Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same

Abstract

To provide methods for delivering polypeptide cargos to the cytosol and / or nucleus of target eukaryotic cells from extracellular space.SOLUTION: The method comprises contacting a cell with a polypeptide cargo in the presence of a peptide shuttle agent at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo. Also described here are parameters that may be used in the rational design of such synthetic peptide shuttle agents, peptide shuttle agents that satisfy one or more of these design parameters, as well as methods and compositions relating to the use of the synthetic peptide shuttle agents for delivery of a variety of polypeptide cargos (such as transcription factors, antibodies, CRISPR-associated nucleases and functional genome editing complexes) from an extracellular space to the cytosol and / or nucleus of the target eukaryotic cells. Applications and targets for genome-editing NK cells for improved immunotherapy are also described.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This description relates to synthetic peptide shuttle agents useful for delivering various polypeptide cargoes from the extracellular space to the cytosol and / or nucleus of target eukaryotic cells. More specifically, this description relates to parameters useful for the rational design of such synthetic peptide shuttle agents. [Background technology]

[0002] Cellular delivery techniques for transporting large molecules into the interior of eukaryotic cells have wide applications, particularly in the biopharmaceutical industry. While some soluble chemicals (e.g., small molecule drugs) can passively diffuse through eukaryotic cell membranes, large cargoes (e.g., biologics, polynucleotides, and polypeptides) require the assistance of shuttle agents to reach their intracellular targets.

[0003] Areas that are likely to benefit greatly from advances in cell delivery technology include the fields of genome editing and cell therapy, which have made significant progress over the past two decades. Deciphering the various growth factors and molecular cues that govern cell proliferation, differentiation, and reprogramming will open up numerous therapeutic possibilities to address unmet medical needs. For example, direct derivation of pluripotent stem cells from mature cells, direct cell transformation (transdifferentiation), and genome editing (zinc finger nucleases, TALEN™, and CRISPR-linked endonuclease technologies) are examples of methods developed to maximize the therapeutic value of cells for clinical applications. Currently, producing cells with high therapeutic activity typically requires ex vivo manipulation, primarily achieved by viral transduction, raising significant safety and economic concerns for human applications. The ability to directly deliver active proteins, such as transcription factors or engineered nucleases, into these cells would advantageously circumvent the safety concerns and regulatory barriers associated with more risky gene transfer methods. In particular, methods for delivering active genome editing complexes directly into immune cells would be highly desirable for improving immunotherapy.

[0004] Protein transduction techniques involving direct fusion of recombinant protein cargo with cell membrane-permeable peptides (e.g., the HIV transactivator protein TAT) require large amounts of recombinant protein and often fail to deliver the cargo to the appropriate intracellular location, leading to massive endosomal trapping and eventual degradation. Several endosomal membrane-disrupting peptides have been developed to facilitate the escape of endosome-trapped cargo into the cytosol. However, many of these endosomolytic peptides are used to mitigate endosomal trapping of cargo already delivered into cells and do not themselves support the initial shuttling step of transporting the cargo across the cell membrane into the cell (Salomone et al., 2012; Salomone et al., 2013; Erazo-Oliveras et al., 2014; Fasoli et al., 2014).

[0005] In particular, Salomone et al. (2012) 11 The chimeric peptide CM resulting from the fusion of a cell penetration motif to the CM18 hybrid (residues 1-7 of cecropin-A and 2-12 of melittin) 18 -TAT 11 They describe a peptide that is rapidly internalized by cells (due to its TAT ​​motif) and subsequently participates in the destabilization of the membranes of intracellular vesicles (due to the membrane disruptive ability of the CM18 peptide). The peptide CM18 fused to fluorescently labeled Atto-633 (molecular weight: 774 Da; 21% of the molecular weight of the peptide) 18 -TAT 11 is endosomal trapped TAT 11 -EGFP escape into the cytosol (see Figure 3 in Salomone et al., 2012), but CM 18 -TAT 11It has been shown that the peptide (alone or conjugated with Atto-633) does not function as a shuttle agent capable of increasing the delivery of polypeptide cargo from the extracellular space to the cell interior—i.e., across the cell membrane. Indeed, Salomone et al., 2012, demonstrated that co-treatment (TAT 11 -EGFP and CM 18 -TAT 11 -Atto-633 simultaneous treatment) and time-shift treatment (i.e., TAT 11 After incubation of cells with -EGFP alone, fluorescence imaging was performed, followed by CM. 18 -TAT 11 The authors compared the results of the CM assay with the Atto-633 peptide alone (again after incubation of the same cells with the Atto-633 peptide alone, followed by fluorescent imaging), and reported that "both resulted in the same delivery results" (see page 295 of Salomone et al., 2012, the last sentence of the first paragraph under the heading "2.9 Cargo Delivery Assay"). In other words, Salomone et al., 2012 reported that CM 18 -TAT 11 It was noted that the peptide (alone or conjugated to Atto-633) did not affect the delivery of polypeptide cargo from the extracellular space into cells (i.e., protein transduction). Thus, there remains a need for improved shuttle agents that can increase the transduction efficiency of polypeptide cargo and deliver cargo into the cytosol and / or nucleus of target eukaryotic cells.

[0006] This description refers to a number of documents, the contents of which are incorporated herein by reference in their entirety. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Salomone et al., 2012 [Non-patent document 2] Salomone et al., 2013 [Non-patent document 3] Erazo-Oliveras et al., 2014 [Non-patent document 4] Fasoli et al., 2014 Summary of the Invention

[0008] Several different peptides were screened with the aim of identifying polypeptide-based shuttle agents capable of intracellularly delivering independent polypeptide cargos to the cytosol / nucleus of eukaryotic cells. On the one hand, these large-scale screening efforts led to the unexpected discovery that certain domain-based peptide shuttle agents ultimately increase the number and / or proportion of cells that internalize the polypeptide cargo, and also enhance the transduction efficiency of the polypeptide cargo by enabling the internalized cargo to reach the cytosol / nuclear compartment (thus avoiding or reducing endosomal trapping of the cargo). These domain-based shuttle agents comprise an endosomal leakage domain (ELD) operably linked to a cell-permeable domain (CPD), and optionally one or more histidine-rich domains. On the other hand, the above screening efforts also revealed several peptides with no or poor polypeptide cargo transduction activity, excessive toxicity, and / or other undesirable properties (e.g., poor solubility and / or stability). Herein, these empirical data (both favorable and unfavorable) were used to identify successful, less successful, and unsuccessful peptide physiochemical properties to arrive at a set of design parameters that allow for the rational design and / or identification of peptides with protein transduction activity.

[0009] Thus, the present description relates to methods for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell by contacting the cell with a polypeptide cargo in the presence of a peptide shuttle agent described herein at a concentration sufficient to increase transduction efficiency of the polypeptide cargo compared to that in the absence of the shuttle agent. More specifically, the description relates to parameters that can be used for the rational design of such synthetic peptide shuttle agents, peptide shuttle agents that satisfy one or more of these design parameters, and methods and compositions related to the use of the synthetic peptide shuttle agents for delivery of various polypeptide cargoes from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell. The description also relates to machine learning or computer-assisted techniques that can be used to generate peptide variants that satisfy one or more design parameters described herein.

[0010] The present description also relates to the co-transduction of a polypeptide cargo of interest and a marker protein as a means of identifying and / or enriching transduced cells. Surprisingly, it was discovered that a significantly higher proportion of target eukaryotic cells successfully transduced with the polypeptide cargo of interest also successfully transduced the marker protein. Conversely, a significantly higher proportion of cells not transduced with the polypeptide cargo of interest also failed to transduce the marker protein. Isolation of marker protein-positive cells (e.g., by FACS) significantly increased the proportion of cells successfully transduced with the polypeptide cargo of interest, and the correlation was found to be concentration-dependent, in that cell populations exhibiting the highest marker protein fluorescence also tended to exhibit the highest transduction rates of the polypeptide cargo of interest. It was also discovered that, after a first round of transduction of a polypeptide cargo of interest, unsuccessfully transduced cells can be isolated and re-transduced with the polypeptide cargo of interest in a subsequent transduction round. Thus, in some aspects, the present description relates to methods comprising co-transduction of a polypeptide cargo of interest with a marker protein, which may be used to isolate or enrich for cells transduced with the polypeptide cargo of interest. In some embodiments, the present description also relates to methods comprising repeated sequential transduction experiments, e.g., performed on cells that failed to transduce the marker protein after a first or previous transduction reaction. Such methods provide an attractive approach for improving transduction efficiency in useful cell populations (e.g., patient-derived cells for cell therapy) and / or in inherently more difficult-to-transduce cell populations.

[0011] General definition Headings and other identifiers, e.g., (a), (b), (i), (ii), etc., are provided solely for ease of reading of the specification and claims. The use of headings and other identifiers in the specification and 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 presented.

[0012] The use of the words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0013] The term "about" is used to indicate that a value includes the standard deviation of error of the device or method being used to determine the value. Generally, the technical term "about" is intended to specify a possible variation of up to 10%. Thus, variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of a value are encompassed within the term "about." Unless otherwise indicated, the use of the term "about" before a range applies to both ends of the range.

[0014] As used in this specification and the claims, the words "comprising" (and any form of including, 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 further, unrecited elements or method steps.

[0015] As used herein, "protein" or "polypeptide" or "peptide" refers to any peptide-linked chain of amino acids, which may or may not contain any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfation, sumoylation, prenylation, ubiquitination, etc.). For clarity, protein / polypeptide / peptide modifications are contemplated as long as the modification does not destroy the protein transduction activity of the shuttle agents described herein.

[0016] As used herein, "domain" or "protein domain" generally refers to a portion of a protein that has a specific functional group or function. Some domains preserve their function when separated from the rest of the protein and, therefore, can be used in a modular manner. The modular nature of many protein domains can provide flexibility in their placement within the shuttle agents described herein. However, some domains may function better when engineered into specific locations within the shuttle agent (e.g., at or between the N-terminal or C-terminal regions). The location of a domain within its endogenous protein may also guide where the domain should be engineered within the shuttle agent and what type / length of linker should be used. Standard recombinant DNA techniques can be used by those skilled in the art to engineer the placement and / or number of domains within the shuttle agents described herein in light of the present disclosure. Furthermore, assays disclosed herein, as well as other assays known in the art, can be used to evaluate the functionality of each of the domains within the shuttle agent (e.g., their ability to facilitate cell penetration across the cell membrane, endosomal escape, and / or access to the cytosol). Standard methods can also be used to assess whether a domain of a shuttle agent affects the activity of a cargo to be delivered intracellularly. In this regard, the phrase "operably linked" as used herein refers to the ability of a domain in a shuttle agent described herein to perform its intended function (e.g., cell penetration, endosomal escape, and / or intracellular targeting). For clarity, the phrase "operably linked" is intended to define a functional connection between two or more domains, without being limited to a particular order or distance between them.

[0017] As used herein, the term "synthetic," as used in expressions such as "synthetic peptide" or "synthetic polypeptide," refers 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 preparations can be assessed, for example, by high-performance liquid chromatography analysis and mass spectrometry. Chemical synthesis approaches can be advantageous over cellular expression systems (e.g., yeast or bacterial protein expression systems) because they can eliminate the need for extensive recombinant protein purification steps (e.g., required for clinical use). In contrast, longer synthetic polypeptides can be more complex and / or expensive to produce by chemical synthesis approaches, and such polypeptides can be more advantageously produced using cellular expression systems. In some embodiments, the peptides or shuttle agents described herein can be chemically synthesized (e.g., by solid-phase or solution-phase peptide synthesis) as opposed to being expressed from a recombinant host cell. In some embodiments, the peptides or shuttle agents described herein can lack an N-terminal methionine residue. One skilled in the art can adapt the described synthetic peptides or shuttle agents to meet particular stability or other needs by using one or more modified amino acids (e.g., non-naturally occurring amino acids) or by chemically modifying the described synthetic peptides or shuttle agents.

[0018] As used herein in connection with the presently described shuttle agents, the phrase "polypeptide-based" is intended to distinguish the presently described shuttle agents from non-polypeptide or non-protein-based shuttle agents, such as lipid- or cationic polymer-based transduction agents, which are often associated with increased cytotoxicity and may not be suitable for use in human therapy.

[0019] As used herein, the term "independent" generally refers to molecules or agents that are not covalently linked to one another. For example, the phrase "independent polypeptide cargo" refers to a polypeptide cargo to be delivered into a cell that is not covalently linked (e.g., not fused) to a shuttle agent described herein. In some embodiments, having a shuttle agent that is independent of (not fused to) the polypeptide cargo can be advantageous, for example, by not having to redesign a new fusion protein for a different polypeptide cargo and / or by providing increased shuttle agent versatility, such that the ratio of shuttle agent to cargo can be easily varied (as opposed to being limited to a 1:1 ratio as is the case with fusion proteins).

