (AMP) such as a linear cationic α-helical antimicrobial peptide (AMP). These peptides play a crucial role in innate immune responses due to their strong ability to interact with bacterial membranes. Unbound by conventional theory, these peptides are thought to exist in a disordered state in aqueous solutions, but adopt an α-helical secondary structure in hydrophobic environments. This latter conformation is believed to contribute to their typical concentration-dependent membrane-disrupting properties. When accumulated in endosomes at specific concentrations, some antimicrobial peptides may induce endosome leakage.

[0134] In some implementations, the ELD can be an antimicrobial peptide (AMP) such as a bactericidal peptide-A / meadow venom peptide hybrid (CM) peptide. Such peptides are considered to be among the smallest and most potent AMP-derived peptides with membrane-disrupting capabilities. Bactericidal peptides are a family of antimicrobial peptides capable of perturbing both Gram-positive and Gram-negative bacteria. Bactericidal peptide A (CA), the first identified antimicrobial peptide, consists of 37 amino acids with a linear structure. Meadow venom peptide (M), a 26-amino acid peptide, is a cell membrane lysing factor found in bee venom. Bactericidal peptide-meadow venom peptide hybrids have shown the production of short, potent antibiotic peptides without cytotoxicity (i.e., non-hemolytic) to eukaryotic cells, a property ideal in any antimicrobial agent. These chimeric peptides construct various combinations of the hydrophilic N-terminal domain of bactericidal peptide A with the hydrophobic N-terminal domain of meadow venom peptide and have been tested on bacterial model systems. Two 26-mers, CA(1-13)M(1-13) and CA(1-8)M(1-18) (Boman et al., 1989), have demonstrated broad-spectrum and improved efficacy of natural bactericidal peptide A without the cytotoxic effects of melitoxin.

[0135] In the work on generating shorter CM series peptides, Andreu et al. (1992) constructed hybrid peptides such as 26-mers (CA(1-8)M(1-18)) and combined them with 20-mers (CA(1-8)M(1-12)), 18-mers (CA(1-8)M(1-10)) and six 15-mers (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) were compared. The 20 and 18-mers maintained similar activity compared to CA(1-8)M(1-18). Among the six 15-mers, CA(1-7)M(1-8) showed low antibacterial activity, while the other five showed similar antibiotic efficacy and no hemolytic activity compared to the 26-mer. Therefore, in some embodiments, the synthetic peptides or peptide-based shuttles of the present invention may comprise ELDs as or derived from CM series peptide variants (such as those described above).

[0136] In some embodiments, the ELD can be a CM-series peptide CM18, [C(1-7)M(2-12)], composed of residues 1-7 of the bactericidal peptide-A (KWKLFKKIGAVLKVLTTG) (SEQ ID NO:347) fused with residues 2-12 (YGRKKRRQRRR) (SEQ ID NO:348) of the melittin peptide. When fused with the cell-penetrating peptide TAT, CM18 exhibits independent transmembrane penetration and endosome membrane destabilization, allowing some of the endosome-captured load to be released into the cytosol (Salomone et al., 2012). However, in some of the authors' experiments, the use of the CM18-TAT11 peptide fused with the fluorophore (atto-633) introduced uncertainty regarding the peptide's contribution relative to the fluorophore, as the fluorophore itself has shown a contribution to endosome dissolution—e.g., via photochemical disruption of the endosome membrane (Erazo-Oliveras et al., 2014).

[0137] In some embodiments, the ELD may be CM18 having the amino acid sequence of SEQ ID NO:1 of WO / 2016 / 161516, 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%, 90%, 91%, 92%, 93%, 94%, or 95% identity with SEQ ID NO:1 of WO / 2016 / 161516 and having endosome lysis activity.

[0138] In some implementations, ELD can be an N-terminal peptide derived from the HA2 subunit of influenza hemagglutinin (HA), which can also cause instability of the endosome membrane when it accumulates in the endosome.

[0139] In some embodiments, the synthetic peptides or polypeptide-based shuttles of the present invention may comprise an ELD as or derived from the ELDs described in Table I, or variants thereof having endosome escape activity and / or pH-dependent membrane disruption activity.

[0140] Table I: Examples of Internal Leakage Domains

[0141]

[0142]

[0143] In some embodiments, the shuttle of the present invention may comprise one or more ELDs or one or more types of ELDs. More particularly, it may comprise at least 2, at least 3, at least 4, at least 5 or more ELDs. In some embodiments, the shuttle may comprise 1-10 ELDs, 1-9 ELDs, 1-8 ELDs, 1-7 ELDs, 1-6 ELDs, 1-5 ELDs, 1-4 ELDs, 1-3 ELDs, etc.

[0144] In some embodiments, the order or position of ELD relative to other domains (CPD, histidine-rich domains) within the shuttle of the present invention can be varied, as long as the shuttle's shuttle capability is maintained.

[0145] In some embodiments, the ELD may be a variant or fragment of any of those listed in Table I that have endosome lysis activity. In some embodiments, the ELD may comprise or consist of an amino acid sequence of any one of SEQ ID NO:1-15, 63, or 64 of WO / 2016 / 161516, or a sequence 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 or constitutes endosome lysis activity of any one of SEQ ID NO:1-15, 63, or 64 of WO / 2016 / 161516.

[0146] In some embodiments, the shuttle of this specification does not contain one or more of the amino acid sequences of any one of SEQ ID NO:1-15, 63 or 64 of WO / 2016 / 161516.

[0147] Cell Penetration Domain (CPD)

[0148] In some aspects, the shuttle of the present invention may comprise a cell-penetrating domain (CPD). As used herein, "cell-penetrating domain" refers to an amino acid sequence that confers the ability of a macromolecule (e.g., a peptide or protein) containing a CPD to be transduced into a cell.

[0149] In some embodiments, the CPD may be (or may be derived from) a cell-penetrating peptide or a protein transduction domain of a cell-penetrating peptide. Cell-penetrating peptides can function as carriers to successfully deliver various loads intracellularly (e.g., polynucleotides, peptides, small molecule compounds, or other macromolecules / compounds that are otherwise impermeable to the membrane). Cell-penetrating peptides typically comprise short peptides rich in basic amino acids, which mediate their internalization into the cell upon fusion (or otherwise operatively linked) with a macromolecule (Shaw, Catchpole et al., 2008). The first cell-penetrating peptide was identified by analyzing the cell-penetrating ability of the HIV-1 transcription cis-activator (Tat) protein (Green and Loewenstein 1988, Vives, Brodin et al., 1997). This protein contains a short hydrophilic amino acid sequence called “TAT,” which facilitates its insertion into the plasma membrane and the formation of pores. Since this discovery, a variety of other cell-penetrating peptides have been described. In this respect, in some embodiments, the CPD may be a cell-penetrating peptide as listed in Table II or a variant thereof having cell-penetrating peptide activity.

[0150] Table II: Examples of Cell-Penetrating Peptides

[0151]

[0152] Unbound by conventional theories, it is believed that cell-penetrating peptides interact with the cell membrane and then cross it via pinocytosis or endocytosis. In the case of TAT peptides, it is thought that their hydrophilicity and charge facilitate their insertion into the plasma membrane and the formation of pores (Herce and Garcia 2007). It is also believed that the α-helical motif within hydrophobic peptides (such as SP) forms pores within the plasma membrane (Veach, Liu et al., 2004).

[0153] In some embodiments, the shuttle of the present invention may comprise one or more CPDs or one or more types of CPDs. More particularly, it may comprise at least 2, at least 3, at least 4, or at least 5 or more CPDs. In some embodiments, the shuttle may comprise between 1 and 10 CPDs, between 1 and 6 CPDs, between 1 and 5 CPDs, between 1 and 4 CPDs, between 1 and 3 CPDs, etc.

[0154] In some embodiments, the CPD may be a TAT having the amino acid sequence of SEQ ID NO:17 of WO / 2016 / 161516, 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:17 of WO / 2016 / 161516 and having cell-penetrating activity; or a transmembrane peptide having the amino acid sequence of SEQ ID NO:18 of WO / 2016 / 161516, or a transmembrane peptide having the amino acid sequence of SEQ ID NO:18 of WO / 2016 / 161516. NO:18 has variants of it that have 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 and cell-penetrating activity.

[0155] In some embodiments, CPD may be PTD4 having the amino acid sequence of SEQ ID NO:65 of WO / 2016 / 161516, 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 WO / 2016 / 161516.

[0156] In some embodiments, the order or position of the CPD relative to other domains (ELD, histidine-rich domains) within the shuttle as described in this specification may be varied, as long as the shuttle's shuttling capability is maintained.

[0157] In some embodiments, the CPD may be a variant or fragment of any of those listed in Table II that have cell-penetrating activity. In some embodiments, the CPD may comprise, or consist of, an amino acid sequence of any one of SEQ ID NO:16-27 or 65 of WO / 2016 / 161516, or a sequence 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, or constitutes a cell-penetrating sequence of any one of SEQ ID NO:16-27 or 65 of WO / 2016 / 161516.

[0158] In some embodiments, the shuttle of this specification does not contain any of the amino acid sequences of SEQ ID NO:16-27 or 65 of WO / 2016 / 161516.

[0159] Methods, kits, uses, compositions and cells

[0160] In some embodiments, this specification relates to a method for delivering protein and / or non-protein loads from extracellular space to the cytosol and / or nucleus of target eukaryotic cells. The method includes contacting the target eukaryotic cells with the load in the presence of a shuttle agent, wherein the concentration of the shuttle agent is sufficient to increase the transduction efficiency of the load compared to the absence of the shuttle agent. In some embodiments, contacting the target eukaryotic cells with the load in the presence of the shuttle agent results in an increase in the transduction efficiency of the non-protein load of at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold compared to the absence of the shuttle agent.

[0161] In some embodiments, this specification relates to a method for increasing the transduction efficiency of protein and / or non-protein loads into the cytosol and / or nucleus of target eukaryotic cells. As used herein, the expression "increase transduction efficiency" means the ability of the shuttle of this specification to improve the percentage or proportion of the target cell population to which the target load (e.g., non-protein load) is delivered intracellularly. Immunofluorescence microscopy, flow cytometry, and other suitable methods can be used to assess load transduction efficiency. In some embodiments, the shuttle of this specification can achieve 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 of this specification can achieve one of the above-described transduction efficiencies and at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% cell viability, for example as determined by the assay described in Example 3.3a of WO / 2018 / 068135 or by another suitable assay known in the art.

[0162] In addition to increasing target cell transduction efficiency, the shuttles of this specification may also facilitate the delivery of target loads (e.g., protein and / or non-protein loads) to the cytosol and / or nucleus of target cells. In this respect, efficiently delivering extracellular loads to the cytosol and / or nucleus of target cells using peptides can be challenging because loads tend to be trapped in intracellular endosomes after crossing the plasma membrane, which may limit their intracellular availability and potentially lead to their eventual metabolic degradation. For example, the use of protein transduction domains from the HIV-1 Tat protein has been reported to result in a significant amount of load being isolated into intracellular vesicles. In some respects, the shuttles of this specification may facilitate the ability of endosome-trapped loads to escape from endosomes and enter the cytoplasmic compartment. In this respect, the expression "to the cytosol" in the phrase "increases the transduction efficiency of non-protein loads to the cytosol" is intended to mean that the shuttles of this specification allow intracellular delivery of target loads to evade endosome trapping and enter the cytosol and / or nuclear compartment. Once the target load enters the cytosol, it can freely bind to its intracellular targets (e.g., the nucleus, nucleolus, mitochondria, peroxisomes). In some embodiments, therefore, the expression "to the cytosol" is intended to encompass not only cytosol delivery but also delivery to other subcellular compartments where the load must first enter the cytoplasmic compartment.