[0020] As used herein, the phrase "is or derived from" or "derived from" includes functional variants of a given protein domain (e.g., a CPD or ELD), such as conservative amino acid substitutions, deletions, modifications, as well as variants or functional derivatives that do not abrogate the activity of the protein domain.

[0021] Other objects, advantages and features of the present description will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of example only, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0022] [Figure 1] Figure 1A shows typical results of a calcein endosomal escape assay in which HEK293A cells were loaded with the fluorescent dye calcein ("100 μM calcein") and then treated (or not) with a shuttle agent that promotes endosomal escape of calcein ("100 μM calcein + CM18-TAT 5 μM"). Figure 1A shows the results of a fluorescence microscopy experiment, and Figure 1B shows the results of a flow cytometry experiment. [Figure 2]FIG. 1 shows the results of a calcein endosomal escape flow cytometry assay in which HeLa cells were loaded with calcein ("Calcein 100 μM") and then treated with increasing concentrations of the shuttle agent CM18-TAT-Cys (labeled "CM18-TAT"). [Figure 3] FIG. 1 shows the results of a calcein endosomal escape flow cytometry assay in HeLa cells loaded with calcein ("Calcein 100 μM") and then treated with 5 μM or 8 μM of the shuttle agents CM18-TAT-Cys or CM18-Penetratin-Cys (labeled "CM18-TAT" and "CM18-Penetratin," respectively). [Figure 4] FIG. 1 shows the results of a calcein endosomal escape flow cytometry assay in HeLa cells loaded with calcein ("Calcein 100 μM") and then treated with 5 μM or 8 μM of the shuttle agents CM18-TAT-Cys or CM18-Penetratin-Cys (labeled "CM18-TAT" and "CM18-Penetratin," respectively). [Figure 5]

[0023] Figure 1 shows the results of a GFP transduction experiment visualized by fluorescence microscopy in which a GFP cargo protein was co-incubated with 0, 3, or 5 μM CM18-TAT-Cys (labeled "CM18-TAT") and then exposed to HeLa cells. Cells were observed by bright field (upper panel) and fluorescence microscopy (lower panel). [Figure 6] Figure 6 shows the results of a GFP transduction efficiency experiment in which GFP cargo protein (10 μM) was co-incubated with various concentrations of CM18-TAT-Cys (labeled "CM18-TAT") and then exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown in Figure 6A, and the corresponding cytotoxicity data is shown in Figure 6B. [Figure 7]Figure 7 shows the results of a GFP transduction efficiency experiment in which various concentrations of GFP cargo protein (10, 5, or 1 μM) were co-incubated with either 5 μM CM18-TAT-Cys (Figure 7A, labeled "CM18TAT") or 2.5 μM dCM18-TAT-Cys (Figure 7B, labeled "dCM18TAT") and then exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown. [Figure 8]

[0033] Figure 1 shows the results of a GFP transduction efficiency experiment in which GFP cargo protein (10 μM) was co-incubated with various concentrations and combinations of CM18-TAT-Cys (labeled "CM18TAT"), CM18-Penetratin-Cys (labeled "CM18Penetratin"), and their respective dimers (dCM18-TAT-Cys (labeled "dCM18TAT"), dCM18-Penetratin-Cys (labeled "dCM18Penetratin")) and then exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown. [Figure 9]

[0033] Figure 1 shows the results of a GFP transduction efficiency experiment in which GFP cargo protein (10 μM) was co-incubated with various concentrations and combinations of CM18-TAT-Cys (labeled "CM18TAT"), CM18-Penetratin-Cys (labeled "CM18Penetratin"), and their respective dimers (dCM18-TAT-Cys (labeled "dCM18TAT"), dCM18-Penetratin-Cys (labeled "dCM18Penetratin")) and then exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown. [Figure 10]This figure shows a typical result of a TAT-GFP transduction experiment in which TAT-GFP cargo protein (5 μM) was co-incubated with 3 μM CM18-TAT-Cys (labeled "CM18-TAT") and then exposed to HeLa cells. Cells and GFP fluorescence were visualized by bright-field and fluorescence microscopy at 10× and 40× magnification. Arrows indicate endosomal delivery of TAT-GFP in the absence of CM18-TAT-Cys and its nuclear delivery in the presence of CM18-TAT-Cys. [Figure 11] Figure 11 shows the results of a TAT-GFP transduction efficiency experiment in which TAT-GFP cargo protein (5 μM) was co-incubated with various concentrations of CM18-TAT-Cys (labeled "CM18TAT") and then exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown in Figure 11A, and the corresponding cytotoxicity data is shown in Figure 11B. [Figure 12] Figure 1 shows a typical result of a GFP-NLS transduction experiment in which GFP-NLS cargo protein (5 μM) was co-incubated with 5 μM CM18-TAT-Cys (labeled "CM18-TAT") and then exposed to HeLa cells for 5 minutes. Cells and GFP fluorescence were visualized by bright-field and fluorescence microscopy at 10x, 20x, and 40x magnification. Arrows indicate the area of ​​nuclear delivery of GFP-NLS. [Figure 13] Figure 13 shows the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μM) was co-incubated with various concentrations of CM18-TAT-Cys (labeled "CM18TAT") and then exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown in Figure 13A, and the corresponding cytotoxicity data is shown in Figure 13B. [Figure 14]

[0033] Figure 1 shows the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μM) was co-incubated with various concentrations and combinations of CM18-TAT (labeled "CM18TAT"), CM18-Penetratin (labeled "CM18Penetratin"), and their respective dimers (dCM18-TAT-Cys, dCM18-Penetratin-Cys, labeled "dCM18TAT," and "dCM18Penetratin," respectively), followed by exposure to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown. [Figure 15]

[0033] Figure 1 shows the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μM) was co-incubated with various concentrations and combinations of CM18-TAT (labeled "CM18TAT"), CM18-Penetratin (labeled "CM18Penetratin"), and their respective dimers (dCM18-TAT-Cys, dCM18-Penetratin-Cys, labeled "dCM18TAT," and "dCM18Penetratin," respectively), followed by exposure to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown. [Figure 16] Figure 1 shows the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μM) was co-incubated with CM18-TAT-Cys (3.5 μM, labeled "CM18TAT") alone or with dCM18-penetratin-Cys (1 μM, labeled "dCM18pen") in plain DMEM medium ("DMEM") or DMEM containing 10% FBS ("FBS") for 5 minutes or 1 hour, followed by flow cytometry analysis. The percentage of fluorescent (GFP-positive) cells is shown. Cells not treated with shuttle agent or GFP-NLS ("ctrl") and cells treated with GFP-NLS but without shuttle agent ("GFP-NLS 5 μM") were used as controls. [Figure 17]Figure 17 shows the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μM) was co-incubated with or without 1 μM CM18-TAT-Cys (labeled "CM18TAT") and then exposed to THP-1 cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown in Figure 17A, and the corresponding cytotoxicity data is shown in Figure 17B. [Figure 18] Figure 1 shows the results of a transduction efficiency experiment in which the cargo protein, a FITC-labeled anti-tubulin antibody (0.5 μM), was co-incubated with 5 μM CM18-TAT-Cys (labeled "CM18-TAT") and then exposed to HeLa cells. Functional antibody delivery was visualized by brightfield (20x) and fluorescence microscopy (20x and 40x), which visualized fluorescent tubulin filaments in the cytoplasm. [Figure 19] 19A and 19B show the results of a FITC-labeled anti-tubulin antibody transduction efficiency experiment in which antibody cargo protein (0.5 μM) was co-incubated with 3.5 μM CM18-TAT-Cys (labeled "CM18TAT"), CM18-Penetratin-Cys (labeled "CM18pen"), or dCM18-Penetratin-Cys (labeled "dCM18pen"), or a combination of 3.5 μM CM18-TAT-Cys and 0.5 μM dCM18-Penetratin-Cys, and then exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (FITC-positive) cells is shown in FIG. 19A, and the corresponding cytotoxicity data is shown in FIG. 19B. [Figure 20]

[0023] Figure 1 shows the results of a DNA transfection efficiency experiment in which plasmid DNA (pEGFP) was labeled with Cy5™ dye and co-incubated with 0, 0.05, 0.5, or 5 μM CM18-TAT-Cys (labeled "CM18-TAT"), followed by exposure to HEK293A cells. Flow cytometry analysis allowed quantification of Cy5™ emission (corresponding to intracellular DNA delivery, y-axis) and GFP emission (corresponding to successful nuclear delivery of DNA, percentage shown above each bar). [Figure 21] Figure 21A shows the results of a GFP-NLS transduction efficiency experiment in which GFP-NLS cargo protein (5 μM) was co-incubated with 1, 3, or 5 μM CM18-TAT-Cys (labeled "CM18TAT"), His-CM18-TAT (labeled "His-CM18TAT"), and exposed to HeLa cells. Cells were assessed by flow cytometry, and the percentage of fluorescent (GFP-positive) cells is shown in Figure 21A, and the corresponding cytotoxicity data is shown in Figure 21B. [Figure 22] Figure 22 shows the results of a transduction efficiency experiment in which a GFP-NLS cargo protein was delivered intracellularly using the shuttle His-CM18-PTD4 in HeLa cells. GFP-NLS transduction efficiency was assessed by flow cytometry, and the percentage of GFP-fluorescent cells ("Pos Cells (%)") as well as the corresponding cell viability data ("Viability (%)") are shown. Figure 22A shows a comparison of GFP-NLS transduction efficiency using different transduction protocols (Protocol A vs. B). Figure 22B shows the effect of different concentrations of the shuttle His-CM18-PTD4 using Protocol B. [Figure 23]These are microscopic images showing the results of transduction experiments in which GFP-NLS-labeled (Figures 24A, 24B, 25, and 26) or FITC-labeled (Figures 24C and 24D) anti-tubulin antibody cargo proteins were delivered intracellularly using the shuttle His-CM18-PTD4 in HeLa cells. Bright-field and fluorescent images of live cells are shown in Figures 23, 24, and 26. In Figure 25, cells were fixed, permeabilized, and subjected to immunolabeling with anti-GFP and fluorescent secondary antibodies. The white triangle window shows an example of a co-labeled area between the nuclear (DAPI) and GFP-NLS signals. Figure 26 shows images captured by confocal microscopy. [Figure 24-1] These are microscopic images showing the results of transduction experiments in which GFP-NLS-labeled (Figures 24A, 24B, 25, and 26) or FITC-labeled (Figures 24C and 24D) anti-tubulin antibody cargo proteins were delivered intracellularly using the shuttle His-CM18-PTD4 in HeLa cells. Bright-field and fluorescent images of live cells are shown in Figures 23, 24, and 26. In Figure 25, cells were fixed, permeabilized, and subjected to immunolabeling with anti-GFP and fluorescent secondary antibodies. The white triangle window shows an example of a co-labeled area between the nuclear (DAPI) and GFP-NLS signals. Figure 26 shows images captured by confocal microscopy. [Figure 24-2] Same as above. [Figure 25] These are microscopic images showing the results of transduction experiments in which GFP-NLS-labeled (Figures 24A, 24B, 25, and 26) or FITC-labeled (Figures 24C and 24D) anti-tubulin antibody cargo proteins were delivered intracellularly using the shuttle His-CM18-PTD4 in HeLa cells. Bright-field and fluorescent images of live cells are shown in Figures 23, 24, and 26. In Figure 25, cells were fixed, permeabilized, and subjected to immunolabeling with anti-GFP and fluorescent secondary antibodies. The white triangle window shows an example of a co-labeled area between the nuclear (DAPI) and GFP-NLS signals. Figure 26 shows images captured by confocal microscopy. [Figure 26]These are microscopic images showing the results of transduction experiments in which GFP-NLS-labeled (Figures 24A, 24B, 25, and 26) or FITC-labeled (Figures 24C and 24D) anti-tubulin antibody cargo proteins were delivered intracellularly using the shuttle His-CM18-PTD4 in HeLa cells. Bright-field and fluorescent images of live cells are shown in Figures 23, 24, and 26. In Figure 25, cells were fixed, permeabilized, and subjected to immunolabeling with anti-GFP and fluorescent secondary antibodies. The white triangle window shows an example of a co-labeled area between the nuclear (DAPI) and GFP-NLS signals. Figure 26 shows images captured by confocal microscopy. [Figure 27] Figure 27 shows microscopic images of a kinetic (time course) transduction experiment in HeLa cells, where the fluorescence of the GFP-NLS cargo protein was tracked 45 (Figure 27A), 75 (Figure 27B), 100 (Figure 27C), and 120 (Figure 27D) seconds after intracellular delivery using the shuttle His-CM18-PTD4. The diffuse cytoplasmic fluorescence pattern observed after 45 seconds (Figure 27A) gradually becomes a more condensed nuclear pattern at 120 seconds (Figure 27D). [Figure 28] Figure 28 shows microscopic images of a co-delivery transduction experiment in which two cargo proteins (GFP-NLS and mCherry™-NLS) were simultaneously delivered intracellularly by the shuttle His-CM18-PTD4 in HeLa cells. Cells and fluorescent signals were visualized by bright-field (Figure 28A) and fluorescent (Figures 28B-28D) microscopy. The open triangle windows show examples of areas of co-labeling between the nucleus (DAPI) and GFP-NLS or mCherry™. [Figure 29-1]Figures 29A, 29B, 29D-29G, and 29I show the results of GFP-NLS transduction efficiency experiments in HeLa cells using various shuttle agents or single domain / control peptides. GFP-NLS transduction efficiency was assessed by flow cytometry, and the percentage of GFP-fluorescent cells ("Pos Cells (%)") and corresponding cell viability data ("Viability (%)") are shown in Figures 29A, 29B, 29D-29G, and 29I. In Figures 29A and 29D-29F, cells were exposed to the cargo / shuttle agent for 10 seconds. In panel I, cells were exposed to the cargo / shuttle agent for 1 minute. In Figures 29B, 29C, 29G, and 29H, cells were exposed to the cargo / shuttle agent for 1, 2, or 5 minutes. "Relative fluorescence intensity (FL1-A)" or "signal intensity" corresponds to the average of all fluorescence intensities from each cell with a GFP fluorescent signal after delivery of the GFP-NLS fluorescent protein using the shuttle agent. FIG. 29D shows the results of a control experiment in which only a single domain peptide (ELD or CDP) or the peptide His-PTD4 (His-CPD) was used for GFP-NLS transduction instead of a multi-domain shuttle agent. [Figure 29-2] Same as above. [Figure 29-3] Same as above. [Figure 29-4] Same as above. [Figure 29-5] Same as above. [Figure 30] Figure 30 shows microscopic images of HeLa cells transduced with GFP-NLS using the shuttle agents Figure 30A: TAT-KALA, Figure 30B: His-CM18-PTD4, Figure 30C: His-C(LLKK)3C-PTD4, Figure 30D: PTD4-KALA, Figure 30E: EB1-PTD4, and Figure 30F: His-CM18-PTD4-His. The inset in the bottom panel shows the results of the corresponding flow cytometry analysis, indicating the percentage of cells showing GFP fluorescence. [Figure 31]Figure 1 shows the results of a transduction efficiency experiment in which a GFP-NLS cargo protein was delivered intracellularly using the shuttle His-CM18-PTD4 in THP-1 cells using different protocols (Protocol A vs. C). GFP-NLS transduction efficiency was assessed by flow cytometry, and the percentage of GFP-fluorescent cells ("Pos Cells (%)") as well as the corresponding cell viability data ("Viability (%)") are shown. "Ctrl" corresponds to THP-1 cells exposed to the GFP-NLS cargo protein in the absence of a shuttle agent. [Figure 32] Figures 32A-32C show microscopic images of THP-1 cells transduced with the GFP-NLS cargo protein using the shuttle His-CM18-PTD4. Images captured at 4x, 10x, and 40x magnification are shown in Figures 32A-32C, respectively. The white triangular window in Figure 32C shows an example of an area of ​​co-labeling between cells (bright field) and GFP-NLS fluorescence. Figure 32D shows the results of the corresponding flow cytometry analysis, showing the percentage of cells exhibiting GFP fluorescence. [Figure 33] 33A and 33B show microscopy images of THP-1 cells transduced with the GFP-NLS cargo protein using the shuttle His-CM18-PTD4. The open triangle window shows an example of an area of ​​co-labeling between the cells (bright field; Figures 33A and 33B) and the GFP-NLS fluorescence (Figures 33C and 33D). [Figure 34] Figure 34A shows the results of GFP-NLS transduction efficiency experiments in THP-1 cells using the shuttles TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4. The cargo protein / shuttle agent was exposed to THP-1 cells for 15, 30, 60, or 120 seconds. GFP-NLS transduction efficiency was assessed by flow cytometry, and the percentage of GFP-fluorescent cells ("Pos Cells (%)") as well as the corresponding cell viability data ("Viability (%)") are shown in Figure 34A. In Figure 34B, "Relative Fluorescence Intensity (FL1-A)" corresponds to the average of all fluorescence intensities from each cell with a GFP fluorescent signal after delivery of the GFP-NLS fluorescent protein using the shuttle agent. [Figure 35] Figures 35A-35E show the results of a transduction efficiency experiment in which THP-1 cells were exposed daily to GFP-NLS cargo for 2.5 hours in the presence of a shuttle agent. In Figures 35A-35E, His-CM18-PTD4 was used, and in Figure 35F, His-C(LLKK)3C-PTD4 was used. GFP-NLS transduction efficiency was determined by flow cytometry on day 1 or day 3, and the results are presented in Figures 35A-35C and 35F as the percentage of GFP-fluorescent cells ("Pos Cells (%)") and corresponding cell viability data ("Viability (%)"). Figure 35D shows the metabolic activity index of THP-1 cells after 1, 2, 4, and 24 hours, and Figure 35E shows the metabolic activity index of THP-1 cells after 1 to 4 days of exposure to the His-CM18-PTD4 shuttle. [Figure 36]