[0163] 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 include topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of non-protein loaded and synthetic peptide shuttles. In some embodiments, compositions for topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of non-protein loaded and synthetic peptide shuttles are described herein.

[0164] In some embodiments, the methods described herein may include contacting the target eukaryotic cells with a shuttle or composition as defined herein, as well as the protein and / or non-protein load. In some embodiments, the shuttle or composition may be pre-incubated with the load to form a mixture, and then the target eukaryotic cells may be exposed to the mixture. In some embodiments, the type of shuttle may be selected based on the characteristics and / or physicochemical properties of the load to be delivered intracellularly. In other embodiments, the type of shuttle may be selected taking into account the characteristics and / or physicochemical properties of the load to be delivered intracellularly, cell type, tissue type, etc.

[0165] In some embodiments, the method may include treating the target cells multiple times with the shuttle agent or composition (e.g., 1, 2, 3, 4 or more times per day, and / or according to a predetermined schedule). In this case, 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 will be able to adapt the teachings of this specification to different combinations of different shuttle agents, domains, uses, and methods to suit specific needs for delivering protein and / or non-protein loads to specific cells with desired viability.

[0166] In some embodiments, the methods described herein can be applied to the delivery of proteins and / or non-protein-loaded cells into cells in vivo. Such methods can be achieved through parenteral administration or direct injection into tissues, organs, or systems.

[0167] In some respects, the synthetic peptide shuttles of this specification can be used in in vitro or in vivo methods to increase the transduction efficiency of protein and / or non-protein loads (e.g., therapeutic or bioactive protein and / or non-protein loads) into target eukaryotic cells, wherein the synthetic peptide shuttle or a variant thereof is used at a concentration or formulated for use at a concentration sufficient to increase the transduction efficiency of the load into the target eukaryotic cells and cytosol and / or nuclear delivery, compared to the absence of the synthetic peptide shuttle or a variant thereof.

[0168] In some embodiments, the synthetic peptide shuttles of this specification can be used in therapy, wherein the synthetic peptide shuttle or a variant thereof transduces a therapeutically or biologically active protein and / or non-protein load to the cytosol and / or nucleus of a target eukaryotic cell, wherein the synthetic peptide shuttle or a variant thereof is used (or formulated for use at a concentration sufficient to increase the transduction efficiency of the load to the target eukaryotic cell compared to the absence of the synthetic peptide shuttle.

[0169] In some aspects, this document describes a composition for transducing a non-protein load into target eukaryotic cells, the composition comprising a synthetic peptide shuttle formulated with a pharmaceutically suitable excipient, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the load in the target eukaryotic cells and cytosol and / or nuclear delivery after administration. In some embodiments, the composition further comprises the load. In some embodiments, the composition may be mixed with the load prior to administration or therapeutic use.

[0170] In some respects, this document describes a composition for therapy comprising the synthetic peptide shuttle formulated together with a protein and / or non-protein load to be transduced into target eukaryotic cells via a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the load in the target eukaryotic cells and cytosol and / or nuclear delivery after administration.

[0171] In some embodiments, the shuttle or composition and the protein and / or non-protein load can be exposed to target cells in the presence or absence of serum. In some embodiments, the method may be suitable for clinical or therapeutic use.

[0172] In some embodiments, this specification relates to a kit for delivering protein and / or non-protein loads from the extracellular space to the cytosol and / or nucleus of target eukaryotic cells. In some embodiments, this specification relates to a kit for increasing the transduction efficiency of protein and / or non-protein loads into the cytosol of target eukaryotic cells. The kit may contain a shuttle agent or composition as defined herein, and a suitable container.

[0173] 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 include those cells possessing cellular mechanisms for endocytosis (i.e., the production of endosomes).

[0174] In some embodiments, this specification relates to an isolated cell comprising a synthetic peptide shuttle as defined herein. In some embodiments, the cell may be a protein-induced pluripotent stem cell. It should be understood that cells that are typically resistant to DNA transfection or unsuitable for DNA transfection may be candidates of interest for the synthetic peptide shuttle of this specification.

[0175] In some embodiments, this specification relates to a method for producing a synthetic peptide shuttle that delivers a protein and / or non-protein load from extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, the method comprising synthesizing a peptide that is: (1) a peptide of at least 17, 18, 19 or 20 amino acids in length, comprising (2) an amphiphilic α-helical motif having (3) a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein at least five of the parameters (4) to (15) defined herein are followed.

[0176] In some embodiments, this specification relates to a method for identifying or selecting a shuttle agent that delivers a protein and / or non-protein load from extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, the method comprising: (a) synthesizing a peptide as defined herein; (b) contacting the target eukaryotic cell with the load in the presence of the peptide; (c) measuring the transduction efficiency of the load in the target eukaryotic cell; and (d) identifying or selecting the peptide as a shuttle agent for transducing the load when an increase in the transduction activity (e.g., transduction efficiency) of the load in the target eukaryotic cell is observed.

[0177] In some aspects, this specification relates to a composition for transducing a non-protein load into target eukaryotic cells, the composition comprising a synthetic peptide shuttle formulated with a pharmaceutically suitable excipient, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency and cytosol delivery of the load in the target eukaryotic cells after administration. In some embodiments, the composition further comprises the load.

[0178] In some embodiments, this specification relates to oral formulations comprising the shuttle agent described herein and the load as described herein, such as enteric-coated oral dosage forms.

[0179] In some embodiments, the shuttles described herein are envisioned for use in the food, agriculture, and / or agro-industry. In some embodiments, the shuttles described herein can be formulated as feed additives to aid in weight gain and / or nutrient absorption.

[0180] In some respects, this document describes a method for generating a candidate synthetic peptide shuttle for transduction of a target protein and / or non-protein load in target eukaryotic cells, the method comprising synthesizing a peptide that is: (1) a peptide of at least 17, 18, 19, or 20 amino acids, the peptide comprising (2) an amphiphilic α-helical motif having (3) a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein at least five of the parameters (4) to (15) defined herein are satisfied, and wherein, in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, the shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators by at least 3, 3.5, 4, 4.5, 5, 5.5, or 6 compared to a corresponding negative control lacking the shuttle. 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times, and / or capable of achieving at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of a propidium iodide or other membrane-impermeable fluorescent DNA intercalating agent. 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% transduction efficiency (e.g., as determined by flow cytometry).

[0181] In some aspects, this document describes an in vitro or in vivo method for identifying or selecting synthetic peptide shuttles expected to have transduction activity for protein and / or non-protein loads in target eukaryotic cells, the method comprising: providing a model eukaryotic cell or model organism suitable for evaluating load transduction in the target eukaryotic cells; providing a candidate synthetic peptide shuttle (e.g., any shuttle as defined herein); and measuring the transduction activity (e.g., load transduction efficiency, such as by flow cytometry) of the candidate synthetic peptide shuttle in transducing propidium iodide or other membrane-impermeable fluorescent DNA intercalators into the eukaryotic cell line model, wherein when the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators in the model eukaryotic cells or model organism is increased by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 compared to a corresponding negative control lacking the candidate synthetic peptide shuttle, the transduction efficiency is increased by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8. 8.5, 9, 9.5, or 10 times, and / or 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 When the transduction efficiency is 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., as determined by flow cytometry), the candidate shuttle is expected to have transduction activity against both protein and non-protein loads in the target eukaryotic cells.

[0182] Project I

[0183] In some respects, one or more of the following items are described here:

[0184] 1. A method for non-protein load transduction, the method comprising contacting a target eukaryotic cell with a non-protein load and a concentration of a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle is sufficient to increase the transduction efficiency of the non-protein load compared to the absence of the synthetic peptide shuttle.

[0185] 2. The method according to Item 1, wherein the non-protein load: (a) is an organic compound; (b) has a molecular weight of less than 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 Da, or between 50 and 5,000, 50 and 4,000, 50 and 3,000, 50 and 2,000, or 50 and 1,000 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 biopolymer, such as a polynucleotide or polysaccharide; (e) is not covalently linked to the synthetic peptide shuttle during transduction; or (f) any combination of (a) to (e).

[0186] 3. The method according to Project 1 or Project 2, wherein the non-protein load is a medicine used to treat cancer (e.g., skin cancer, basal cell carcinoma, nevus-like basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye disease, dry or wet 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).

[0187] 4. The method according to any one of items 1 to 3, wherein the non-protein load is or contains hedgehog inhibitors (e.g., itraconazole, posaconazole, arsenic trioxide (ATO), Gant61, PF-4708671, HPI-1, HPI-4), pain inhibitors such as voltage-gated sodium (Nav) channel inhibitors (e.g., QX-314), and / or inhibitors of inflammation (e.g., inhibitors of inflammatory cytokine production or NF-κB pathway inhibitors).

[0188] 5. The method according to any one of items 1 to 4, wherein the shuttle is: (1) a peptide of at least 20 amino acids in length, the peptide comprising (2) an amphiphilic α-helical motif having (3) a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein at least five of the following parameters (4) to (15) are obeyed: (4) an open cylindrical representation based on an α-helix having 3.6 residues per turn, the hydrophobic outer surface comprising a highly hydrophobic core composed of spatially adjacent L, I, F, V, W and / or M amino acids, the amino acids comprising 12% to 50% of the amino acids of the peptide; (5) the peptide having a hydrophobic torque (μ) of 3.5 to 11; (6) the peptide having a predicted net charge of at least +4 at physiological pH; (7) the peptide having an isoelectric point (pI) of 8 to 13; (8) the peptide consisting of 35% to 65% of any combination of the following amino acids: A, C, G, I, L, M, F, (9) The peptide is composed of 0% to 30% of any combination of the following amino acids: N, Q, S, and T; (10) The peptide is composed of 35% to 85% of any combination of the following amino acids: A, L, K, or R; (11) The peptide is composed of 15% to 45% of any combination of the following amino acids: A and L, provided that at least 5% L is present in the peptide; (12) The peptide is composed of 20% to 45% of any combination of the following amino acids. (13) The peptide is composed of 0% to 10% of any combination of the following amino acids: D and E; (14) The difference between the percentage of A and L residues (A+L%) and the percentage of K and R residues (K+R) in the peptide is less than or equal to 10%; and (15) The peptide is composed of 10% to 45% of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H.

[0189] 6. The method according to item 5, wherein: (a) the shuttle conforms to 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); (b) the 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; and (c) the amphiphilic α-helical motif has a length of 3.5, 3 The hydrophobic torque (μ) between the lower limit of 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 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 α-helical motif contains a positively charged A hydrophilic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, wherein the positively charged hydrophilic outer surface comprises: (i) at least two, three, or four adjacent positively charged K and / or R residues when projected onto the helical wheel; and / or (ii) a segment containing three to five K and / or R residues when projected onto the helical wheel; (e) the amphiphilic α-helical motif comprises a hydrophobic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, wherein the hydrophobic outer surface comprises: (i) at least two adjacent L residues when projected onto the helical wheel; and / or (ii) a segment containing three to five K and / or R residues when projected onto the helical wheel. (f) The hydrophobic outer surface comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W and / or M amino acids, wherein the amino acids constitute 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 in the shuttle; (g) The shuttle has 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.The following are possible values: (a) a hydrophobic moment (μ) between the lower limit of 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 the 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; (b) the shuttle has a predicted net charge between +4, +5, +6, +7, +8, +9 and +10, +11, +12, +13, +14 or +15; (c) the shuttle has a predicted pI of 10 to 13; or (d) any combination of (a) to (i).