[0023] Figure 1 shows a comparison of GFP-NLS transduction efficiency in several different cell types (e.g., adherent and suspension, and cell lines and primary cells) using the shuttle His-CM18-PTD4, as measured by flow cytometry. Results are expressed as the percentage of GFP-fluorescent cells ("Pos Cells (%)") and corresponding cell viability data ("Viability (%)"). [Figure 37] Figures 37A-37H show fluorescence microscopy images of various cell types transduced with the GFP-NLS cargo using the shuttle His-CM18-PTD4. GFP fluorescence was visualized by fluorescence microscopy at 10x magnification. In the inset, the results of a parallel flow cytometry experiment are also provided (viability and percentage of GFP-fluorescent cells). [Figure 38] Figure 38 shows fluorescence microscopy images of primary human myoblasts transduced with GFP-NLS using the shuttle His-CM18-PTD4. Cells were fixed and permeabilized, and then immunolabeled for GFP-NLS using an anti-GFP antibody and a fluorescent secondary antibody. The immunolabeled GFP is shown in Figure 38A, and in Figure 38B, nuclear (DAPI) labeling is overlaid on the image. [Figure 39]Schematics (Figures 39A, 39B, and 39C) and example fluorescent images (Figures 39D and 39E) of the transfection plasmid surrogate assay used to evaluate the activity of CRISPR / Cas9-NLS complexes delivered into cells. (Figure 39A) On day 1, cells are transfected with an expression plasmid encoding the fluorescent proteins mCherry™ and GFP, with a stop codon separating the two open reading frames. Transfection of cells with the expression plasmid results in mCherry™ expression only (Figure 39D). A CRISPR / Cas9-NLS complex, designed / programmed to cleave plasmid DNA at the stop codon, is then delivered intracellularly to the transfected cells expressing mCherry™, resulting in a double-stranded break in the plasmid DNA at the stop codon (Figure 39B). In some cells, random non-homologous DNA repair of the cut plasmid occurs, resulting in removal of the stop codon (Figure 39C) and thus GFP expression and fluorescence (Figure 39E). The open triangle window shows an example of an area of ​​co-labeling of mCherry™ and GFP fluorescence. [Figure 40]Fluorescence microscopy images of HeLa cells expressing mCherry™ and GFP demonstrate CRISPR / Cas9-NLS-mediated cleavage of the plasmid replacement DNA. In panels A-D, HeLa cells were cotransfected with three plasmids: the plasmid replacement as described in the brief description of Figures 39A-39F, and two other expression plasmids encoding the Cas9-NLS protein and crRNA / tracrRNA, respectively. CRISPR / Cas9-mediated cleavage of the plasmid replacement at the stop codon and subsequent DNA repair by the cell allows expression of GFP (Figures 40B and 40D) in addition to mCherry™ (Figures 40A and 40C). In Figures 40E-40H, HeLa cells were transfected with the plasmid replacement and then transduced with the active CRISPR / Cas9-NLS complex using the shuttle His-CM18-PTD4. Alternative CRISPR / Cas9-NLS-mediated cleavage of the plasmid at the stop codon and subsequent DNA repair by the cell allows expression of GFP (Figures 40F and 40H) in addition to mCherry™ (Figures 40E and 40G). [Figure 41A] (Lanes A-D) Products from a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR / Cas9-mediated cleavage of cellular genomic DNA. HeLa cells were transduced with a CRISPR-Cas9-NLS complex programmed to cleave the PPIB gene. The presence of the cleavage product, enclosed in white boxes 1 and 2, indicates cleavage of the PPIB gene by the CRISPR-Cas9-NLS complex delivered into cells using the shuttle His-C(LLKK)3C-PTD4 (Figure 41A, lane B) or a lipid transfection agent used as a positive control (Figure 41A, lane D). This cleavage product is absent in the negative control (Figure 41A, lanes A and C). [Figure 41B]Figure 1 shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR / Cas9-mediated cleavage of cellular genomic DNA (PPIB DNA sequence). The left panel shows the cleavage products of the amplified PPIB DNA sequence by the CRISPR / Cas9 complex after delivery of the complex with the shuttle agent His-CM18-PTD4 in HeLa cells. The right panel shows the amplified DNA sequence before the T7E1 digestion procedure as a negative control. [Figure 41C] Figure 1 shows the products of a DNA cleavage assay (T7E1 assay) separated by agarose gel electrophoresis, which is used to measure CRISPR / Cas9-mediated cleavage of cellular genomic DNA (PPIB DNA sequence). The left panel shows the PPIB DNA sequence amplified after incubation of HeLa cells with Cas9 / RNA complexes in the presence of a lipid transfection agent (DharmaFect™ Transfection Reagent #T-20XX-01) (positive control). The right panel shows the amplified DNA sequence before the T7E1 digestion procedure as a negative control. [Figure 42] Figure 1 shows the transcriptional activity of THP-1 cells transduced with the transcription factor HOXB4 using various concentrations of the shuttle His-CM18-PTD4 and different cargo / shuttle exposure times. Successful nuclear delivery of HOXB4 was determined by monitoring target gene mRNA levels by real-time PCR. Results were normalized to those in the negative control (HOXB4 without shuttle agent) and expressed as "fold above control" (left bar). Total cellular RNA (ng / μL) was quantified, as was a marker of cell viability (right bar). "Φ" or "Ctrl" means "no treatment," "TF" means "transcription factor alone," and "FS" means "shuttle alone." [Figure 43]Figure 1 shows the transcriptional activity of THP-1 cells transduced with the transcription factor HOXB4 using various concentrations of the shuttle His-CM18-PTD4 and different cargo / shuttle exposure times. Successful nuclear delivery of HOXB4 was determined by monitoring target gene mRNA levels by real-time PCR. Results were normalized to those in the negative control (HOXB4 without shuttle agent) and expressed as "fold above control" (left bar). Total cellular RNA (ng / μL) was quantified, as was a marker of cell viability (right bar). "Φ" or "Ctrl" means "no treatment," "TF" means "transcription factor alone," and "FS" means "shuttle alone." [Figure 44] Figure 1 shows the transcriptional activity of THP-1 cells transduced with the transcription factor HOXB4 using various concentrations of the shuttle His-CM18-PTD4 and different cargo / shuttle exposure times. Successful nuclear delivery of HOXB4 was determined by monitoring target gene mRNA levels by real-time PCR. Results were normalized to those in the negative control (HOXB4 without shuttle agent) and expressed as "fold above control" (left bar). Total cellular RNA (ng / μL) was quantified, as was a marker of cell viability (right bar). "Φ" or "Ctrl" means "no treatment," "TF" means "transcription factor alone," and "FS" means "shuttle alone." [Figure 45] Figures 45A and 45C show fluorescence microscopy images of HeLa cells transduced with wild-type HOXB4 cargo using the shuttle His-CM18-PTD4. After a 30-minute incubation to allow transduced HOXB4-WT to accumulate in the nucleus, cells were fixed and permeabilized, and HOXB4-WT was labeled using a primary anti-HOXB4 monoclonal antibody and a fluorescent secondary antibody (Figures 45B and 45D). Nuclei were labeled with DAPI (Figures 45A and 45C). The open triangle window shows an example of an area of ​​co-labeling between the nucleus and HOXB4; compare Figures 45A vs. 45B (x20 magnification) and Figures 45C vs. 45D (x40 magnification). [Figure 46] Figure 46 shows the products of a DNA cleavage assay separated by agarose gel electrophoresis, which is used to measure CRISPR / Cas9-mediated cleavage of cellular genomic DNA (HPTR sequence) after intracellular delivery of the complex with different shuttle agents. Figure 46A shows the results in HeLa cells using the shuttle agents His-CM18-PTD4, His-CM18-PTD4-His, and His-C(LLKK)3C-PTD4. Figure 46B shows the results in Jurkat cells using His-CM18-PTD4-His and His-CM18-L2-PTD4. The negative control (lane 4 in panels A and B) shows the HPTR DNA sequence amplified after incubating cells with the CRISPR / Cas9 complex without the presence of a shuttle agent. The positive control (lane 5 in panels A and B) shows the HPTR DNA sequence amplified after incubating cells with the Cas9 / RNA complex in the presence of a commercially available lipid transfection agent. [Figure 47] Figure 1 shows the transcriptional activity of THP-1 cells transduced with the transcription factor HOXB4 using the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4, His-C(LLKK)3C-PTD4, and His-CM18-PTD4-His. Successful nuclear delivery of HOXB4 was determined by monitoring target gene mRNA levels by real-time PCR. Results were normalized to those in the negative control (HOXB4 without shuttle agent) and expressed as "fold over control" (left bar). Total cellular RNA (ng / μL) was quantified, as was a marker of cell viability (right bar). "Φ" or "Ctrl" means "no treatment," "TF" means "transcription factor alone," and "FS" means "shuttle alone." [Figure 48]Figure 48B shows in vivo GFP-NLS delivery in the rat parietal cortex by His-CM18-PTD4. Briefly, GFP-NLS (20 μM) was injected into the rat parietal cortex for 10 minutes in the presence of the shuttle agent His-CM18-PTD4 (20 μM). Dorsoventral rat brain sections were collected and analyzed by fluorescence microscopy at 4x (Figure 48A), 10x (Figure 48C), and 20x (Figure 48D) magnifications. The injection site was located in the deepest layer of the parietal cortex (PCx). In the presence of the His-CM18-PTD4 shuttle agent, GFP-NLS diffused into the nuclei of the PCx, corpus callus (Cc), and striatum (Str) (white curves mark the boundaries between brain structures). Figure 48B shows the stereotaxic coordinates of the injection site (black arrow) from the Franklin and Paxinos rat brain atlas. Injections of GFP-NLS in the presence of His-CM18-PTD4 were performed on the left side of the brain, and negative controls (injections of GFP-NLS alone) were performed on the contralateral side. The black circles and black connecting lines in Figure 48B indicate the areas observed in the fluorescent photographs (Figures 48A, 48C, and 48D). [Figure 49-1] Figures 49A and 49B show helical wheel (left panel) and "open cylinder" (right panel) representations of the peptides FSD5 and VSVG-PTD4, respectively. The geometric shape of each amino acid residue corresponds to its biochemical properties (i.e., hydrophobicity, charge, or hydrophilicity) based on the residue's side chain. One of the major differences between the two open cylinder representations of FSD5 and VSVG-PTD4 is the presence of a highly hydrophobic core in FSD5 (outlined in Figure 49A, left and right panels), which is absent in VSVG-PTD4. The cylinder in the bottom center panel of Figures 49A and 49B is a simplified version of the open cylinder representation in the right panel, where "H" represents a highly hydrophobic surface area; "h" represents a hydrophobic surface area; "+" represents a positively charged residue; and "h" represents a hydrophilic residue. [Figure 49-2] 49C to 49F show three-dimensional models predicted for the structures of peptides FSD5, FSD18, VSVG-PTD4, and FSD44, respectively. [Figure 49-3]Figure 49G shows a multiple sequence alignment of peptides His-CM18-PTD4; EB1-PTD4; His-C(LLKK)3C-PTD4; FSD5; FSD10; FSD19; FSD20; FSD21; FSD44; FSD46; and FSD63, along with the "consistency" score for each residue position. The histidine-rich domain was intentionally excluded during the alignment. [Figure 50-1] Figures 50A and 50C show microscopic images of viable HeLa cells successfully transduced with the shuttle agent FSD5 using a fluorescently labeled antibody as cargo. Figure 50A shows cytoplasmic transduction of goat anti-rabbit IgG H&L (Alexa Fluor® 594) antibody visualized by bright field (top panel) and fluorescence microscopy (bottom panel) at 20x magnification. Figures 50B and 50C show cytoplasmic transduction of goat anti-mouse IgG H&L (Alexa Fluor® 488) antibody visualized by bright field (top panel) and fluorescence microscopy (bottom panel) at 10x and 20x magnification, respectively. [Figure 50-2] Figure 50D shows the results of a transduction experiment in which HeLa cells were transduced with an anti-NUP98 antibody that recognizes an antigen in the perinuclear membrane using the shuttle agent FSD19. After transduction, HeLa cells were fixed, permeabilized, and labeled with a fluorescent (Alexa™ 488) secondary antibody that recognizes the anti-NUP98 antibody (left panel) and Hoechst nuclear stain (right panel). The upper and lower panels show images taken at 20x and 40x magnification, respectively. [Figure 51-1]Figures 51A-51F show the results of genome editing experiments in which CRISPR / Cas9-NLS genome editing complexes were transduced into various cell types (HeLa, NK cells, NIC-H196 cells, HCC-78 cells, and REC-1 cells) using various shuttle agent peptides (FSD5, FSD8, FSD10, and FSD18). Successful genome editing was confirmed by genomic DNA cleavage assays. The CRISPR / Cas9-NLS complexes consist of recombinant Cas9-NLS complexed with crRNA / tracrRNA designed to cleave the HPRT genomic DNA sequence. Successful genome editing was observed by the detection of genomic DNA cleavage products (thick solid arrows) compared to the uncleaved genomic target gene (thin dotted arrows). The negative control ("-ctrl") was from a transduction experiment performed in the absence of the shuttle agent peptide. Imaging software was used to directly quantify the relative signal intensity of the cleavage product bands on the gel. The sum of all bands in a given lane corresponds to 100% signal, and the italicized numbers at the bottom of each lane are the relative % signal sum of only the two cleavage product bands (dark solid arrows). [Figure 51-2]Figure 51G shows cleavage of the target HPRT genomic sequence by the CRISPR / Cas9-NLS complex transduced with FSD5 in the absence ("No template") or presence ("+500 ng") of a short DNA template (72 bp). The light dotted arrow indicates the band corresponding to the target gene, and the dark solid arrow indicates the band corresponding to the CRISPR / Cas9-NLS-mediated cleavage product of this target gene, indicating successful transduction of a fully functional genome editing complex in both the presence and absence of a DNA template. The italicized numbers at the bottom of each lane are the sum of the relative signal percentages of only the two cleavage product bands (dark solid arrows). Figure 51H shows the results of PCR amplification of the sample in Figure 51G using primers specific for the short DNA template, indicating the genomic insert of the DNA template sequence (see arrow in Figure 51H). Figure 51I shows cleavage of the target HPRT genomic sequence by the CRISPR / Cas9-NLS complex transduced with FSD5 in the absence ("No Template") or presence ("+500 ng") of a long linear DNA template (1631 bp). The light dotted arrow indicates the band corresponding to the target gene, and the dark solid arrow indicates the band corresponding to the CRISPR / Cas9-NLS-mediated cleavage product of this target gene, demonstrating successful transduction of a fully functional genome editing complex in both the presence and absence of a DNA template. The italicized numbers at the bottom of each lane represent the sum of the relative signal percentages of only the two cleavage product bands (dark solid arrows). Figure 51J shows the results of PCR amplification of the sample in Figure 51I using primers designed to amplify across the genomic cleavage site. The genomic insert of the long DNA template sequence is visualized by the presence of larger bands in the "+500 ng" (faint) and "+1000 ng" (darker) lanes. See arrow in Figure 51J.Figures 51K and 51L show the results of cleavage of the target genomic DNMT1 DNA sequence using the CRISPR / Cpf1-NLS genome editing complex in the absence ("-ctrl") or presence of the shuttle agent FSD18 in HeLa (Figure 51K) and NK cells (Figure 51L) after PCR amplification and separation by agarose gel electrophoresis. The light dotted arrow indicates the band corresponding to the target gene, and the dark solid arrow indicates the band corresponding to the CRISPR / Cpf1-NLS-mediated cleavage product of this target gene, indicating successful transduction of a fully functional genome editing complex. Imaging software was used to quantify the relative signal intensity of each of the various bands directly on the gel. The sum of all bands in a given lane corresponds to 100% signal, and the italicized numbers at the bottom of each lane are the sum of the relative signal % of only the two cleavage product bands (dark solid arrows). When the lipofectamine-based gene transfer reagent CRISPRMAX™ was used to transduce the CRISPR / Cpf1-NLS genome editing complex, no genomic DNA cleavage was observed. [Figure 52-1]Figure 52 shows the results of cleavage of the target genomic B2M DNA sequence using CRISPR / Cas9-NLS and crRNA / tracrRNA, or CRISPR / Cpf1-NLS and single-guide RNA, in various cell types: THP-1 (Figure 52A); NK (Figure 52B-D); and HeLa (Figure 52E), following separation by agarose gel electrophoresis. Cells were incubated with the CRISPR / Cpf1 complex and FSD shuttle agent for the indicated time and concentration. The light dotted arrow indicates the band corresponding to the target gene, and the darker solid arrow indicates the band corresponding to the CRISPR-mediated cleavage product of this target gene, indicating successful transduction of a fully functional CRISPR / Cas9-NLS genome editing complex. Imaging software was used to quantify the relative signal intensity of each of the various bands directly on the gel. The sum of all bands in a given lane corresponds to 100% signal, and the italicized numbers at the bottom of each lane are the % relative signal sum of only the two cleavage product bands (dark solid arrows). [Figure 52-2] Figure 52 shows the results of cleavage of target genomic GSK3 (Figure 52F), CBLB (Figure 52G), and DNMT1 (Figure 52H-I) DNA sequences using CRISPR / Cpf1-NLS and single-guide crRNA in the absence ("-ctrl") or presence of shuttle agents FSD10, FSD18, FSD19, or FSD23 at various concentrations and exposure times in various cell types: NK (Figure 52F-G); THP-1 (Figure 52H); and primary myoblasts (Figure 52I), following separation by agarose gel electrophoresis. Cells were incubated with the CRISPR / Cpf1 complex and FSD for the indicated times and concentrations. Light dotted arrows indicate bands corresponding to the target genes, and darker solid arrows indicate bands corresponding to the CRISPR-mediated cleavage products of these target genes, indicating successful transduction of fully functional CRISPR / Cpf1-NLS genome editing complexes. [Figure 52-3]Figures 52J-52N show the results of cleavage of the target genomic NKG2A DNA sequence using CRISPR / Cpf1-NLS and single-guide crRNA in NK cells (Figures 52F-52G) and NK-92 cells (Figure 52H) in the absence ("-ctrl") or presence of shuttle agents FSD10, FSD21, FSD22, or FSD23 at various concentrations and exposure times, following separation by agarose gel electrophoresis. Cells were incubated with the CRISPR / Cpf1 complex for the indicated times and concentrations. The light dotted arrow indicates the band corresponding to the target gene, and the darker solid arrow indicates the band corresponding to the CRISPR-mediated cleavage product of this target gene, indicating successful transduction of the fully functional CRISPR / Cpf1-NLS genome editing complex. [Figure 53] The results of CRISPR-mediated cleavage of target genomic HPRT, DNMT1, and B2M DNA sequences are shown. Two genomic HPRT and DNMT1 (Figure 53A) DNA sequences or two DNA loci on genomic B2M exon 2 (Figure 53B) were targeted in HeLa cells using CRISPR / Cas9-NLS and CRISPR / Cpf1-NLS genome editing complexes designed for these purposes, which were then transduced with the shuttle peptide FSD18. Two DNA loci on genomic B2M exon 2 (Figure 53C) were targeted in NK cells using CRISPR / Cpf1-NLS and single-guide crRNA-1, crRNA-2, or both in the absence ("-ctrl") or presence of shuttle agents FSD10, FSD21, or FSD23 at various concentrations and exposure times, following separation by agarose gel electrophoresis. The light dotted arrow indicates the band corresponding to the target gene, and the darker solid arrow indicates the band corresponding to the CRISPR / Cpf1-mediated cleavage product in the presence of crRNA-1 or crRNA2 or both for B2M exon 2 (crRNA1+2), indicating successful transduction of a fully functional CRISPR / Cpf1-NLS genome editing complex. [Figure 54]Figures 54A-54D show the results of flow cytometry assays in which T cells were treated with increasing concentrations of the shuttle agent FSD21 (8 μM: Figure 54B, 10 μM: Figure 54C, and 12 μM: Figure 54D), 1.33 μM of the CRISPR / Cpf1-NLS system, and 2 μM of single-guide crRNA targeting the B2M genomic DNA sequence. HLA-positive and HLA-negative (B2M knockout) cells were identified 72 hours after treatment using an APC-labeled mouse anti-human HLA-ABC antibody. The left-shifted cell population indicates successful inactivation of cell-surface HLA receptors due to inactivation of the B2M gene. [Figure 55] Figures 55A-55D show the results of flow cytometry assays in which T cells were treated with increasing concentrations of the shuttle agent FSD18 (8 μM: Figure 55B, 10 μM: Figure 55C, and 12 μM: Figure 55D), 1.33 μM of the CRISPR / Cpf1-NLS system, and 2 μM of single-guide crRNA targeting the B2M DNA sequence. HLA-positive and HLA-negative (B2M knockout) cells were identified 72 hours after treatment using an APC-labeled mouse anti-human HLA-ABC antibody. The left-shifted cell population indicates successful inactivation of cell-surface HLA receptors due to inactivation of the B2M gene. [Figure 56] Flow cytometry assay results are shown for THP-1 cells treated with 1.33 μM of the CRISPR / Cpf1-NLS system, 2 μM of one or three guide crRNAs each targeting different sites within the B2M genomic DNA sequence, and 3 μM of FSD18 (Figures 56B-56E), or not ("untreated"; Figure 56A). HLA-positive and HLA-negative (B2M knockout) cells in untreated cells (Figure 56A), crRNA E-treated cells (Figure 56B), crRNA G-treated cells (Figure 56C), crRNA J-treated cells (Figure 56D), and crRNA E+G+J-treated cells (Figure 56E) were identified 48 hours after treatment using an APC-labeled mouse anti-human HLA-ABC antibody. The left-shifted cell population indicates successful inactivation of cell surface HLA receptors due to inactivation of the B2M gene. [Figure 57] Figure 57A shows the results of an experiment in which NK-92 cells were genome-edited to determine whether inactivation of the endogenous NKG2A gene could enhance their ability to kill target HeLa cells. Briefly, NK-92 cells were transduced with a CRISPR / Cpf1-NLS genome-editing complex designed to cleave the NKG2A gene using the shuttle peptide FSD23. After transduction, NK-92 cells were immunolabeled with a phycoerythrin (PE)-labeled anti-NKG2A antibody and then analyzed by flow cytometry, as shown in Figure 57A, to confirm successful NKG2A inactivation. As a control, unlabeled wild-type NK-92 cells ("unlabeled WT cells") had no antibody signal, while labeled wild-type NK-92 cells ("labeled WT cells") had a complete immunolabeling signal. In the case of NKG2A-KO NK-92 cells, two cell populations (peaks) were observed: one with a complete knockout of NKG2A receptor expression on the cell surface ("complete NKG2A KO cells"), and one with a partial lack of expression ("partial NKG2A KO cells"). Figure 57B shows the results of a cytotoxicity assay in which target HeLa cells, previously loaded with an intracellular fluorescent dye (calcein), were exposed to wild-type (solid line) or genome-edited NKG2A-KO (dotted line) effector NK-92 cells at various effector:target ratios (E:T ratios). Cytotoxicity was assessed by the relative release of intracellular calcein into the extracellular space resulting from disruption of the target HeLa cell plasma membrane ("% cell lysis"). [Figure 58-1]Flow cytometry assay results are shown for T cells treated with a mixture of GFP-NLS, a CRISPR / Cpf1-NLS complex designed to cleave the B2M genomic DNA sequence, and the shuttle agent FSD18 (Figures 58A-58F), or not (Figure 58A). Figures 58B and 58C show the results of two-dimensional flow cytometry analysis based on GFP fluorescence (x-axis) and cell surface HLA expression (y-axis) 5 hours after treatment. Fluorescently labeled cell sorting of cells into GFP-negative (Figure 58E) and GFP-positive (Figure 58F) fractions based on their GFP fluorescence (Figure 58D) increased the proportion of genome-edited (HLA-negative) cells in the GFP-positive fraction to 29.7% (Figure 58F). Each cell fraction was then subjected to a standard T7E1 cleavage assay followed by agarose gel electrophoresis to assess the efficacy of genome editing. The results are shown in Figure 58G, where the thin dotted arrow indicates the band corresponding to the target gene, and the thicker solid arrow indicates the band corresponding to the CRISPR system-mediated cleavage product of this target gene. The values ​​at the bottom of lanes 2, 3, and 4 correspond to the relative signal intensity (%) of the cleavage product band (solid arrow) in that lane. [Figure 58-2] Same as above. [Figure 58-3] Same as above. [Figure 59-1]Flow cytometry assay results are shown in which T cells were treated (Figures 59C-59E) or not (Figures 59A and 59B) with a mixture of GFP-NLS, a CRISPR / Cpf1-NLS complex designed to cleave the endogenous B2M gene, and the shuttle agent FSD18. Figures 59A and 59B show the results of "untreated" negative control cells that were not exposed to the peptide shuttle agent, GFP, or CRISPR / Cpf1, and were analyzed by flow cytometry for GFP fluorescence (Figure 59A) and cell surface HLA expression (Figure 59B). T cells were co-transduced with both GFP-NLS and CRISPR / Cpf1 via the peptide FSD18, and the resulting GFP fluorescence distribution is shown in Figure 59C. The two gates shown in Figure 59C represent the cell fraction considered GFP-positive ("GFP+"; 93.2%; solid gate) and the subfraction of cells considered to exhibit high GFP fluorescence ("GFP-high"; 33.1%; dotted gate). Fluorescence-activated cell sorting was performed to quantify the level of cell surface HLA expression in cells considered GFP-positive (Figure 59D) compared to cells considered to exhibit high GFP fluorescence (Figure 59E). The co-transduction experiment described above was repeated using 12 μM or 15 μM FSD18, followed by fluorescence-activated cell sorting into GFP-positive and GFP-negative cell fractions. Each fraction was subjected to a standard T7E1 cleavage assay, and various samples were subjected to agarose gel electrophoresis. [Figure 59-2] Same as above. [Figure 60] Flow cytometry analysis results are shown for T cells that were either subjected to a first transduction (Figure 60B) or not ("untreated"; Figure 60A) with the cargo GFP-NLS using peptide FSD18. The first transduction resulted in a GFP-NLS transduction efficiency of 55.4% (Figure 60B). Fluorescence-activated cell sorting was performed to isolate GFP-negative cells (Figure 60C) after the first transduction, and this GFP-negative cell population was subjected to a second transduction of GFP-NLS using peptide FSD18. The results of this second transduction are shown in Figure 60D, revealing a GFP-NLS transduction efficiency of 60.6%. DETAILED DESCRIPTION OF THE INVENTION