[0190] 7. The method according to any one of items 1 to 6, wherein the shuttle complies with 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 is composed of any combination of the following amino acids in amounts of 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60%: A, C, G, I, L, M, F, P, W, Y, and V; (9) The shuttle consists 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% of any combination of the following amino acids: N, Q, S, and T; (10) the shuttle consists of 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60% of the following amino acids. (11) The shuttle consists of 15% to 40%, 20% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: A and L; (12) The shuttle consists of 20% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: K and R; (13) The shuttle consists of 5% to 10% of any combination of the following amino acids: D (14) The difference between the percentage of A and L residues (A+L%) in the shuttle and the percentage of K and R residues (K+R) in the shuttle is less than or equal to 9%, 8%, 7%, 6% or 5%; and (15) The shuttle is composed of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H, in amounts of 15% to 40%, 20% to 35% or 20% to 30%.

[0191] 8. The method according to any one of items 1 to 7, wherein the shuttle comprises a histidine-rich domain, optionally wherein the histidine-rich domain is: (i) oriented toward the N-terminus and / or C-terminus of the shuttle; (ii) is an extension comprising at least 3, at least 4, at least 5, or 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% of histidine residues; and / or comprises 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 of (i) and (ii).

[0192] 9. The method according to any one of items 1 to 8, wherein the shuttle comprises a flexible connector domain rich in serine and / or glycine residues.

[0193] 10. The method according to any one of items 1 to 9, wherein the shuttle comprises or is composed of the following amino acid sequences: (a) [X1]-[X2]-[connector]-[X3]-[X4] (Formula 1); (b) [X1]-[X2]-[connector]-[X4]-[X3] (Formula 2); (c) [X2]-[X1]-[connector]-[X3]-[X4] (Formula 3); (d) [X2]-[X1]-[connector]-[X4]-[X3] (Formula 4); (e) [X3]-[X4]-[connector]-[X1]-[X2] (Formula 5); (f) [X3]-[X4]-[connector]-[X2]-[X1] (Formula 6); (g) [X4]-[ X3]-[connector]-[X1]-[X2](Equation 7); or (h)[X4]-[X3]-[connector]-[X2]-[X1](Equation 8), wherein [X1] is selected from: 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 -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 -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 -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 [link] is selected from -Gn-;-Sn-;-(GnSn)n-;-(GnSn)nGn-;-(GnSn)nSn-;-(GnSn)nGn(GnSn)n-;and -(GnSn)nSn(GnSn)n-;and -(GnSn)nSn(GnSn)n-;where: [Φ] is an amino acid, which is: Leu, P he, Trp, Ile, Met, Tyr, or Val, preferably Leu, Phe, Trp, or Ile; [+] is an amino acid, namely Lys or Arg; [ζ] is an amino acid, namely Gln, Asn, Thr, or Ser; A is the amino acid Ala; G is the amino acid Gly; S is the amino acid Ser; and n is an integer from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 1 to 4, or 1 to 3.

[0194] 11. The method according to any one of items 1 to 10, wherein the shuttle comprises or is composed of a peptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of any one of SEQ ID NO:19-50.

[0195] 12. The method according to any one of items 1 to 11, wherein the shuttle comprises an endosomal leakage domain (ELD) and / or a cell penetration domain (CPD).

[0196] 13. The method according to any one of items 1 to 12, wherein: (i) the ELD is or is derived from: endosome-dissolving peptide; antimicrobial peptide (AMP); linear cationic α-helical antimicrobial peptide; bactericidal peptide-A / bee venom peptide hybrid (CM) peptide; pH-dependent membrane-active peptide (PAMP); peptide amphiphile; N-terminal peptide derived from the HA2 subunit of influenza hemagglutinin (HA); CM18; diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB 1; VSVG; Pseudomonas toxin; melitoxin; KALA; JST-1; C(LLKK)3C; G(LLKK)3G; or any combination thereof; (ii) the CPD is or is derived from: a cell-penetrating peptide or a protein transduction domain derived from a cell-penetrating peptide; TAT; PTD4; a membrane-penetrating peptide; pVEC; M918; Pep-1; Pep-2; Xentry; an arginine extension; a transporter; SynB1; SynB3; or any combination thereof; or (iii) both of (i) and (ii).

[0197] 14. The method according to any one of items 1 to 13, wherein the shuttle is a cyclic peptide and / or contains one or more D-amino acids.

[0198] 15. The method according to any one of items 1 to 14, wherein the method is an in vitro method, such as for therapeutic and / or diagnostic purposes.

[0199] 16. The method according to any one of items 1 to 14, wherein the method is an in vivo method, such as for therapeutic and / or diagnostic purposes.

[0200] 17. The method according to item 16, the method comprising the topical, enteral / gastrointestinal (e.g., oral) or parenteral administration of the non-protein load and the synthetic peptide shuttle.

[0201] 18. A composition for transducing a non-protein load into target eukaryotic cells, the composition comprising a synthetic peptide shuttle formulated with a pharmaceutically suitable excipient, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the non-protein load into the target eukaryotic cells and cytosol delivery after administration.

[0202] 19. The composition according to item 17, wherein the composition further comprises the non-protein load.

[0203] 20. The composition according to item 18 or 19, wherein: (a) the synthetic peptide shuttle is according to any one of items 1 or 5 to 14; (b) the non-protein load is according to any one of items 2 to 4; (c) the composition is used in an in vitro or in vivo method according to any one of items 15 to 17; or (d) any combination of (a) to (c).

[0204] 21. A kit for use in the method according to any one of items 1 to 17, the kit comprising a synthetic peptide shuttle according to any one of items 1 or 5 to 14 and a non-protein load according to any one of items 2 to 4.

[0205] 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.

[0206] 23. A synthetic peptide shuttle having transduction activity for both protein and non-protein loads, said shuttle comprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of any one of SEQ ID NO:19-50.

[0207] 24. The synthetic peptide shuttle according to item 23, wherein the synthetic peptide shuttle is a shuttle according to any one of items 5 to 13.

[0208] Project II

[0209] In some respects, one or more of the following items are described here:

[0210] 1. A method for non-protein load transduction, the method comprising contacting a target eukaryotic cell with a non-protein load and a concentration of a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle is sufficient to increase the transduction efficiency of the non-protein load compared to the absence of the synthetic peptide shuttle.

[0211] 2. The method according to Item 1, wherein the non-protein load: (a) is an organic compound; (b) has a molecular weight of less than 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 Da, or between 50 and 5,000, 50 and 4,000, 50 and 3,000, 50 and 2,000, or 50 and 1,000 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 biopolymer, such as a polynucleotide or polysaccharide; (e) is not covalently linked to the synthetic peptide shuttle during transduction; or (f) any combination of (a) to (e).

[0212] 3. The method according to Project 1 or Project 2, wherein the non-protein load is a medicine used to treat cancer (e.g., skin cancer, basal cell carcinoma, nevus-like basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye disease, dry or wet 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).

[0213] 4. The method according to any one of items 1 to 3, wherein the non-protein load is or contains hedgehog inhibitors (e.g., itraconazole, posaconazole, arsenic trioxide (ATO), Gant61, PF-4708671, HPI-1, HPI-4), pain inhibitors such as voltage-gated sodium (Nav) channel inhibitors (e.g., QX-314), and / or inhibitors of inflammation (e.g., inhibitors of inflammatory cytokine production or NF-κB pathway inhibitors).

[0214] 5. The method according to any one of items 1 to 4, wherein the shuttle is: (1) a peptide of at least 20 amino acids in length, the peptide comprising (2) an amphiphilic α-helical motif having (3) a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein at least five of the following parameters (4) to (15) are obeyed: (4) an open cylindrical representation based on an α-helix having 3.6 residues per turn, the hydrophobic outer surface comprising a highly hydrophobic core composed of spatially adjacent L, I, F, V, W and / or M amino acids, the amino acids comprising 12% to 50% of the amino acids of the peptide; (5) the peptide having a hydrophobic torque (μ) of 3.5 to 11; (6) the peptide having a predicted net charge of at least +4 at physiological pH; (7) the peptide having an isoelectric point (pI) of 8 to 13; (8) the peptide consisting of 35% to 65% of any combination of the following amino acids: A, C, G, I, L, M, F, (9) The peptide is composed of 0% to 30% of any combination of the following amino acids: N, Q, S, and T; (10) The peptide is composed of 35% to 85% of any combination of the following amino acids: A, L, K, or R; (11) The peptide is composed of 15% to 45% of any combination of the following amino acids: A and L, provided that at least 5% L is present in the peptide; (12) The peptide is composed of 20% to 45% of any combination of the following amino acids. (13) The peptide is composed of 0% to 10% of any combination of the following amino acids: D and E; (14) The difference between the percentage of A and L residues (A+L%) and the percentage of K and R residues (K+R) in the peptide is less than or equal to 10%; and (15) The peptide is composed of 10% to 45% of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H.

[0215] 6. The method according to item 5, wherein: (a) the shuttle conforms to 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); (b) the 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; and (c) the amphiphilic α-helical motif has a length of 3.5, 3 The hydrophobic torque (μ) between the lower limit of 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 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 α-helical motif contains a positively charged A hydrophilic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, wherein the positively charged hydrophilic outer surface comprises: (i) at least two, three, or four adjacent positively charged K and / or R residues when projected onto the helical wheel; and / or (ii) a segment containing three to five K and / or R residues when projected onto the helical wheel; (e) the amphiphilic α-helical motif comprises a hydrophobic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, wherein the hydrophobic outer surface comprises: (i) at least two adjacent L residues when projected onto the helical wheel; and / or (ii) a segment containing three to five K and / or R residues when projected onto the helical wheel. (f) The hydrophobic outer surface comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W and / or M amino acids, wherein the amino acids constitute 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 in the shuttle; (g) The shuttle has 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.The following are possible values: (a) a hydrophobic moment (μ) between the lower limit of 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 the 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; (b) the shuttle has a predicted net charge between +4, +5, +6, +7, +8, +9 and +10, +11, +12, +13, +14 or +15; (c) the shuttle has a predicted pI of 10 to 13; or (d) any combination of (a) to (i).

[0216] 7. The method according to any one of items 1 to 6, wherein the shuttle complies with 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 is composed of any combination of the following amino acids in amounts of 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60%: A, C, G, I, L, M, F, P, W, Y, and V; (9) The shuttle consists of any combination of the following amino acids: N, Q, S, and T, at 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 shuttle consists of any combination of the following amino acids: N, Q, S, and T, at 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60%. (11) The shuttle consists of any combination of the following amino acids: A, L, K, or R; (12) The shuttle consists of any combination of the following amino acids: A and L, at 15% to 40%, 20% to 40%, 20% to 35%, or 20% to 30%; (13) The shuttle consists of any combination of the following amino acids: K and R, at 20% to 40%, 20% to 35%, or 20% to 30%; (14) The shuttle consists of any combination of the following amino acids: at 5% to 10%. D and E; (14) the difference between the percentage of A and L residues (A+L%) in the shuttle and the percentage of K and R residues (K+R) in the shuttle is less than or equal to 9%, 8%, 7%, 6% or 5%; and (15) the shuttle is composed of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H, in amounts of 15% to 40%, 20% to 35% or 20% to 30%.