[0023] Sequence Listing This application contains a sequence listing in computer readable format having a size of approximately 5 MB, created on October 10, 2017. This computer readable format sequence listing is incorporated herein by reference. [Table 1] [Table 1-2]

[0024] Extensive screening efforts have led to the discovery that domain-based peptide shuttle agents comprising an endosomal leakage domain (ELD) operably linked to a cell-permeable domain (CPD), and optionally one or more histidine-rich domains, increase the transduction efficiency of independent polypeptide cargo in eukaryotic cells, thereby allowing the cargo to reach the cytosolic / nuclear compartment (see, e.g., Examples 1-15). Conversely, the screening efforts have also revealed several peptides with no or reduced polypeptide cargo transduction capacity, excessive toxicity, and / or other undesirable properties (e.g., poor solubility and / or stability).

[0025] Based on these empirical data (both favorable and unfavorable), we compared the amino acid sequences and properties of successful, less successful, and unsuccessful peptides to better understand the physicochemical properties common to the more successful shuttle agents. This comparison involved two main approaches: first, manual stratification of the various screened peptides according to their transduction performance based on our reference biological characterization data; and second, a simpler "transduction score" approach that considers only the transduction efficiency and cytotoxicity of various peptides for a given polypeptide cargo and cell line.

[0026] For manual stratification, the proteins may be selected based on, for example, their solubility / stability / ease of synthesis; their ability to facilitate the escape of endosome-trapped calcein (see, e.g., Example 2); their ability to deliver one or more types of independent polypeptide cargo into cells as assessed by flow cytometry in various cell types and cell lines (e.g., primary, immortalized, adherent, suspension, etc.) and under various transduction protocols (see, e.g., Examples 3-6 and 8-15); fluorescence microscopy (e.g., for fluorescently labeled cargo), increased transcriptional activity (e.g., The screened peptides were individually evaluated according to their transduction performance, taking into due consideration their ability to deliver polypeptide cargo to the cytosol and / or nucleus, as assessed by their activity (e.g., for transcription factor cargo) or genome editing capacity (e.g., for nuclease cargo or genome editing complexes such as CRISPR / Cas9 or CRISPR / Cpf1) (see, e.g., Examples 3-6 and 8-15), as well as toxicity to various cell types and cell lines (e.g., primary, immortalized, adherent, suspension, etc.) under various transduction protocols.

[0027] In the "transduction score" approach, each peptide was assigned a score corresponding to a given cell line and fluorescently labeled polypeptide cargo, which combined both transduction efficiency and cellular toxicity data into a single numerical value. The transduction score was calculated by simply multiplying the highest percentage transduction efficiency observed by flow cytometry for a given peptide, cargo, and cell type by the percent cell viability of the peptide in the cell line tested. Peptides were then sorted by their transduction score as a screening tool to stratify peptides as successful, less successful, or unsuccessful shuttle agents.

[0028] The above manual curation and "transduction score"-based analysis revealed several parameters commonly shared by successful domain-based shuttle agents (see, e.g., Example A). These parameters were then used to manually design novel peptide shuttle agents that possess polypeptide cargo transduction activity and lack and / or are not based on known putative CPDs and / or ELDs (see, e.g., Example B). Furthermore, it was observed that peptides satisfying the greatest number of design parameters typically had the highest transduction scores, while peptides satisfying the fewest number of design parameters typically had the lowest transduction scores.

[0029] The design parameters described herein were further confirmed by testing multiple synthetic peptides whose amino acid sequences were generated using a machine learning algorithm (see, e.g., Example C.1). This machine learning algorithm was "trained" using transduction efficiency and cellular toxicity data of domain-based peptides (but without the design parameters described herein). Interestingly, the peptides generated by the machine learning algorithm that exhibited the highest transduction scores were typically peptides that satisfied all of the design parameters described herein, thereby embodying their use in the proactive design and / or prediction of the transduction activity of novel peptide shuttle agents (e.g., tailored to specific polypeptide cargoes and / or cell types). Furthermore, computer-assisted random peptide sequence generation, followed by descriptor filtering, was used to generate a list of 10,000 peptide variants that satisfied nearly all of the design parameters described herein (see Example C.2).

[0030] Rationally designed peptide shuttling agents are shown herein to facilitate the escape of endosome-trapped fluorescent dyes, suggesting endosomolytic activity (see, e.g., Example D). Furthermore, the ability of rationally designed peptide shuttling agents to transduce various polypeptide cargoes (e.g., fluorescent proteins, transcription factors, antibodies, and entire CRISPR-bound genome editing complexes with or without DNA templates) in a variety of different cell types (both adherent and suspension) is also demonstrated herein (see, e.g., Examples E-G).

[0031] Rational design parameters and peptide shuttle agents In some embodiments, the present description relates to methods for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, the method comprising contacting the target eukaryotic cell with the polypeptide cargo in the presence of a shuttle agent at a concentration sufficient to increase the transduction efficiency of said polypeptide cargo compared to that in the absence of the shuttle agent. In some embodiments, the shuttle agent relates to a peptide that satisfies one or more of the following parameters:

[0032] (1) In some embodiments, the shuttle agent is a peptide at least 20 amino acids in length. For example, the peptide may comprise a minimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues and a maximum length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues. In some embodiments, shorter peptides (e.g., in the range of 20-50 amino acids) may be particularly advantageous because they may be more easily synthesized and purified by chemical synthesis methods and may be more suitable for clinical use (as opposed to recombinant proteins, which must be purified from cell expression systems). While numbers and ranges in this description are often recited as multiples of 5, this description should not be so limited. For example, the maximum lengths described herein also encompass lengths of 56, 57, 58, ... 61, 62, etc., and it should be understood that the absence of recitations in this description is solely for the sake of brevity. The same reasoning applies to the percent identities described herein.

[0033] (2) In some embodiments, the peptide shuttle agent comprises an amphipathic alpha-helical motif. As used herein, the phrase "alpha-helical motif" or "alpha helix" refers to a right-handed coiled or spiral structure (helix) with an alpha helix having a rotation angle between consecutive amino acids of 100 degrees and / or 3.6 residues per turn, unless otherwise specified. As used herein, the phrase "comprising an alpha-helical motif" or "amphipathic alpha-helical motif," etc., refers to the three-dimensional structure that the described peptide (or a segment of a peptide) is predicted to adopt in a biological setting based on the peptide's primary amino acid sequence, regardless of whether the peptide actually adopts it when used in a cell as a shuttle agent. Furthermore, the described peptides may contain one or more alpha-helical motifs at various peptide positions. For example, the shuttle agent FSD5 is predicted to adopt an alpha-helical structure throughout its length (see Figure 49C), while the shuttle agent FSD18 is predicted to contain two separate alpha-helices toward the N- and C-terminal regions of the peptide (see Figure 49D). In some embodiments, the shuttle agent described herein is not predicted to contain a beta-sheet motif, for example, as shown in Figures 49E and 49F. Methods for predicting the presence of alpha helices and beta-sheets in proteins and peptides are well known in the art. For example, one such method is based on 3D modeling using PEP-FOLD™, an online resource for de novo peptide structure prediction (http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD / ) (Lamiable et al., 2016; Shen et al., 2014; Thevenet et al., 2012). Other methods for predicting the presence of alpha helices in peptides and proteins are known and readily available to those skilled in the art.