[0217] 8. The method according to any one of items 1 to 7, wherein the shuttle comprises a histidine-rich domain, optionally wherein the histidine-rich domain is: (i) oriented toward the N-terminus and / or C-terminus of the shuttle; (ii) is an extension comprising at least 3, at least 4, at least 5, or 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% of histidine residues; and / or comprises 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 of (i) and (ii).

[0218] 9. The method according to any one of items 1 to 8, wherein the shuttle comprises a flexible connector domain rich in serine and / or glycine residues.

[0219] 10. The method according to any one of items 1 to 9, wherein the shuttle comprises or is composed of the following amino acid sequences: (a) [X1]-[X2]-[connector]-[X3]-[X4] (Formula 1); (b) [X1]-[X2]-[connector]-[X4]-[X3] (Formula 2); (c) [X2]-[X1]-[connector]-[X3]-[X4] (Formula 3); (d) [X2]-[X1]-[connector]-[X4]-[X3] (Formula 4); (e) [X3]-[X4]-[connector]-[X1]-[X2] (Formula 5); (f) [X3]-[X4]-[connector]-[X2]-[X1] (Formula 6); (g) [X4]-[ X3]-[connector]-[X1]-[X2](Equation 7); or (h)[X4]-[X3]-[connector]-[X2]-[X1](Equation 8), wherein [X1] is selected from: 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 -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 -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 -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 [link] is selected from -Gn-;-Sn-;-(GnSn)n-;-(GnSn)nGn-;-(GnSn)nSn-;-(GnSn)nGn(GnSn)n-;and -(GnSn)nSn(GnSn)n-;and -(GnSn)nSn(GnSn)n-;where: [Φ] is an amino acid, which is: Leu, P he, Trp, Ile, Met, Tyr, or Val, preferably Leu, Phe, Trp, or Ile; [+] is an amino acid, namely Lys or Arg; [ζ] is an amino acid, namely Gln, Asn, Thr, or Ser; A is the amino acid Ala; G is the amino acid Gly; S is the amino acid Ser; and n is an integer from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 1 to 4, or 1 to 3.

[0220] 11. The method according to any one of items 1 to 10, wherein the shuttle comprises or consists of the following amino acid sequences: an amino acid sequence of any one of SEQ ID NO: 1 to 50; an amino acid sequence that is approximately equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids (e.g., excluding any linker domain) of any one of SEQ ID NO: 1 to 50; or an amino acid sequence that is approximately equal to or consists of SEQ ID NO: 1 to 50. NO: Any one of the amino acid sequences from 1 to 50 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 (e.g., excluding any linker domains calculated) to be identical.

[0221] 12. The method according to any one of items 1 to 10, wherein the shuttle comprises or consists of the following amino acid sequences: SEQ ID NO: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344; and the amino acid sequence of any one of SEQ ID NO: NO: An amino acid sequence that is approximately equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., excluding any linker domain) in any of the following sequences: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344; or an amino acid sequence that is approximately equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., excluding any linker domains); or an amino acid sequence that is similar to SEQ ID NO. NO: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, or 58% of any one of these values. 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 (e.g., excluding any linker domains calculated) amino acid sequences.

[0222] 13. The method according to any one of items 1 to 12, wherein the shuttle comprises an endosomal leakage domain (ELD) and / or a cell penetration domain (CPD).

[0223] 14. The method according to any one of items 1 to 13, wherein: (i) the ELD is or is derived from: endosomal lysing peptide; antimicrobial peptide (AMP); linear cationic α-helical antimicrobial peptide; bactericidal peptide-A / bee venom peptide hybrid (CM) peptide; pH-dependent membrane-active peptide (PAMP); peptide amphiphile; N-terminal peptide derived from the HA2 subunit of influenza hemagglutinin (HA); CM18; diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB 1; VSVG; Pseudomonas toxin; melitoxin; KALA; JST-1; C(LLKK)3C; G(LLKK)3G; or any combination thereof; (ii) the CPD is or is derived from: a cell-penetrating peptide or a protein transduction domain derived from a cell-penetrating peptide; TAT; PTD4; a membrane-penetrating peptide; pVEC; M918; Pep-1; Pep-2; Xentry; an arginine extension; a transporter; SynB1; SynB3; or any combination thereof; or (iii) both of (i) and (ii).

[0224] 15. The method according to any one of items 1 to 14, wherein the shuttle is a cyclic peptide and / or contains one or more D-amino acids.

[0225] 16. The method according to any one of items 1 to 15, wherein in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, the shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators 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, and / or is capable of achieving at least 10%, 11%, 12%, 13%, or 10-fold increase in transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. 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% transduction efficiency (e.g., as determined by flow cytometry).

[0226] 17. The method according to any one of items 1 to 16, wherein in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, 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-fold compared to a corresponding negative control lacking the shuttle agent, and / or achieves at least 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16-fold of GFP-NLS transduction efficiency. %, 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% of the transduction efficiency (e.g., as determined by flow cytometry).

[0227] 18. The method according to any one of items 1 to 17, wherein the shuttle further comprises a chemical modification of one or more amino acids, wherein the chemical modification does not impair the transduction activity of the synthetic peptide shuttle.

[0228] 19. The method according to item 18, wherein the chemical modification is at the N and / or C end of the shuttle.

[0229] 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 cysteine ​​group (e.g., a C-terminal cysteine ​​group), or a fatty acid (e.g., a C4-C16 fatty acid, preferably N-terminal).

[0230] 21. The method according to any one of items 1 to 20, wherein the method is an in vitro method, such as for therapeutic and / or diagnostic purposes.

[0231] 22. The method according to any one of items 1 to 20, wherein the method is an in vivo method, such as for therapeutic and / or diagnostic purposes.

[0232] 23. The method according to item 22, the method comprising the topical, enteral / gastrointestinal (e.g., oral) or parenteral administration of the non-protein load and the synthetic peptide shuttle.

[0233] 24. A composition for transducing a non-protein load into target eukaryotic cells, the composition comprising a synthetic peptide shuttle formulated with a pharmaceutically suitable excipient, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the non-protein load into the target eukaryotic cells and cytosol and / or nuclear delivery after administration.

[0234] 25. The composition according to item 24, wherein the composition further comprises the non-protein load.

[0235] 26. A composition for a therapy comprising the synthetic peptide shuttle formulated together with a non-protein load to be transduced into target eukaryotic cells via a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the non-protein load into the target eukaryotic cells and cytosol and / or nuclear delivery after administration.

[0236] 27. The composition according to any one of items 24 to 26, wherein: (a) the synthetic peptide shuttle is according to any one of items 1 or 5 to 20; (b) the non-protein load is according to any one of items 2 to 4; (c) the composition is used in an in vitro or in vivo method according to any one of items 21 to 23; or (d) any combination of (a) to (c).

[0237] 28. A kit for use in the method according to any one of items 1 to 23, the kit comprising a synthetic peptide shuttle according to any one of items 1 or 5 to 20 and a non-protein load according to any one of items 2 to 4.

[0238] 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.

[0239] 30. A synthetic peptide shuttle having transduction activity for both protein and non-protein loads, said shuttle comprising or consisting of the following amino acid sequences: an amino acid sequence of any one of SEQ ID NO: 19 to 50; an amino acid sequence that is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., excluding any linker domain) similar to any one of SEQ ID NO: 19 to 50; or an amino acid sequence similar to SEQ ID NO: 19 to 50. NO:19 to 50 contains 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 amino acid sequences (e.g., excluding any linker domains).

[0240] 31. The synthetic peptide shuttle according to item 30, wherein the synthetic peptide shuttle is a shuttle according to any one of items 5 to 20.

[0241] 32. A synthetic peptide shuttle having transduction activity in target eukaryotic cells for both protein and non-protein loads, said shuttle being: (1) a peptide of at least 17, 18, 19 or 20 amino acids, said peptide comprising (2) an amphiphilic α-helical motif having (3) a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein at least five of the following parameters (4) to (15) are obeyed: (4) an open cylindrical shape based on an α-helix having 3.6 residues per turn, said hydrophobic outer surface comprising spatially adjacent L, I, F, V, W and / or M amino groups. (5) The peptide has a highly hydrophobic core composed of an acid, wherein the amino acid constitutes 12% to 50% of the amino acids in the peptide; (6) The peptide has a hydrophobic moment (μ) of 3.5 to 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 to 13; (9) The peptide is composed of any combination of 35% to 65% of the following amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (10) The peptide is composed of any combination of 0% to 30% of the following amino acids: N, Q, S, and T; (11) The peptide is composed of any combination of the following amino acids from 15% to 45%: A and L, provided that at least 5% L is present in the peptide; (12) The peptide is composed of any combination of the following amino acids from 20% to 45%: K and R; (13) The peptide is composed of any combination of the following amino acids from 0% to 10%: D and E; (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 less than or equal to 10%. And (15) the peptide is composed of 10% to 45% of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H, wherein in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, the shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9 compared to the corresponding negative control lacking the shuttle.5 or 10 times, and / or capable of achieving 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% transduction efficiency (e.g., as determined by flow cytometry) of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. .

[0242] 33. The synthetic peptide shuttle agent according to item 32, wherein in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, 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-fold compared to a corresponding negative control lacking the shuttle agent, and / or achieves at least 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of GFP-NLS. 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% transduction efficiency (e.g., as determined by flow cytometry).

[0243] 34. A synthetic peptide shuttle according to item 32 or 33, wherein: (a) the shuttle conforms to 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); (b) the shuttle is a peptide having 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, 100, 110, 120, 130, 140, or 150 amino acids; (c) the The amphiphilic α-helical motifs are located at 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... The hydrophobic moment (μ) between the lower limits of 0.3, 6.4, 6.5, 6.6, 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; (d) The amphiphilic α-helical motif comprises a positively charged hydrophilic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, wherein the positively charged hydrophilic outer surface comprises: (i) at least two, three, or four adjacent positively charged K and / or R residues in the helical wheel projection; and / or (ii) a segment of six adjacent residues containing three to five K and / or R residues in the helical wheel projection; (e) the amphiphilic α-helical motif comprises a hydrophobic outer surface, based on an α-helix with a rotation angle of 100 degrees between consecutive amino acids and / or an α-helix having 3.6 residues per turn, wherein the hydrophobic outer surface comprises: (i) at least two adjacent L residues in the helical wheel projection; and / or (ii) a segment comprising ten adjacent residues of at least five hydrophobic residues selected from L, I, F, V, W, and M, as projected onto the helical wheel; (f) the hydrophobic outer surface comprising a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids, the amino acids comprising 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 in the shuttle; (g) the shuttle having 4.0, 4.1, 4.2, 4.3, 4.4, 4.The lower limits of 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, and 7.0 are the same as the lower limits of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, and 10. 1. A hydrophobic torque (μ) between the upper limits of 10.2, 10.3, 10.4, or 10.5; (h) The shuttle has a predicted net charge between +4, +5, +6, +7, +8, +9 and +10, +11, +12, +13, +14, or +15; (i) The shuttle has a predicted pI of 10 to 13; or (j) Any combination of (a) to (i).

[0244] 35. A synthetic peptide shuttle according to any one of items 32 to 34, wherein the shuttle complies with 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 is composed of any combination of the following amino acids in 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60%: A, C, G, I, L, M, F, P, W, Y (9) The shuttle consists 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% of any combination of the following amino acids: N, Q, S, and T; (10) The shuttle consists of 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60% of the following amino acids: N, Q, S, and T. (11) The shuttle consists of 15% to 40%, 20% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: A and L; (12) The shuttle consists of 20% to 40%, 20% to 35%, or 20% to 30% of any combination of the following amino acids: K and R; (13) The shuttle consists of 5% to 10% of any combination of the following amino acids: (14) The difference between the percentage of A and L residues (A+L%) in the shuttle and the percentage of K and R residues (K+R) in the shuttle is less than or equal to 9%, 8%, 7%, 6% or 5%; and (15) The shuttle is composed of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H, in amounts of 15% to 40%, 20% to 35% or 20% to 30%.

[0245] 36. A synthetic peptide shuttle according to any one of items 32 to 35, wherein the shuttle: (i) comprises a histidine-rich domain according to item 8; (ii) comprises a flexible linker domain according to item 9; (iii) is a shuttle according to any one of items 10 to 14; or (iv) any combination of (i) to (iii).

[0246] 37. The synthetic peptide shuttle according to any one of items 32 to 36, wherein the synthetic peptide shuttle further comprises a chemical modification of one or more amino acids, wherein the chemical modification does not impair the transduction activity of the synthetic peptide shuttle.

[0247] 38. The synthetic peptide shuttle according to item 37, wherein the chemical modification is at the N and / or C terminus of the shuttle.

[0248] 39. The 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 cysteine ​​group (e.g., a C-terminal cysteine ​​group), or a fatty acid (e.g., a C4-C16 fatty acid, preferably N-terminal).

[0249] 40. A synthetic peptide shuttle according to any one of items 32 to 39, wherein the shuttle comprises or consists of the following amino acid sequences: SEQ ID NO: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344; and the amino acid sequence of any one of SEQ ID NO: NO: An amino acid sequence that is approximately equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., excluding any linker domain) in any of the following sequences: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344; or an amino acid sequence that is approximately equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., excluding any linker domains); or an amino acid sequence that is similar to SEQ ID NO. NO: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, or 58% of any one of these values. 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 (e.g., excluding any linker domains calculated) amino acid sequences.

[0250] 41. A synthetic peptide shuttle having transduction activity in target eukaryotic cells for both protein and non-protein loads, wherein the shuttle comprises or consists of the following amino acid sequences: (a) an amino acid sequence of any one of SEQ ID NO: 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344; or (b) an amino acid sequence consistent with ... NO: Any one of 1 to 50, 58 to 78, 80 to 107, 109 to 139, 141 to 146, 149 to 161, 163 to 169, 171, 174 to 234, 236 to 240, 242 to 260, 262 to 285, 287 to 294, 296 to 300, 302 to 308, 310, 311, 313 to 324, 326 to 332, 338 to 342, or 344 The amino acid sequence differs only from conserved amino acid substitutions (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, preferably excluding any linker domains), wherein, in a eukaryotic cell line model suitable for evaluating load transduction in the target eukaryotic cells (e.g., HeLa), the shuttle agent renders propidium iodide or other membrane-impermeable fluorescent DNA intercalators transduced more efficiently than the corresponding negative pair lacking the shuttle agent. The concentration is increased 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 is capable of achieving at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, or 27% of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. 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% transduction efficiency (e.g., as determined by flow cytometry).

[0251] 42. The synthetic peptide shuttle according to item 41, wherein the synthetic peptide shuttle is the synthetic peptide shuttle according to any one of items 32 to 39.

[0252] 43. A synthetic peptide shuttle having protein loading transduction activity in target eukaryotic cells, wherein the shuttle comprises or consists of the following amino acid sequences: (a) an amino acid sequence of any one of SEQ ID NO: 52, 57, 79, 108, 140, 147, 148, 173, 241, 261, 286, 295, 301, 309, 312, 325, 333-337 or 343; or (b ... NO: any one of the following amino acid sequences differing only from a conserved amino acid substitution (e.g., differing by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, preferably excluding any linker domains): 52, 57, 79, 108, 140, 147, 148, 173, 241, 261, 286, 295, 301, 309, 312, 325, 333-337, or 343, wherein the shuttle induces transduction of GFP-NLS in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells. The efficiency is increased 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, and / or is able to achieve 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% of the transduction efficiency of GFP-NLS (e.g., as determined by flow cytometry).

[0253] 44. The synthetic peptide shuttle according to any one of items 41 to 43, wherein each conserved amino acid substitute is selected from amino acids within the same amino acid class, said amino acid class being: aliphatic: G, A, V, L and I; hydroxyl-containing or sulfur / selenium-containing: S, C, U, T and M; aromatic: F, Y and W; basic: H, K and R; acidic and its amide: D, E, N and Q.

[0254] 45. A synthetic peptide shuttle variant having transduction activity for protein and / or non-protein loads in target eukaryotic cells, said synthetic peptide shuttle variant being identical to the synthetic peptide shuttle according to any one of items 32 to 44, except that at least one amino acid is replaced with a corresponding synthetic amino acid containing a side chain having similar physiological and chemical properties (e.g., structure, hydrophobicity, or charge) to the substituted amino acid, wherein said shuttle variant increases the transduction efficiency of said load in the target eukaryotic cells compared to the absence of said shuttle variant.

[0255] 46. ​​A synthetic peptide shuttle variant according to item 45, wherein the synthetic amino acid is replaced by:

[0256] (a) Replace the basic amino acid with any of the following: α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, 2,6-diamino-4-hexynic acid, β-(1-piperazinyl)-alanine, 4,5-dehydro-lysine, δ-hydroxylysine, ω,ω-dimethylarginine, homoarginine, ω,ω'-dimethylarginine, ω-methylarginine, β-(2-quinolinyl)-alanine, 4-aminopiperidin-4-carboxylic acid, α-methylhistidine, 2,5-diiodohistidine, 1-methylhistidine, 3-methylhistidine, spinachin, 4-aminophenylalanine, 3-aminotyrosine, β-(2-pyridyl)-alanine or β-(3-pyridyl)-alanine;

[0257] (b) Replace the nonpolar (hydrophobic) amino acid with any of the following: dehydroalanine, β-fluoroalanine, β-chloroalanine, β-iodoalanine, α-aminobutyric acid, α-aminoisobutyric acid, β-cyclopropylalanine, aziridine-2-carboxylic acid, α-allylglycine, propargylglycine, tert-butylalanine, β-(2-thiazolyl)-alanine, thioproline, 3,4-dehydroproline, tert-butylglycine, β-cyclopentylalanine, β-cyclohexylalanine, α-methylproline, pentylalanine, α-methylvaline, penicillamine, β,β-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, piperacillic acid, 4,5-dehydroleucine, alloleucine, noreleucine, α-methylleucine, cyclohexylglycine, cis-octahydroindole-β-2-carboxylic acid, β-(2-thiophene) β-(3-benzothiophene)-alanine, phenylglycine, α-methylphenylalanine, homophenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-(3-benzothiophene)-alanine, 4-nitrophenylalanine, 4-bromophenylalanine, 4-tert-butylphenylalanine, α-methyltryptophan, β-(2-naphthyl)-alanine, β-(1-naphthyl)-alanine, 4-iodophenylalanine, 3-fluorophenylalanine Acids, 4-fluorophenylalanine, 4-methyltryptophan, 4-chlorophenylalanine, 3,4-dichlorophenylalanine, 2,6-difluorophenylalanine, n-in-methyltryptophan, 1,2,3,4-tetrahydronorhalman-3-carboxylic acid, β,β-diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine or 4-benzoylphenylalanine;

[0258] (c) Replace the polar, uncharged amino acid with any of the following: β-cyanoalanine, β-ureidoalanine, homocysteine, alletine, pyroglutamic acid, 2-oxothiazolidin-4-carboxylic acid, citrulline, thiocitrulline, homocitrulline, hydroxyproline, 3,4-dihydroxyphenylalanine, β-(1,2,4-triazol-1-yl)-alanine, 2-mercaptohistidine, β-(3,4-dihydroxyphenyl)-serine, β-(2-thienyl)-serine 4-Azide-phenylalanine, 4-cyanophenylalanine, 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, thyroxine, β-(7-methoxycoumarin-4-yl)-alanine or 4-(7-hydroxy-4-coumarinyl)-aminobutyric acid; and / or

[0259] (d) Replace the acidic amino acid with any of the following: γ-hydroxyglutamic acid, γ-methyleneglutamic acid, γ-carboxyglutamic acid, α-aminoadipic acid, 2-aminopimelic acid, α-aminooctanoic acid, 4-carboxyphenylalanine, sulfoalanine, 4-phosphonophenylalanine or 4-sulfomethylphenylalanine.

[0260] 47. A synthetic peptide shuttle or a variant thereof according to any one of items 32 to 46, used in an in vitro or in vivo method to increase the transduction efficiency of protein and / or non-protein loads (e.g., therapeutically active protein and / or non-protein loads) into target eukaryotic cells, wherein the synthetic peptide shuttle or variant thereof is used at a concentration sufficient to increase the transduction efficiency of the load into the target eukaryotic cells and cytosol and / or nuclear delivery, compared to the absence of the synthetic peptide shuttle or variant thereof.

[0261] 48. A synthetic peptide shuttle or a variant thereof according to any one of items 32 to 47, said synthetic peptide shuttle or variant thereof used in a therapy, wherein said synthetic peptide shuttle or variant thereof transduces a therapeutically active protein and / or non-protein load to the cytosol and / or nucleus of a target eukaryotic cell, wherein said synthetic peptide shuttle or variant thereof is used at a concentration sufficient to increase the transduction efficiency of said load to the target eukaryotic cell compared to the absence of said synthetic peptide shuttle.

[0262] 49. An in vitro or in vivo method for transduction of protein and / or non-protein loads, the method comprising contacting target eukaryotic cells with the load and a concentration of a synthetic peptide shuttle or a variant thereof according to any one of items 32 to 46, wherein the concentration of the synthetic peptide shuttle or the variant thereof is sufficient to increase the transduction efficiency of the load into the target eukaryotic cells compared to the absence of the synthetic peptide shuttle.

[0263] 50. The in vitro or in vivo method according to item 49, wherein the in vitro or in vivo method is a method for therapeutic and / or diagnostic purposes.

[0264] 51. A composition for therapy comprising a synthetic peptide shuttle or a variant thereof according to any one of items 32 to 46, formulated together with a protein and / or non-protein load to be transduced into target eukaryotic cells by a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle or the variant thereof in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency and cytosol delivery of the load into the target eukaryotic cells after administration.

[0265] 52. The composition according to item 51, wherein the composition is formulated for topical, enteral / gastrointestinal (e.g., oral) or parenteral application.

[0266] 53. A kit comprising a synthetic peptide shuttle or a variant thereof according to any one of items 32 to 46, and a protein and / or non-protein load to be transduced by said synthetic peptide shuttle or variant thereof.

[0267] 54. A synthetic peptide shuttle or a variant thereof according to any one of items 32 to 48, an in vitro or in vivo method according to item 49 or 50, a composition according to item 51 or 52, or a kit according to item 53, 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.

[0268] 55. A synthetic peptide shuttle or a variant thereof according to any one of items 32 to 48 or 54, an in vitro or in vivo method according to item 49 or 50 or 54, a composition according to item 51, 52 or 54, or a kit according to item 53 or 54, wherein the non-protein load is according to any one of items 2 to 4.