[0034] As used herein, the phrase "amphipathic" refers to a peptide that has both hydrophobic and hydrophilic elements (e.g., based on the side chains of the amino acids that make up the peptide). For example, the phrase "amphipathic alpha helix" or "amphipathic alpha helix motif" refers to a peptide that is predicted to adopt an alpha helix motif, with a nonpolar hydrophobic face and a polar hydrophilic face, based on the nature of the side chains of the amino acids that form the helix.

[0035] (3) In some embodiments, the peptide shuttle agents described herein comprise an amphipathic alpha-helical motif with a positively charged hydrophilic exterior, such as one rich in R and / or K residues. As used herein, the phrase "positively charged hydrophilic exterior" refers to the presence of at least three lysine (K) and / or arginine (R) residues clustered on one side of the amphipathic alpha-helical motif, based on an alpha-helical wheel projection (see, e.g., Figure 49A, left panel). Such helical wheel projections can be generated using a variety of programs, such as the online helical wheel projection tool available at http: / / rzlab.ucr.edu / scripts / wheel / wheel.cgi. In some embodiments, the amphipathic alpha helix motif comprises a positively charged hydrophilic exterior surface comprising (a) at least two, three, or four adjacent positively charged K and / or R residues; and / or (b) a segment of six adjacent residues containing three to five K and / or R residues on a helical wheel projection, based on an alpha helix having a rotation angle between consecutive amino acids of 100 degrees and / or 3.6 residues per turn.

[0036] In some embodiments, the peptide shuttle agents described herein comprise an amphipathic alpha-helical motif comprising a hydrophobic exterior, the hydrophobic exterior comprising: (a) at least two adjacent L residues on a helical wheel projection; and / or (b) a segment of 10 adjacent residues comprising at least five hydrophobic residues selected from L, I, F, V, W, and M, based on an alpha helix having a rotation angle between consecutive amino acids of 100 degrees and / or 3.6 residues per turn on a helical wheel projection.

[0037] (4) In some embodiments, the peptide shuttle agents described herein comprise an amphipathic alpha-helical motif with 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 cylinder representation of an alpha-helix with 3.6 residues per turn, as shown, for example, in Figure 49A, right panel, the highly hydrophobic core may be composed of spatially adjacent L, I, F, V, W, and / or M amino acids representing 12-50% of the peptide's amino acids, calculated excluding the histidine-rich domain (see below). In some embodiments, the highly hydrophobic core can be composed 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 of the peptide. More specifically, the highly hydrophobic core parameters can be calculated by first placing the amino acids of the peptide in an open cylinder representation and then delineating the region of contiguous highly hydrophobic residues (L, I, F, V, W, M) as shown in Figure 49A, right panel. The number of highly hydrophobic residues contained in this delineated highly hydrophobic core is then divided by the total amino acid length of the peptide, excluding histidine-rich domains (e.g., N- and / or C-terminal histidine-rich domains). For example, for the peptide shown in Figure 49A, there are 8 residues in the depicted highly hydrophobic core and 25 total residues in the peptide (excluding the 12 terminal histidines). The highly hydrophobic core is therefore 32% (8 / 25).

[0038] (5) Hydrophobic moment is a measure of the amphipathicity of a helix, peptide, or portion thereof, calculated from the vector sum of the hydrophobicities of amino acid side chains (Eisenberg et al., 1982). An online tool for calculating the hydrophobic moment of a polypeptide is available at http: / / rzlab.ucr.edu / scripts / wheel / wheel.cgi. A high hydrophobic moment indicates strong amphipathicity, while a low hydrophobic moment indicates poor amphipathicity. In some embodiments, the peptide shuttle agents described herein may consist of or include a peptide or alpha-helical domain with a hydrophobic moment of 3.5-11 μm. In some embodiments, the shuttle agent is selected from the group consisting of: 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, The peptide may comprise an amphipathic alpha helix motif having a hydrophobic moment between a lower limit of 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, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0. In some embodiments, a shuttle agent can be a peptide having a hydrophobic moment between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5. In some embodiments, the hydrophobic moment is calculated excluding any histidine-rich domain that may be present in the peptide.

[0039] (6) In some embodiments, the peptide shuttle agents described herein may have a predicted net charge of at least +4 at physiological pH, calculated from the side chains K, R, D, and E. For example, the net charge of the peptide may be at least +5, +6, +7, at least +8, at least +9, at least +10, at least +11, at least +12, at least +13, at least +14, or at least +15 at physiological pH. These positive charges are generally conferred by the greater presence of positively charged lysine and / or arginine residues, as opposed to negatively charged aspartic acid and / or glutamic acid residues.

[0040] (7) In some embodiments, the peptide shuttle agents described herein may have a predicted isoelectric point (pI) of 8 to 13, preferably 10 to 13. Programs and methods for calculating and / or measuring the isoelectric point of peptides or proteins are known in the art. For example, pI may be calculated using Prot Param software, available at http: / / web.expasy.org / protparam / .

[0041] (8) In some embodiments, the peptide shuttle agents described herein may be composed of 35-65% hydrophobic residues (A, C, G, I, L, M, F, P, W, Y, V). In certain embodiments, the peptide shuttle agents may be composed of 36%-64%, 37%-63%, 38%-62%, 39%-61%, or 40%-60% of any combination of the following amino acids: A, C, G, I, L, M, F, P, W, Y, and V.

[0042] (9) In some embodiments, the peptide shuttle agents described herein may be composed of 0-30% neutral hydrophilic residues (N, Q, S, T). In certain embodiments, the peptide shuttle agents may be composed of any combination of 1%-29%, 2%-28%, 3%-27%, 4%-26%, 5%-25%, 6%-24%, 7%-23%, 8%-22%, 9%-21%, or 10%-20% of the amino acids: N, Q, S, and T.

[0043] (10) In some embodiments, the peptide shuttle agents described herein may be composed of 35-85% of the following amino acids: A, L, K, and / or R. In certain embodiments, the peptide shuttle agents may be composed of 36%-80%, 37%-75%, 38%-70%, 39%-65%, or 40%-60% of any combination of the following amino acids: A, L, K, or R.

[0044] (11) In some embodiments, the peptide shuttle agents described herein may be composed of 15-45% amino acids: A and / or L, provided that at least 5% L is present in the peptide. In specific embodiments, the peptide shuttle agents may be composed of 15-40%, 20-40%, 20-35%, or 20-30% amino acids: any combination of A and L, provided that at least 5% L is present in the peptide.

[0045] (12) In some embodiments, the peptide shuttle agents described herein may be composed of 20-45% amino acids: K and / or R. In certain embodiments, the peptide shuttle agents may be composed of 20-40%, 20-35%, or 20-30% amino acids: any combination of K and R.

[0046] (13) In some embodiments, the peptide shuttle agents described herein may be composed of 0-10% amino acids: D and / or E. In certain embodiments, the peptide shuttle agents may be composed of 5-10% amino acids: any combination of D and E.

[0047] (14) In some embodiments, the absolute difference between the percentage of A and / or L and the percentage of K and / or R in a peptide shuttle agent can be 10% or less. In certain embodiments, the absolute difference between the percentage of A and / or L and the percentage of K and / or R in a peptide shuttle agent can be 9%, 8%, 7%, 6%, or 5% or less.

[0048] (15) In some embodiments, the peptide shuttle agents described herein may be composed of 10%-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., other than A, L, K, or R). In certain embodiments, the peptide shuttle agents may be composed of 15-40%, 20-35%, or 20-30% 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.

[0049] In some embodiments, the peptide shuttle agents described herein satisfy at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or all of parameters (1) through (15) described herein. In certain embodiments, the peptide shuttle agents described herein satisfy all of parameters (1) through (3) and also satisfy at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all of parameters (4) through (15) described herein.

[0050] In some embodiments, the peptide shuttle agents described herein contain only one histidine-rich domain, and the residues of that one histidine-rich domain may be included in the calculation / evaluation of parameters (1)-(15) described herein. In some embodiments, when the peptide shuttle agents described herein contain two or more histidine-rich domains, the residues of only one histidine-rich domain may be included in the calculation / evaluation of parameters (1)-(15) described herein. For example, when the peptide shuttle agents described herein contain 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)-(15) described herein.

[0051] In some embodiments, machine learning or computer-aided design techniques can be implemented to generate peptides that satisfy one or more of the parameters (1) through (15) described herein. Some parameters, such as parameters (1) and (5) through (15), are more amenable to computer-aided design, while structural parameters, such as parameters (2), (3), and (4), are more amenable to manual design. Thus, in some embodiments, peptides that satisfy one or more of parameters (1) through (15) can be generated using a combination of computer-aided and manual design techniques. For example, multiple sequence alignment analysis of several peptides shown herein (and elsewhere) to function as effective shuttle agents revealed the presence of several consensus sequences—i.e., commonly recognized patterns of alternating hydrophobic, cationic, and hydrophilic alanine and glycine amino acids. The presence of these consensus sequences can induce structural parameters (2), (3), and (4) to be satisfied (i.e., amphipathic alpha-helix formation, positively charged faces, and a highly hydrophobic core of 12% to 50%). These and other consensus sequences may therefore be employed in machine learning and / or computer-aided design techniques to generate peptides that satisfy one or more of parameters (1)-(15).

[0052] Thus, in some embodiments, the peptide shuttle agents described herein may comprise or consist of the following amino acid sequence: (a) [X1]-[X2]-[linker]-[X3]-[X4] (Formula 1); (b) [X1]-[X2]-[linker]-[X4]-[X3] (Formula 2); (c) [X2]-[X1]-[linker]-[X3]-[X4] (Formula 3); (d) [X2]-[X1]-[linker]-[X4]-[X3] (Formula 4); (e) [X3]-[X4]-[linker]-[X1]-[X2] (Formula 5); (f) [X3] - [X4] - [Linker] - [X2] - [X1] (Formula 6); (g) [X4] - [X3] - [Linker] - [X1] - [X2] (Formula 7); or (h) [X4] - [X3] - [Linker] - [X2] - [X1] (Formula 8); In the formula, [X1] is selected from the following: 2[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; 2[Φ] - 1[+] - 2[Φ] - 2[+] -; 1[+] - 1[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; and 1[+] - 1[Φ] - 1[+] - 2[Φ] - 2[+] -; [X2] is selected from the following: -2[Φ] - 1[+] - 2[Φ] - 2[ζ] -; -2[Φ] - 1[+] - 2[Φ] - 2[+] -; -2[Φ] - 1[+] - 2[Φ] - 1[+] - 1[ζ] -; -2[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; -2[Φ] - 2[+] - 1[Φ] - 2[+] -; -2[Φ] - 2[+] - 1[Φ] - 2[ζ] -; -2[Φ] - 2[+] - 1[Φ] - 1[+] - 1[ζ] -; and -2[Φ] - 2[+] - 1[Φ] - 1[ζ] - 1[+] -; [X3] is selected from the following: -4[+] - A -; -3[+] - G - A -; -3[+] - A - A -; -2[+] - 1[Φ] - 1[+] - A -; -2[+] - 1[Φ] - G - A -; -2[+] - 1[Φ] - A - A -; or -2[+] - A - 1[+] - A; -2[+] - A - G - A; -2[+] - A - A - A -; -1[Φ] - 3[+] - A -; -1[Φ] - 2[+] - G - A -; -1[Φ] - 2[+] - A - A -; -1[Φ] - 1[+] - 1[Φ] - 1[+] - A; -1[Φ] - 1[+] - 1[Φ] - G - A; -1[Φ] - 1[+] - 1[Φ] - A - A; -1[Φ] - 1[+] - A - 1[+] - A; -1[Φ] - 1[+] - A - G - A; -1[Φ] - 1[+] - A - A - A; -A - 1[+] - A - 1[+] - A; -A - 1[+] - A - G - A; and -A - 1[+] - A - A - A; [X4] is selected from the following: -1[ζ]-2A-1[+]-A; -1[ζ]-2A-2[+]; -1[+]-2A-1[+]-A; -1[ζ]-2A-1[+]-1[ζ]-A-1[+]; -1[ζ]-A-1[ζ]-A-1[+]; -2[+]-A-2[+]; -2[+]-A-1[+]-A; -2[ +]-A-1[+]-1[ζ]-A-1[+];-2[+]-1[ζ]-A-1[+];-1[+]-1[ζ]-A-1[+]-A;-1[+]-1[ζ] -A-2[+];-1[+]-1[ζ]-A-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-A-1[+];-1[+]-2[ζ]-2[+ ];-1[+]-2[ζ]-1[+]-A;-1[+]-2[ζ]-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-1[ζ]-A-1[+ ];-3[ζ]-2[+];-3[ζ]-1[+]-A;-3[ζ]-1[+]-1[ζ]-A-1[+];-1[ζ]-2A-1[+]-A;-1[ζ] -2A-2[+];-1[ζ]-2A-1[+]-1[ζ]-A-1[+];-2[+]-A-1[+]-A;-2[+]-1[ζ]-1[+]-A;-1 [+]-1[ζ]-A-1[+]-A;-1[+]-2A-1[+]-1[ζ]-A-1[+]; and -1[ζ]-A-1[ζ]-A-1[+]; and [Linker] is selected from the following: -Gn-; -Sn-; -(GnSn)n-; -(GnSn)nGn-; -(GnSn)nSn-; -(GnSn)nGn(GnSn)n-; and -(GnSn)nSn(GnSn)n-; In the formula, [Φ] is an amino acid: Leu, Phe, Trp, Ile, Met, Tyr, or Val; [+] is an amino acid: Lys or Arg; [ζ] is an amino acid: Gln, Asn, Thr, or Ser; A is an amino acid: Ala; G is an amino acid: Gly; S is an amino acid: Ser; and n is an integer of 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.