[0269] 56. A method for generating a candidate synthetic peptide shuttle intended to transduce a target load in target eukaryotic cells, the method comprising synthesizing a peptide that is: (1) a peptide of at least 17, 18, 19 or 20 amino acids, the peptide comprising (2) an amphiphilic α-helical motif having (3) a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein at least five of the following parameters (4) to (15) are obeyed: (4) an open cylindrical shape based on an α-helix having 3.6 residues per turn, the hydrophobic outer surface comprising spatially adjacent L, I, F, (5) The peptide has a highly hydrophobic core composed of amino acids V, W, and / or M, which constitute 12% to 50% of the amino acids in the peptide; (6) The peptide has a hydrophobic moment (μ) of 3.5 to 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 to 13; (9) The peptide is composed of any combination of 35% to 65% of the following amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (10) The peptide is composed of any combination of 0% to 30% of the following amino acids: N, Q, S, and T; The peptide is composed of 35% to 85% of any combination of the following amino acids: A, L, K, or R; (11) The peptide is composed of 15% to 45% of any combination of the following amino acids: A and L, provided that at least 5% L is present in the peptide; (12) The peptide is composed of 20% to 45% of any combination of the following amino acids: K and R; (13) The peptide is composed of 0% to 10% of any combination of the following amino acids: D and E; (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 less than or equal to 1. 0%; and (15) the peptide is composed of 10% to 45% of any combination of the following amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H, wherein in a eukaryotic cell line model (e.g., HeLa) suitable for evaluating load transduction in the target eukaryotic cells, the shuttle increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators by 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.5 or 10 times, and / or capable of achieving 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% transduction efficiency (e.g., as determined by flow cytometry) of propidium iodide or other membrane-impermeable fluorescent DNA intercalators. .

[0270] 57. The method according to item 56, wherein the candidate synthetic peptide shuttle is a synthetic peptide shuttle or a variant of a synthetic peptide shuttle according to any one of items 32 to 46.

[0271] 58. An in vitro or in vivo method for identifying, selecting, or defining synthetic peptide shuttles expected to have transduction activity for both protein and non-protein loads in target eukaryotic cells, the method comprising: providing a model eukaryotic cell or model organism suitable for evaluating load transduction in the target eukaryotic cells; providing a candidate synthetic peptide shuttle (e.g., as described in any one of items 5 to 20 or 32 to 46); and measuring the transduction activity of the candidate synthetic peptide shuttle in transducing propidium iodide or other membrane-impermeable fluorescent DNA intercalators into the model eukaryotic cell or model organism (e.g., load transduction efficiency, such as by flow cytometry), wherein the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators in the model eukaryotic cell or model organism is increased by at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 compared to a corresponding negative control lacking the candidate synthetic peptide shuttle. 7.5, 8, 8.5, 9, 9.5, or 10 times, and / or 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%, or 36% of the said propidium iodide or other membrane-impermeable fluorescent DNA intercalating agent. When the transduction efficiency reaches 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., as determined by flow cytometry), the candidate shuttle is expected to have transduction activity against both protein and non-protein loads in the target eukaryotic cells.

[0272] Example

[0273] Example 1: Materials and Methods

[0274] All materials and methods not described or specified herein are generally used as in WO / 2016 / 161516 and / or WO / 2018 / 068135.

[0275] 1.1 Materials and Reagents

[0276]

[0277] 1.3 Cell lines and culture conditions

[0278] Culture the cells according to the manufacturer's instructions.

[0279]

[0280] FBS: Fetal bovine serum

[0281] BCS: Fetal serum

[0282] 1.4 Propidium iodide transduction protocol

[0283] The day before the experiment, HeLa cells were plated in 96-well dishes (20,000 cells / well). Each delivery mixture containing a synthetic peptide shuttle (10 μM) and either propidium iodide (PI) (10 μg / mL) or GFP-NLS (10 μM) was prepared and brought to a final volume of 50 μL with phosphate-buffered saline (PBS). Cells were washed once with PBS, and then the shuttle / PI or shuttle / GFP-NLS was added to the cells over one minute. 100 μL of DMEM containing 10% FBS was then added to the mixture and removed. Cells were washed once with PBS and incubated in DMEM containing 10% FBS. After 2 hours of incubation, cells were analyzed by flow cytometry. For the “FS then PI” condition, only the synthetic peptide shuttle (10 μM) was added to the HeLa cells over one minute, and PI (10 μg / mL) was added over one minute after the same washing step one hour later. Cells were analyzed one hour after PI or GFP-NLS treatment.

[0284] 1.5Gli Reporting Hedgehog Pathway Inhibitor Transduction Protocol in NIH3T3 Cells

[0285] The loading stock solutions were prepared as follows: Gant61 stock solution (20 mM in DMSO); HPI4 stock solution (40 mM in DMSO); itraconazole stock solution (4.8 mM (4 mg / mL) in DMSO); and arsenic trioxide (ATO) stock solution (40 mM in H2O). The peptide shuttle (5 μM) and hedgehog pathway inhibitor (100 μM) were mixed, and the volume was brought to 50 μL with PBS.

[0286] Hedgehog signaling pathway Gli reporter NIH 3T3 cells were cultured in DMEM containing 10% fetal bovine serum (FBS). Cells were trypsinized, centrifuged, and resuspended in PBS at 10 million cells / mL. 50 μL of cells (500,000 cells / well) were distributed in untreated 96-well round-bottom plates. The resuspended cells were mixed with a delivery mixture containing a peptide shuttle (5 μM) and a hedgehog pathway inhibitor (100 μM). Cells were incubated with the delivery mixture at room temperature for 90 seconds, and 200 μL of DMEM containing 10% FBS was added to each well. Cells were centrifuged (400 g, 4 min) and washed with 200 μL of PBS. Cells were then resuspended in 200 μL of DMEM and transferred to the wells of a 6-well plate containing 1 mL of DMEM containing 10% FBS, and incubated at 37°C for 2 h. The culture medium was gently removed, and 1 mL of control medium (Opti-MEM) was added. TM Add either 1000 μg / mL mShh or 1000 μg / mL Opti-MEM to each well. Incubate the cells at 37°C for 24–30 hours.

[0287] For analysis, cells were trypsinized and resuspended in 200 μL Opti-MEM. TM The sample was then divided into two equal wells in a round-bottom 96-well plate. Viability was assessed using flow cytometry, and luminescence was measured using the ONE-Step luciferase assay according to the manufacturer's instructions.

[0288] 1.6 In vivo transduction protocol for hedgehog pathway inhibitors

[0289] The recommended suspension composition was: Gant61, 20 mM in DMSO; itraconazole stock solution, 4.8 mM (4 mg / mL) in DMSO. Female C57BL6 mice aged 6 to 7 weeks were shaved and treated with a hair removal product (Nair). TM Hair removal. Five days after hair removal, 30 μL of a mixture containing PBS, synthetic peptide shuttle FSD250D (SEQ ID NO:36), and / or a loading solution was applied to a 3 cm area. 2 On hairless skin. Mice were imaged at 3, 10, and 17 days post-treatment.

[0290] 1.7QX-314 and GFP-NLS cotransduction and patch-clamp technique

[0291] Cell culture. HEK293 cells stably expressing Nav1.7 were grown in Duchenne minimum essential medium (DMEM, GibcoBRL Life Technologies) supplemented with fetal bovine serum (FBS, 10%), L-glutamine (2 mM), penicillin (100 U / mL), and streptomycin (10 mg / mL). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2.

[0292] Delivery. Cells were seeded in 24-well plates 24 hours before the experiment. Cells were washed twice with PBS. A solution containing 1 mM QX-314, 5 μM FSD194, and 15 μM GFP-NLS protein was applied to the cells over 90 seconds and removed by aspiration. In the presence of GFP-NLS, 5 μM FSD194 or 2.5 mM QX-314 was used as a control. Cells were washed with 800 μL of DMEM containing 10% FBS and transferred to recording solution for electrophysiology. GFP-positive cells were identified by microscopic examination and then selected for patch-clamp analysis.

[0293] Electrophysiology. Frequency protocols using a QX-314 were recorded within 30 seconds of forming the whole-cell configuration. The frequency protocol consisted of 10 ms pulses at -20 mV from a hold potential of -140 mV at 10 Hz. Whole-cell Na+ currents in HEK293 cells were recorded at room temperature using an Axopatch 200B (Molecular Devices) with patch-clamp technology in a whole-cell configuration. pClamp v10.0 (Molecular Devices) was used for pulse stimulation and recording. The currents were filtered at 5 kHz, digitized at 100 kHz using a Digidata 1550AD converter (Molecular Devices), and stored on a computer for subsequent analysis. Series resistance was compensated for 70%–80%. Linear leakage current artifacts were removed using in-line leakage subtraction where necessary. Low-resistance electrodes (2 MΩ) were drawn from 8161 glass (Corning) and polished.

[0294] Record the solutions. Electrolyte: 35 mM NaCl, 115 mM NMDG, 2 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 10 mM glucose, 10 mM HEPES. Adjust the pH to 7.3 with 1 M NaOH. Electrode solution: 35 mM NaCl, 105 mM CsF, 10 mM EGTA, and 10 mM HEPES. Adjust the pH to 7.4 with 1 M CsOH.

[0295] Example 2: Synthetic peptide shuttle enables intracellular delivery of propidium iodide.

[0296] Propidium iodide (PI) is a fluorescent DNA-intercalating dye commonly used as a nuclear staining agent in fluorescence microscopy and flow cytometry applications. PI binding to DNA results in a 20- to 30-fold increase in fluorescence and a shift in its maximum excitation / emission spectrum. Because PI typically cannot cross the plasma membrane of living cells, it is routinely used to detect dead cells in cell populations. Surprisingly, this paper reveals that synthetic peptide shuttles, including the shuttle peptides for protein load transduction described in WO / 2016 / 161516 and WO / 2018 / 068135, are capable of transducing PI as well as other non-protein loads.

[0297] HeLa cells were cultured as described in Example 1.3 and subjected to the PI transduction protocol as described in Example 1.4, wherein in some experiments, the protein load GFP-NLS was transduced alone as a control. Results were obtained by flow cytometry two hours after delivery and expressed as the percentage of fluorescent cells (PI+ cells % or GFP+ cells %), as shown in Figures 1A-1D and summarized in the table shown in Figure 2.

[0298] Figures 1 and 2 show the delivery and viability results of HeLa cells co-incubated for 1 min with either a synthetic peptide shuttle or a control peptide along with a non-protein load PI (Figure 1A and Figure 1B) or a protein load GFP-NLS (Figure 1C and Figure 1D). Several members of different families of peptide shuttles or control peptides were tested. The first group of synthetic peptide shuttles tested contained an endosome leakage domain (ELD) operatively linked to a cell penetration domain (CPD) (as previously described in WO / 2016 / 161516 for its ability to transduce protein loads). The second and third groups of synthetic peptide shuttles tested corresponded to those shuttles rationally designed and optimized for delivering protein loads; the second group consisted of peptides previously described in WO / 2018 / 068135. The fourth group of synthetic peptide shuttles tested corresponded to cyclic peptides having an amide bond between their C and N ends (e.g., "FSD268 cyclic amide"; SEQ ID NO:49) or having a disulfide bridge between two side-attached cysteine ​​residues added at the N and C ends (e.g., "FSD268 cyclic disulfide"; SEQ ID NO:50). The fifth group of peptides was a negative control peptide that did not comply with several rational design parameters for synthetic peptide shuttles described in WO / 2018 / 068135 (e.g., FSN3, FSN4, and FSN8; SEQ ID NO:54, 55, and 57, respectively). These negative control peptides also include “FSD10 scrambled” (SEQ ID NO:51), “FSD268 scrambled” (SEQ ID NO:52), and “FSD174 scrambled” (SEQ ID NO:53), which have the same amino acid composition as the peptide shuttles FSD10, FSD268, and FSD174 (SEQ ID NO:13, 43, and 32, respectively), but in which the amino acid sequence is altered to deviate from several reasonable design parameters described in WO / 2018 / 068135. In Figures 1A and 1B, “FS then PI” indicates that PI is added 1 hour after treatment with the synthetic peptide shuttle, thereby ensuring that the PI positive signal is not due to cell death. Finally, the rightmost bars in Figures 1A-1D correspond to negative controls, where cells are incubated only with the load (“PI” in Figures 1A and 1B or “GFP-NLS” in Figures 1C and 1D), or the cells are untreated cells that are not exposed to the load or shuttle peptide (“NT”, Figures 1A-1D).