[0053] In some embodiments, the peptide shuttle agents described herein may comprise or consist of any one of the amino acid sequences of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242. In some embodiments, the peptide shuttle agents described herein may comprise the amino acid sequence motif of SEQ ID NO: 158 and / or 159, which is found in any of peptides FSD5, FSD16, FSD18, FSD19, FSD20, FSD22, and FSD23. In some embodiments, the peptide shuttle agents described herein may comprise the amino acid sequence motif of SEQ ID NO: 158 operably linked to the amino acid sequence motif of SEQ ID NO: 159. In some embodiments, the peptide shuttle agents described herein may comprise or consist 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 NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242, or a peptide that is a functional variant of any one of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242. As used herein, "functional variant" means a peptide that has polypeptide cargo transduction activity and differs from a reference peptide by one or more conservative amino acid substitutions. As used herein, a "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.Families of amino acid residues having similar side chains are well defined in the 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, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0054] In some embodiments, the peptide shuttle agents described herein may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 57-59, 66-72, or 82-102, or a functional variant thereof having at least 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%, or 95% identity to any one of SEQ ID NOs: 57-59, 66-72, or 82-102. In some embodiments, the peptide shuttle agents described herein do not comprise one or more amino acid sequences of any one of SEQ ID NOs: 57-59, 66-72, or 82-102.

[0055] In some embodiments, the shuttle agents described herein may comprise oligomers (e.g., dimers, trimers, etc.) of the peptides described herein. Such oligomers may be constructed by covalently linking the same or different types of shuttle agent monomers (e.g., using disulfide bridges linking cysteine ​​residues introduced into the monomer sequences). In some embodiments, the shuttle agents described herein may comprise N- and / or C-terminal cysteine ​​residues.

[0056] Histidine-rich domain In some embodiments, the peptide shuttle agents described herein may further comprise one or more histidine-rich domains. In some embodiments, the histidine-rich domain may be a stretch of at least two, at least three, at least four, at least five, or at least six amino acids containing at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues. In some embodiments, the histidine-rich domain may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive histidine residues. Without being bound by theory, the histidine-rich domain in the shuttle agent may act as a proton sponge in the endosome by protonating its imidazole group under the acidic conditions of the endosome, providing another mechanism for endosomal membrane destabilization, thus further facilitating the ability of cargo captured by the endosome to reach the cytosol. In some embodiments, the histidine-rich domain may be located at or toward the N- and / or C-terminus of the peptide shuttle agent.

[0057] Linker In some embodiments, the peptide shuttle agents described herein may include one or more suitable linkers (e.g., flexible polypeptide linkers). In some embodiments, such linkers may separate two or more amphipathic alpha-helical motifs (see, e.g., shuttle agent FSD18 in Figure 49D). In some embodiments, a linker can be used to further separate two domains (CPD, ELD, or histidine-rich domain) from each other. In some embodiments, a linker may be formed by adding a sequence of small, hydrophobic amino acids (such as glycine) that lack rotational capacity and polar serine residues that confer stability and flexibility. The linker may be flexible, allowing the shuttle agent domains to move. In some embodiments, proline may be avoided because it may add significant conformational rigidity. In some embodiments, the linker may be a serine / glycine-rich linker (e.g., GGS, GGSGGGS, GGSGGGSGGGS, etc.). In some embodiments, the use of shuttle agents containing suitable linkers may be advantageous for delivering free-standing polypeptide cargo to suspension cells rather than adherent cells. In some embodiments, the linker comprises or consists of -Gn-;-Sn-;-(GnSn)n-;-(GnSn)nGn-;-(GnSn)nSn-;-(GnSn)nGn(GnSn)n-; or -(GnSn)nSn(GnSn)n-, where G is the amino acid Gly; S is the amino acid Ser, and n is an integer from 1 to 5.

[0058] Endosomal leakage domain (ELD) In some aspects, the described peptide shuttle agents may contain an endosomal leakage domain (ELD) to facilitate endosomal escape and access to the cytoplasmic compartment. As used herein, the phrase "endosomal leakage domain" refers to a sequence of amino acids that confers the ability of macromolecules trapped by endosomes to reach the cytoplasmic compartment. Without being bound by theory, endosomal leakage domains are short sequences (often derived from viral or bacterial peptides) that are believed to induce endosomal membrane destabilization and release of endosomal contents into the cytoplasm. As used herein, the phrase "endosomolytic peptide" refers to this general class of peptides that have endosomal membrane destabilizing properties. Thus, in some embodiments, the described synthetic peptide or polypeptide-based shuttle agents may contain an ELD, an endosomolytic peptide. The activity of such peptides can be assessed, for example, using the calcein endosomal escape assay described in Example 2.

[0059] In some embodiments, the ELD can be a peptide that disrupts membranes at acidic pH, such as a pH-dependent membrane-active peptide (PMAP) or a pH-dependent lytic peptide. For example, the peptides GALA and INF-7 are amphipathic peptides that form alpha helices when a decrease in pH modifies the charge of the amino acids they contain. Without being bound by theory, it has been suggested that ELDs such as GALA induce endosomal leakage by forming membrane lipid pores and flip-flops after conformational changes due to a decrease in pH (Kakudo, Chaki et al., 2004; Li, Nicol et al., 2004). In contrast, it has been suggested that ELDs such as INF-7 induce endosomal leakage by accumulating in and destabilizing the endosomal membrane (El-Sayed, Futaki et al., 2009). Therefore, during the process of endosomal maturation, the concomitant decrease in pH triggers a conformational change in the peptide, which destabilizes the endosomal membrane and leads to the release of endosomal contents. The same principle is thought to apply to Pseudomonas toxin A (Varkouhi, Scholte et al., 2011). After a decrease in pH, the structure of the toxin's translocation domain changes, allowing its insertion into the endosomal membrane where a pore is formed (London 1992, O'Keefe 1992). This ultimately leads to endosomal destabilization and translocation of the complex to the outside of the endosome. The above ELDs are encompassed within the ELDs described here as well as other mechanisms of endosomal leakage whose mechanisms of action may not be fully elucidated.

[0060] In some embodiments, the ELD can be an antimicrobial peptide (AMP), such as a linear cationic alpha-helical antimicrobial peptide (AMP). These peptides play an important role in the innate immune response due to their ability to strongly interact with bacterial membranes. Without being bound by theory, these peptides are thought to be disordered in aqueous solution but adopt an alpha-helical secondary structure in a hydrophobic environment. The latter structure is thought to contribute to their typical concentration-dependent membrane-disrupting properties. When accumulated at certain concentrations in endosomes, some antimicrobial peptides can induce endosomal leakage.

[0061] In some embodiments, the ELD can be an antimicrobial peptide (AMP), such as a cecropin-A / melittin hybrid (CM series) peptide. Such peptides are believed to be among the smallest and most potent AMP-derived peptides with membrane-disrupting ability. Cecropins are a family of antimicrobial peptides with membrane-disrupting activity against both Gram-positive and Gram-negative bacteria. Cecropin A (CA), the first identified antimicrobial peptide, is composed of 37 amino acids with a linear structure. Melittin (M), a 26-amino acid peptide, is a membrane-lytic factor found in bee venom. Cecropin-melittin hybrid peptides have been shown to produce short, efficient antibiotic peptides that are non-cytotoxic to eukaryotic cells (i.e., non-hemolytic), a desirable characteristic of any antimicrobial agent. These chimeric peptides have been constructed from various combinations of the hydrophilic N-terminal domain of cecropin A with the hydrophobic N-terminal domain of melittin and tested in bacterial model systems. Two 26-mers, CA(1-13)M(1-13) and CA(1-8)M(1-18) (Boman et al., 1989), have been shown to demonstrate the broader and improved potency of native cecropin A without the cytotoxic effects of melittin.

[0062] In an attempt to generate shorter CM series peptides, the authors of Andreu et al. (1992) constructed hybrid peptides such as the 26-mer (CA,1-8)M, 1-18 and compared them with the 20-mer (CA,1-8)M, 1-12, the 18-mer (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). 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 potency compared to the 26-mer, which lacked hemolytic effect. Thus, in some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may include ELDs that are or are derived from CM series peptide variants, such as those described above.

[0063] In some embodiments, the ELD can be the CM series peptide CM18, composed of residues 1–7 of cecropin-A (KWKLFKKIGAVLKVLTTG) fused to residues 2–12 of melittin (YGRKKRRQRRR) [C(1–7)M(2–12)]. CM18, when fused to the cell-penetrating peptide TAT, independently crosses the plasma membrane and destabilizes endosomal membranes, allowing some endosome-trapped cargo to be released into the cytosol (Salomone et al., 2012). However, the use of the CM18-TAT11 peptide fused to a fluorophore (atto-633) in some of the authors' experiments adds uncertainty about the contribution of the peptide to the fluorophore, as the use of the fluorophore itself has been shown to contribute, for example, to endosomolysis by photochemical disruption of the endosomal membrane (Erazo-Oliveras et al., 2014).

[0064] In some embodiments, the ELD can be CM18 having the amino acid sequence of SEQ ID NO:1, 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 to SEQ ID NO:1 and having endosomolytic activity.

[0065] In some embodiments, the ELD can be a peptide derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA), which can cause endosomal membrane destabilization upon accumulation in endosomes.

[0066] In some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may comprise an ELD that is or is derived from an ELD shown in Table I, or a variant thereof that has endosomal escape activity and / or pH-dependent membrane disruption activity. [Table 2]

[0067] In some embodiments, the shuttle agents described herein can include one or more ELDs or types of ELDs. More particularly, they can include at least two, at least three, at least four, at least five, or more ELDs. In some embodiments, the shuttle agents can include 1 to 10 ELDs, 1 to 9 ELDs, 1 to 8 ELDs, 1 to 7 ELDs, 1 to 6 ELDs, 1 to 5 ELDs, 1 to 4 ELDs, 1 to 3 ELDs, etc.

[0068] In some embodiments, the order or placement of the ELD relative to other domains (CPD, histidine-rich domain) within the shuttle agents described herein may be varied as long as the shuttle agent's ability to fold back and transport is maintained.

[0069] In some embodiments, the ELD is a variant or fragment of any one of those listed in Table I and may have endosomolytic activity. In some embodiments, the ELD may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 1-15, 63, or 64, 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 any one of SEQ ID NOs: 1-15, 63, or 64 and has endosomolytic activity.

[0070] In some embodiments, a shuttle agent described herein does not comprise one or more amino acid sequences within any one of SEQ ID NOs: 1-15, 63, or 64.

[0071] Cell-penetrating domain (CPD) In some embodiments, the shuttle agents described herein may comprise a cell-penetrating domain (CPD). As used herein, the phrase "cell-penetrating domain" refers to a sequence of amino acids that confers the ability of a CPD-containing macromolecule (e.g., a peptide or protein) to be transduced into a cell.