[0299] In summary, the results indicate that family members of synthetic peptide shuttles containing an ELD operatively linked to a CPD (as described in WO / 2016 / 161516) and those rationally designed for protein load transduction (as described in WO / 2018 / 068135) can increase the transduction efficiency (and protein transduction activity) of non-protein, relatively low molecular weight loads (such as PIs). Notably, several negative control peptides that failed to adhere to the rational design parameters for protein load delivery described in WO / 2018 / 068135 also failed to transduce PIs, suggesting that the rational design parameters of WO / 2018 / 068135 can also be applied to the design of peptide shuttles for non-protein load delivery.

[0300] Furthermore, the same synthetic peptide shuttle agents, in linear form (FSD268; SEQ ID NO:43), cyclic form using amide bonds (FSD268 cycloamide; SEQ ID NO:49), or cyclic form using disulfides (FSD268 cyclic disulfide; SEQ ID NO:50), increased PI delivery, thus demonstrating that synthetic shuttle peptides do not necessarily need to be linear to be functional.

[0301] Example 3: Synthetic peptide shuttles enable intracellular delivery of small molecule inhibitors of the hedgehog signaling pathway. deliver

[0302] The ability of a rationally designed peptide shuttle, FSD250D (SEQ ID NO:36), with effective transduction activity against protein load, as described in Example 1.5, to transduce a small molecule inhibitor of the hedgehog signaling pathway in cultured cells was evaluated. 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.

[0303] Table 1: Delivery of hedgehog pathway inhibitors to GLI reporter NIH3T3 cells

[0304]

[0305] Previous experiments have shown that the presence of peptide FSD250D and mShh did not significantly lead to changes in luminescence intensity.

[0306] In summary, the NIH3T3 Gli-luciferase reporter cell line was designed to monitor the activity of hedgehog signaling pathways and contains the firefly luciferase gene under the control of Gli-responsive elements stably integrated into NIH3T3 cells. As shown in Figure 3 and Table 1, exposure of NIH3T3 Gli-luciferase reporter cells to recombinant mouse sound hedgehog factor protein as a positive control (“positive control mShh”) resulted in increased luminescence intensity, which was not observed in negative control cells not exposed to mShh (“negative control mShh”). The presence of the peptide shuttle FSD250D had no effect on the luminescence intensity of cells stimulated by mShh (data not shown), which is expected because the receptor (patched) of mShh is located on the cell surface (rather than inside the cell). However, compared to the absence of FSD250D, exposing reporter cells to small molecule inhibitors (Gant61, HPI-4, itraconazole, or ATO) of the hedgehog signaling pathway, which bind to structurally different intracellular targets, in the presence of FSD250D resulted in significantly reduced cellular luminescence intensity, indicating that the small molecules were successfully transduced by the peptide shuttle. Similar results were observed using the peptide FSD19 (data not shown).

[0307] Example 4: Synthetic peptide shuttles enable intracellular delivery of small molecule inhibitors of the hedgehog signaling pathway. deliver

[0308] The ability of a rationally designed peptide shuttle FSD250D (SEQ ID NO:36), which has effective transduction activity against protein load, as described in Example 1.6, to transduce a small molecule inhibitor of the hedgehog signaling pathway in a hairless mouse model was evaluated.

[0309] In summary, hair growth induction in mouse skin was associated with strong induction of the hedgehog pathway and increased expression of Gli1. The experiment consisted of activating the hedgehog pathway in mice through hair removal, followed by measuring the delay in hair regrowth induced by delivery of small molecule hedgehog pathway inhibitors (Gant61 or itraconazole) that bind to intracellular targets into 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 exhibited delayed hair regrowth 10 days post-treatment (*) compared to the absence of FSD250D.

[0310] Example 5: Synthetic peptide shuttle agents can achieve small molecule sodium channel inhibitor (QX-314) in HEK293 cells. and

[0311] Common intracellular delivery of GFP-NLS

[0312] The small molecule compound QX-314 (lidocaine N-ethyl bromide) is a quaternary salt derivative of lidocaine. QX-314 is membrane impermeable. When delivered to the cytoplasm, QX-314 blocks rapid Na+-dependent action potentials and voltage-dependent non-inactivating Na+ conductance (Ilfeld and Yaksh, 2009). To evaluate the simultaneous cotransduction of small molecule and protein loads by the peptide shuttle, HEK293 cells stably expressing the sodium channel Nav1.7 were exposed to a mixture of QX-314 and GFP-NLS in the presence or absence of the peptide shuttle FSD194 (SEQ ID NO:33). As a control, cells were also treated with GFP-NLS and the peptide shuttle FSD194 in the absence of QX-314. The results were evaluated using the patch-clamp technique as described in Example 1.7, and representative whole-cell Na+ currents of the treated HEK293 cells are shown in Figures 5A-5C. The current was induced by a 10 ms depolarization pulse at 10 Hz. A decrease in current amplitude was observed when cells were incubated with QX-314 and GFP-NLS for 90 seconds in the presence of the peptide shuttle FSD194 (i.e., 1 mM QX-314 + 15 μM GFP-NLS + 5 μM FSD194), consistent with the presence of intracellular QX-314 (Fig. 5C). In contrast, no similar decrease in current amplitude was observed when cells were incubated without QX-314 (i.e., 15 μM GFP-NLS + 5 μM FSD194; Fig. 5A) or with QX-314 but without FSD194 (i.e., 2.5 mM QX-314 + 15 μM GFP-NLS; Fig. 5B). Furthermore, GFP-NLS-positive cells were identified under QX-314+GFP-NLS+FSD194 and FSD194+GFP-NLS conditions, but no GFP-NLS-positive cells were identified under QX-314+GFP-NLS conditions, indicating that GFP-NLS was indeed co-transduced with the peptide shuttle along with QX-314.

[0313] Example 6: Robust PI transduction prediction of shuttles with protein loading transduction activity

[0314] High-throughput screening for the identification, selection, and / or limitation of novel peptide shuttles with protein transduction activity can quickly become extremely expensive due to the costly fabrication and purification of large quantities of recombinant proteins (especially complex proteins such as recombinant immunoglobulins) as loadings. Using GFP or GFP-NLS as protein loadings is advantageous because it allows for rapid screening via flow cytometry to assess intracellular delivery. However, the use of GFP-NLS requires validation of each peptide shuttle by parallel microscopic examination and flow cytometry measurements to ensure that candidate shuttles enable GFP-NLS loadings to avoid endosome trapping and enter the cytosol / nucleus—a resource-intensive and time-consuming process. Therefore, there is a strong need for more cost-effective “alternative” loadings that can reliably predict protein transduction activity and endosome escape.

[0315] The results in Example 2 demonstrate that synthetic peptide shuttles with validated transduction activity against GFP (and other protein loads) can also transduce small molecules such as PI. This raises the interesting possibility that PI could be used as a reliable “alternative” load to screen and identify / select / limit novel shuttles with robust transduction activity against protein loads. Commercially, PI is widely available and relatively inexpensive. Furthermore, PI only exhibits a 20-30 fold increase in fluorescence and a detectable shift in the maximum excitation / emission spectrum upon binding to genomic DNA—a characteristic that makes it particularly suitable for distinguishing endosome-captured loads from endosome-escaped loads that can enter the cytosol / nuclear compartment. Therefore, both intracellular delivery and endosome escape can be measured by flow cytometry, as any PI that remains trapped in endosomes will not reach the nucleus and will neither exhibit enhanced fluorescence nor a spectral shift.

[0316] To evaluate the suitability of PI as a novel "alternative" loading, a proprietary library of over 300 candidate peptide shuttlers was screened in parallel using flow cytometry to target the transduction activities of PI and GFP-NLS in HeLa cells, as generally described in Example 1.4. The transduction protocols were identical in all respects except for the loading concentration (i.e., 10 μg / mL for PI versus 10 μM for GFP-NLS).

[0317] Because a large number of peptides were screened, negative controls were performed in parallel for each experimental batch. These negative controls included a "no treatment" (NT) control where cells were not exposed to the shuttle peptide or load, and a "load-only" control where cells were exposed to the load in the absence of the shuttle agent. Results are shown in Figures 6 and 7, where "transduction efficiency" refers to the percentage of all viable cells positive for the load (PI or GFP-NLS). The "average delivery score" provides further indication of the total load delivered to each cell across all load-positive cells. The average PI or GFP-NLS delivery score was calculated by multiplying the measured average fluorescence intensity for viable PI+ or GFP+ cells (at least in duplicate) by the average percentage of viable PI+ or GFP+ cells, and then dividing by 100,000 (for GFP delivery) or 10,000 (for PI delivery). The average delivery scores for PI and GFP-NLS for each candidate shuttle agent were then normalized by dividing by the average delivery score of the "load-only" negative controls performed in parallel for each experimental batch. Therefore, the “normalized average delivery score” in Figures 6 and 7 represents the fold increase in average delivery score relative to the “load only” negative control.

[0318] The observed batch-to-batch variation for negative controls was relatively small for GFP-NLS, but significantly higher when PI was used as a loading. For example, the transduction efficiency of the “loading only” negative control ranged from 0.4% to 1.3% for GFP-NLS and from 0.9% to 6.3% for PI. Furthermore, the transduction efficiency of several negative control peptides tested in parallel (i.e., peptides known to have low or no GFP transduction activity) (e.g., FSD174 scrambled; data not shown) sometimes yielded lower transduction efficiencies for PI than the “loading only” negative control (but not for GFP-NLS), in some cases up to 5% lower transduction efficiencies, likely due to nonspecific interactions between PI and the peptide. This phenomenon was not observed in GFP-NLS transduction experiments. The foregoing suggests that shuttle transduction efficiency, at least for PI, may be more suitable for comparison with the transduction efficiency of negative control peptides than for comparison with “loading only” conditions.

[0319] Screening of over 300 candidate peptide shuttles for PI and GFP-NLS transduction activity revealed that shuttles exhibiting robust PI transduction efficiency generally correlated with robust GFP-NLS transduction efficiency. Notably, progressively higher PI transduction efficiencies were generally associated with progressively higher GFP-NLS transduction efficiencies. This was illustrated by grouping all screened candidate shuttles into incremental windows based on their PI transduction efficiency, and then calculating the average GFP transduction efficiency of all shuttles falling within that PI% window, as shown in the table below.

[0320]

[0321] Figure 6 shows the results for all screened candidate peptide shuttles with an average PI transduction efficiency of 10% or higher, ranked according to their average PI transduction efficiency levels. Notably, among the 306 candidate peptide shuttles with an average PI transduction efficiency of at least 10%, 96% exhibited GFP transduction efficiency of 10% or higher. The thresholds of at least 15% and 20% PI transduction efficiency correspond to values ​​at least 2.5 and 3 times higher than the highest PI transduction efficiency (approximately 6%) observed in the “load-only” negative control across all experimental batches. Of the 273 candidate peptide shuttles listed in Figure 6 with an average PI transduction efficiency of at least 15%, 97% exhibited GFP transduction efficiency of 15% or higher. Furthermore, among the 256 candidate peptide shuttles listed in Figure 6 with an average PI transduction efficiency of at least 20%, 99.6% of the candidate peptide shuttles exhibited GFP transduction efficiency of 10% or higher, and 96% of the candidate peptide shuttles exhibited GFP transduction efficiency of 20% or higher.

[0322] These results strongly suggest that robust PI delivery predicts peptide shuttles with robust protein loading transduction activity, and therefore PI can indeed be used as an “alternative” load to screen and identify / select / limit novel peptide shuttles with dual loading transduction activity (i.e., for small molecules and proteins).

[0323] Among the candidate peptide shuttles with an average PI transduction efficiency of at least 20% in Figure 6 are peptides with a length of less than 20 residues: FSD390 (17 aa), FSD367 (19 aa), and FSD366 (18 aa). Also included among the candidate peptide shuttles with an average PI transduction efficiency of at least 20% in Figure 6 are peptides containing non-physiological amino acid analogs (e.g., FSD435, which corresponds to FSD395, except that the lysine residue (K) is replaced with an L-2,4-diaminobutyric acid residue) or chemically modified peptides (e.g., FSD438, which corresponds to FSD10, except that it has an N-terminal octanoic acid modification; FSD436, which corresponds to FSD222, except that the phenylalanine residue (F) is replaced with a (2-naphthyl)-L-alanine residue; and FSD171, which corresponds to FSD168, except that it has an N-terminal acetyl group and a C-terminal cysteine ​​group). These results confirm the robustness of the peptide shuttle platform technology to the use of non-physiological amino acids or their analogues in place of physiological amino acids and / or the use of chemical modifications.

[0324] Example 7: Lower levels of PI delivery are more predictive of peptide shuttles with protein loading transduction activity. Low

[0325] The results of more than 300 candidate peptide shuttles screened in Example 6 with an average PI transduction efficiency of less than 10% but an average GFP-NLS transduction efficiency of at least 7% are shown in Figure 7, this time sorted according to their average GFP transduction efficiency level.

[0326] For candidate peptides with PI transduction efficiencies of less than 10%, the large-scale nature of the screening method employed in this paper may preclude any definitive conclusions regarding their potential lack of loading transduction efficiency. Indeed, WO / 2016 / 161516 and WO / 2018 / 068135 disclose that shuttle peptides function in a concentration-dependent manner, and multiple factors such as shuttle concentration, loading concentration, exposure time, and cell type can influence the performance of the shuttle in transduction assays. The large-scale screening of candidate peptide shuttles described herein applied a “universal” single shuttle concentration, single loading concentration, and single exposure time / scheme to each individual and every peptide tested. Therefore, it is difficult to draw any definitive conclusions regarding non-protein loading transduction activity based solely on the low PI transduction efficiencies observed in this large-scale screening.

[0327] References

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Claims

1. A synthetic peptide shuttle with transduction activity against both protein and non-protein loads in target eukaryotic cells, wherein the shuttle comprises an amino acid sequence of SEQ ID NO: 35, 37, 38, 39, 40, 45, 46, 47, 48, 171, 174, 175, 177, 179, 180, 181, 182, 185, 186, 187, 189, 192, 193, 194, 211, 212, 214, 217, 218, 230, 231, 253, 254, 255, 256, 257, 259, 260, 262, 264, 265, 266, 267, 268, 280, 281, 283, 289, 310, or 324. In a eukaryotic cell line model suitable for evaluating load transduction in the target eukaryotic cells, the shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators by at least 3-fold compared to a corresponding negative control lacking the shuttle agent, and / or achieves at least 10% transduction efficiency for propidium iodide or other membrane-impermeable fluorescent DNA intercalators; and / or In a eukaryotic cell line model suitable for evaluating load transduction in the target eukaryotic cells, the shuttle agent increases the transduction efficiency of GFP-NLS by at least 3-fold compared to the corresponding negative control lacking the shuttle agent, and / or enables at least 7% transduction efficiency of GFP-NLS.

2. The synthetic peptide shuttle of claim 1, wherein the shuttle further comprises a histidine-rich domain, wherein the histidine-rich domain is: (a) positioned toward the N-terminus and / or C-terminus of the shuttle; (b) an extension comprising at least 3 amino acids containing at least 50% histidine residues; and / or comprising at least 2 consecutive histidine residues; or (c) both (a) and (b).

3. The synthetic peptide shuttle according to claim 1 or 2, 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, wherein the chemical modifications do not impair the transduction activity of the synthetic peptide shuttle.

4. The synthetic peptide shuttle according to claim 3, wherein the chemical modification is at the N and / or C terminus of the shuttle; and / or wherein the chemical modification is the addition of an acetyl group, a cysteine ​​group, or a fatty acid.

5. Use of the synthetic peptide shuttle according to any one of claims 1 to 4 in an in vitro or ex vivo method for increasing the transduction efficiency of protein and / or non-protein loads into target eukaryotic cells, wherein the synthetic peptide shuttle is used at a concentration sufficient to increase the transduction efficiency of the load into the target eukaryotic cells and cytosol and / or nuclear delivery compared to the absence of the synthetic peptide shuttle.

6. Use of the synthetic peptide shuttle according to any one of claims 1 to 4 in the preparation of a medicament for treating a disease by intracellular delivery of a therapeutically active protein and / or non-protein load, wherein the synthetic peptide shuttle transduces the therapeutically active protein and / or non-protein load to the cytosol and / or nucleus of a target eukaryotic cell, wherein the synthetic peptide shuttle is used at a concentration sufficient to increase the transduction efficiency of the load into the target eukaryotic cell compared to the absence of the synthetic peptide shuttle.

7. An in vitro or ex vivo method for transduction of protein and / or non-protein loads, the method comprising contacting target eukaryotic cells with the load and a concentration of a synthetic peptide shuttle according to any one of claims 1 to 4, wherein the concentration of the synthetic peptide shuttle is sufficient to increase the transduction efficiency of the load into the target eukaryotic cells compared to the absence of the synthetic peptide shuttle.

8. Use of the composition in the preparation of a medicament for treating a disease treatable by intracellular delivery of a therapeutically active protein and / or a non-protein load, wherein the composition comprises a synthetic peptide shuttle according to any one of claims 1 to 4, formulated together with a therapeutically active protein and / or a non-protein load to be transduced into target eukaryotic cells by a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency of the load into the target eukaryotic cells and cytosol delivery after administration.

9. A method for generating a candidate synthetic peptide shuttle agent intended to have transduction activity for a target load in target eukaryotic cells, the method comprising synthesizing peptides comprising SEQ ID NO: 35, 37, 38, 39, 40, 45, 46, 47, 48, 171, 174, 175, 177, 179, 180, 181, 182, 185, 186, 187, 189, 192, 193, 194, 211, 212, 214, 217, 218, 230, 231, 253, 254, 255, 256, 257, 259, 260, 262, 264, 265, 266, 267, The amino acid sequence comprises 268, 280, 281, 283, 289, 310, or 324, wherein, in a eukaryotic cell line model suitable for evaluating load transduction in the target eukaryotic cells, the shuttle agent increases the transduction efficiency of propidium iodide or other membrane-impermeable fluorescent DNA intercalators by at least 3-fold compared to a corresponding negative control lacking the shuttle agent, and / or achieves at least 10% transduction efficiency for propidium iodide or other membrane-impermeable fluorescent DNA intercalators; and / or In a eukaryotic cell line model suitable for evaluating load transduction in the target eukaryotic cells, the shuttle agent increases the transduction efficiency of GFP-NLS by at least 3-fold compared to the corresponding negative control lacking the shuttle agent, and / or enables at least 7% transduction efficiency of GFP-NLS.

10. An in vitro or ex vivo method for non-protein load transduction, the method comprising contacting target eukaryotic cells with a non-protein load and a concentration of a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle is sufficient to increase the transduction efficiency of the non-protein load compared to the absence of the synthetic peptide shuttle, wherein the non-protein load: (a) is an organic compound; (b) Having a molecular weight of less than 10,000 Da; (c) is a small molecule; (d) is not a biopolymer; (e) Not covalently linked to the synthetic peptide shuttle during transduction; or Any combination of (f)(a) to (e), The synthetic peptide shuttle is a synthetic peptide shuttle as defined in any one of claims 1 to 4.

11. The method of claim 10, wherein the non-protein load is a small molecule drug that binds to an intracellular biological or therapeutic target.

12. The method of claim 10, wherein the non-protein load is not a polynucleotide or polysaccharide.

13. Use of synthetic peptide shuttles in in vitro or ex vivo methods for increasing the efficiency of transduction of non-protein loads into target eukaryotic cells. The synthetic peptide shuttle is used at a concentration sufficient to increase the transduction efficiency of the load into the target eukaryotic cells and its delivery to the cytosol and / or nucleus, compared to the absence of the synthetic peptide shuttle. The non-protein load mentioned above: (a) is an organic compound; (b) Having a molecular weight of less than 10,000 Da; (c) is a small molecule; (d) is not a biopolymer; (e) Not covalently linked to the synthetic peptide shuttle during transduction; or Any combination of (f)(a) to (e), The synthetic peptide shuttle is the synthetic peptide shuttle as described in any one of claims 1 to 4.

14. The use according to claim 13, wherein the non-protein load is a small molecule drug that binds to an intracellular biological or therapeutic target.

15. The use according to claim 13, wherein the non-protein load is not a polynucleotide or polysaccharide.

16. Use of a synthetic peptide shuttle in the preparation of a medicament for treating a disease treatable by intracellular delivery of a therapeutically active protein and / or non-protein load, wherein the synthetic peptide shuttle transduces the therapeutically active non-protein load into the cytosol and / or nucleus of a target eukaryotic cell, wherein the synthetic peptide shuttle is used at a concentration sufficient to increase the transduction efficiency of the load into the target eukaryotic cell compared to the absence of the synthetic peptide shuttle: The non-protein load mentioned above: (a) is an organic compound; (b) Having a molecular weight of less than 10,000 Da; (c) is a small molecule; (d) is not a biopolymer; (e) Not covalently linked to the synthetic peptide shuttle during transduction; or Any combination of (f)(a) to (e), The synthetic peptide shuttle is the synthetic peptide shuttle as described in any one of claims 1 to 4.

17. The use according to claim 16, wherein the non-protein load is a small molecule drug that binds to an intracellular biological or therapeutic target.

18. The use according to claim 16, wherein the non-protein load is not a polynucleotide or polysaccharide.

19. Use of the composition in the preparation of a medicament for treating a disease treatable by intracellular delivery of a therapeutically active non-protein load, wherein the composition comprises a synthetic peptide shuttle formulated together with a therapeutically active non-protein load to be transduced into target eukaryotic cells by a synthetic peptide shuttle, wherein the concentration of the synthetic peptide shuttle in the composition is sufficient, compared to the absence of the synthetic peptide shuttle, to increase the transduction efficiency and cytosol delivery of the load to the target eukaryotic cells after administration, and wherein the non-protein load: (a) is an organic compound; (b) Having a molecular weight of less than 10,000 Da; (c) is a small molecule; (d) is not a biopolymer; (e) Not covalently linked to the synthetic peptide shuttle during transduction; or Any combination of (f)(a) to (e), The synthetic peptide shuttle is the synthetic peptide shuttle according to any one of claims 1 to 4.

20. The use according to claim 19, wherein the non-protein load is a small molecule drug that binds to an intracellular biological or therapeutic target.

21. The use according to claim 19, wherein the non-protein load is not a polynucleotide or polysaccharide.

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