[0072] In some embodiments, the CPD can be (or can be derived from) a cell membrane-penetrating peptide or a protein transduction domain of a cell membrane-penetrating peptide. Cell membrane-penetrating peptides can function as carriers for successfully delivering various cargoes (e.g., polynucleotides, polypeptides, small molecule compounds, or other otherwise membrane-impermeable macromolecules / compounds) into cells. Cell membrane-penetrating peptides often contain short peptides rich in basic amino acids that, when fused to (or otherwise operatively linked to) a macromolecule, mediate internalization into cells (Shaw, Catchpole et al., 2008). The first cell membrane-penetrating peptide was identified by analyzing the cell-penetrating ability of the HIV-1 transactivator of transcription (Tat) protein (Green and Loewenstein 1988; Vives, Brodin et al., 1997). This protein contains a short hydrophilic amino acid sequence, designated "TAT," that facilitates its insertion into the cell membrane and pore formation. Since this discovery, numerous other cell membrane-penetrating peptides have been reported. In this regard, in some embodiments, the CPD can be a cell membrane permeable peptide as described in Table II or a variant thereof that has cell membrane permeable activity. [Table 3]

[0073] Without being bound by theory, cell membrane-penetrating peptides are thought to interact with the cell membrane and then pass through it by pinocytosis or endocytosis. In the case of the TAT peptide, its hydrophilicity and charge are thought to facilitate its insertion into the cell membrane and the formation of pores (Herce and Garcia 2007). Alpha-helical motifs (e.g., SP) within hydrophobic peptides are also thought to form pores in the cell membrane (Veach, Liu et al. 2004).

[0074] In some embodiments, the shuttle agents described herein may comprise one or more CPDs or types of CPDs. More particularly, they may comprise at least two, at least three, at least four, or at least five or more CPDs. In some embodiments, the shuttle agents may comprise 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.

[0075] In some embodiments, the CPD is a TAT having the amino acid sequence of SEQ ID NO: 17, 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 to SEQ ID NO: 17 and having cell membrane permeabilizing activity, or a TAT having the amino acid sequence of SEQ ID NO: The penetratin may be a penetratin having an amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% identity to SEQ ID NO: 18 and having cell membrane permeability activity.

[0076] In some embodiments, the CPD can be PTD4 having the amino acid sequence of SEQ ID NO:65, 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 to SEQ ID NO:65.

[0077] In some embodiments, the order or placement of the CPD relative to other domains (ELD, histidine-rich domain) within the shuttle agents described herein may be varied as long as the shuttle agent's ability to fold back and transport is maintained.

[0078] In some embodiments, the CPD is a variant or fragment of any one of those listed in Table II and may have cell membrane permeabilizing activity. In some embodiments, the CPD may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65, 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 any one of SEQ ID NOs: 16-27 or 65 and has cell membrane permeabilizing activity.

[0079] In some embodiments, the shuttle agents described herein do not comprise one or more of the amino acid sequences set forth in SEQ ID NOs: 16-27 or 65.

[0080] cargo In some aspects, the peptide shuttle agents described herein are useful for delivering polypeptide cargoes (e.g., independent polypeptide cargoes) from the extracellular space to the cytosol and / or nucleus of target eukaryotic cells, and the synthetic peptide shuttle agents are used at concentrations sufficient to increase the transduction efficiency of the polypeptide cargoes compared to the transduction efficiency in the absence of the synthetic peptide shuttle agent. In some embodiments, the polypeptide cargoes may be fused to one or more CPDs to further facilitate intracellular delivery. In some embodiments, the CPDs fused to the polypeptide cargos may be the same or different from the CPDs that may be present in the shuttle agents described herein. Such fusion proteins may be constructed using standard recombinant techniques. In some embodiments, the independent polypeptide cargoes may be fused, complexed, or covalently linked to a second biologically active cargo (e.g., a biologically active polypeptide or compound). Alternatively, or concurrently, the polypeptide cargo may include an intracellular targeting domain.

[0081] In some embodiments, a polypeptide cargo must be delivered to the nucleus to exert its intended biological effect. One such example is when the cargo is a polypeptide (e.g., a transcription factor) intended for nuclear delivery. In this regard, research into the mechanism of viral DNA translocation has led to the identification of nuclear localization signals (NLSs). NLS sequences are recognized by proteins (importin α and β) that act as transporters and mediators of translocation across the nuclear envelope. NLSs are generally rich in charged amino acids, such as arginine, histidine, and lysine, conferring a positive charge that is partially responsible for their recognition by importins. Thus, in some embodiments, a polypeptide cargo may include an NLS to facilitate nuclear delivery, such as one or more of the NLSs listed in Table III, or a variant thereof with nuclear targeting activity. Of course, it will be understood that in certain embodiments, a polypeptide cargo may include its native NLS. [Table 4]

[0082] Once recombinant proteins are delivered to the cytoplasm, they are exposed to the protein transport system of eukaryotic cells. In fact, all proteins are synthesized in the cytoplasm of cells and then redistributed to their final intracellular location by a transport system based on small amino acid sequences recognized by shuttle proteins (Karniely and Pines 2005, Stojanovski, Bohnert et al., 2012). In addition to NLSs, other localization sequences can mediate intracellular targeting of the described polypeptide cargo to various organelles after intracellular delivery. Thus, in some embodiments, the described polypeptide cargo can include an intracellular localization signal or a variant thereof with corresponding intracellular targeting activity to facilitate the delivery of the shuttle agent and cargo to a specific organelle, such as one or more of the sequences listed in Table IV. [Table 5]

[0083] In some embodiments, the cargo may be a biologically active compound, such as a biologically active (recombinant) polypeptide (e.g., a transcription factor, cytokine, or nuclease) for intracellular delivery. As used herein, the expression "biologically active" refers to the ability of a compound to mediate structural, regulatory, and / or biochemical function when introduced into a target cell.

[0084] In some embodiments, the cargo may be a recombinant polypeptide intended for nuclear delivery, such as a transcription factor. In some embodiments, the transcription factor is HOXB4 (Lu, Feng et al., 2007), NUP98-HOXA9 (Takeda, Goolsby et al., 2006), Oct3 / 4, Sox2, Sox9, Klf4, c-Myc (Takahashi and Yamanaka 2006), MyoD (Sung, Mun et al. al., 2013), Pdx1, Ngn3 and MafA (Akinci, Banga et al., 2012), Blimp-1 (Lin, Chou et al., 2013), Eomes, T-bet (Gordon, Chaix et al., 2012), FOXO3A (Warr, Binnewies et al., 2013), NF-YA (Dolfini, Minuzzo et al. al., 2012), SALL4 (Aguila, Liao et al. al., 2011), ISL1 (Fonoudi, Yeganeh et al., 2013), FoxA1 (Tan, Xie et al., 2010), Nanog, Esrrb, Lin28 (Buganim et al., 2014), HIF1-α (Lord-Dufour et al. al., 2009), Hlf, Runxlt1, Pbx1, Lmo2, Zfp37, Prdm5 (Riddell et al., 2014) or Bcl-6 (Ichii, Sakamoto et al., 2004).

[0085] In some embodiments, the cargo may be a recombinant polypeptide intended for nuclear delivery, such as a nuclease useful for genome editing techniques. In some embodiments, the nuclease can be an RNA-guided endonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, CRISPR-associated protein 9 (Cas9), Cpf1 (Zetsche et al., 2015), CasX and / or CasY (Burstein et al., 2016), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) (Cox et al., 2015), a homing endonuclease, a meganuclease, a DNA-guided nuclease such as Natronobacterium gregorii argonaute (NgAgo; Gao et al., 2016), or any combination thereof. In some embodiments, the nuclease may be a catalytically dead endonuclease, such as catalytically dead CRISPR-associated protein 9 (dCas9), dCpf1, dCasX, dCasY, or any combination thereof. Nevertheless, other nucleases not explicitly mentioned herein may also be encompassed by this description. In some embodiments, the nuclease may be fused to a nuclear localization signal (e.g., Cas9-NLS, Cpf1-NLS, ZFN-NLS, TALEN-NLS). In some embodiments, the nuclease may be complexed with a nucleic acid (e.g., one or more guide RNAs, crRNA, tracrRNA, or both crRNA and tracrRNA). In some embodiments, the nuclease may have DNA or RNA binding activity but lack the ability to cleave DNA.

[0086] In some embodiments, the shuttle agents described herein may be used for intracellular delivery (e.g., nuclear delivery) of one or more CRISPR endonucleases, such as one or more CRISPR endonucleases described below.

[0087] Type I and its subtypes A, B, C, D, E, F, and I, which include the respective Cas1, Cas2, Cas3, Cas4, Cas6, Cas7, and Cas8 proteins, as well as their signature homologs and subunits, including the Cse1, Cse2, Cas7, Cas5, and Cas6e subunits in Escherichia coli (E. coli) (type IE) and the Csy1, Csy2, Csy3, and Cas6f subunits in Pseudomonas aeruginosa (type IF) (Wiedenheft et al., 2011; Makarova et al., 2011). Type II and its subtypes A, B, and C, which include the respective Cas1, Cas2, and Cas9 proteins, as well as their signature homologs and subunits, including the Csn complex (Makarova et al., 2011).

[0088] Type III and its subtypes A, B, and MTH326-like modules contain the respective Cas1, Cas2, Cas6, and Cas10 proteins, as well as signature homologs and subunits of these Cas proteins, including the Csm and CMR complexes (Makarova et al., 2011). Type IV represents the Csf3 family of Cas proteins. Members of this family appear near CRISPR repeats in Acidithiobacillus ferrooxidans ATCC 23270, Azoarcus species (strain EbN1), and Rhodoferax ferrireducens (strain DSM15236 / ATCC BAA-621 / T118). In the latter two species, the CRISPR / Cas loci are found on plasmids. Type V and its subtypes were discovered more recently and include Cpf1, C2c1 and C2c3. Type VI contains the enzyme C2c2, which is reported to share little homology to known sequences.

[0089] In some embodiments, the shuttle agent described herein can be used with one or more of the above-mentioned nucleases, endonucleases, RNA-guided endonucleases, and CRISPR endonucleases for various applications, such as those described herein.CRISPR system interacts with its respective nucleic acid, such as DNA binding, RNA binding, helicase and nuclease motif (Makarova et al., 2011; Barrangou & Marraffini, 2014).CRISPR system can be used for various genome editing applications, including: Cas-mediated genome editing methods that perform non-homologous end joining (NHEJ) and / or homologous recombination (HDR) (Cong et al., 2013), catalytically dead Cas (dCas), which can repress and / or activate transcription initiation when bound to a promoter sequence, one or several gRNAs, and an RNA polymerase, with or without complex formation with other protein partners (Bikard et al., 2013); Catalytically dead Cas (dCas) can also be fused to various functional protein domains as a way to deliver enzymatic activity to specific sites in the genome, including transcriptional repression, transcriptional activation, chromatin remodeling, fluorescent reporters, histone modification, recombinase systems, acetylation, methylation, ubiquitination, phosphorylation, sumoylation, ribosylation, and citrullination (Gilbert et al., 2013).

[0090] Those skilled in the art will understand that while the present shuttle agents are exemplified in this example using Cas9 and Cpf1, they can be used with other nucleases as described herein. Thus, nucleases such as Cpf1, Cas9, and variants of such nucleases or others are encompassed by the present description. In one aspect, it should be understood that the present description broadly encompasses any cargo with nuclease activity, such as an RNA-guided endonuclease, or a variant thereof (e.g., one that can bind to DNA or RNA but has lost nuclease activity; or one that is fused to a transcription factor).

[0091] In some embodiments, the polypeptide cargo may be a cytokine, such as a chemokine, interferon, interleukin, lymphokine, or tumor necrosis factor. In some embodiments, the polypeptide cargo may be a hormone or growth factor. In some embodiments, the cargo may be an antibody (e.g., a labeled antibody, a therapeutic antibody, an anti-apoptotic antibody, an antibody that recognizes an intracellular antigen). In some embodiments, the cargo may be a detectable label (e.g., a fluorescent polypeptide or a reporter enzyme) intended for intracellular delivery for research and / or diagnostic purposes.

[0092] In some embodiments, the cargo may be a globular or fibrous protein. In some embodiments, the cargo may have a molecular weight of any one of about 5, 10, 15, 20, 25, 30, 35, 40, 45-50 to about 150, 200, 250, 300, 350, 400, 450, 500 kDa or more. In some embodiments, the cargo may have a molecular weight of about 20-200 kDa.

[0093] In some embodiments, the polypeptide cargo may be a peptide cargo, such as a peptide that recognizes an intracellular molecule.

[0094] In some embodiments, the polypeptide cargo may be an enzyme and / or an enzyme inhibitor.

[0095] In some embodiments, the described peptide shuttle agents are useful for delivering polypeptide cargos from the extracellular space to the cytosol and / or nucleus of various types of target eukaryotic cells, and the synthetic peptide shuttle agents are used at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to that in the absence of the synthetic peptide shuttle agent. The target eukaryotic cells can be animal, mammalian, or human cells. In some embodiments, the target eukaryotic cells can 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), or immune cells (e.g., NK cells, T cells, dendritic cells, antigen-presenting cells). It will be appreciated that cells that are often resistant or receptive to protein transduction may be interesting candidates for the described synthetic peptide or polypeptide-based shuttle agents.

[0096] Non-toxic, metabolizable shuttle agent In some embodiments, the shuttle agents described herein may be non-toxic to target eukaryotic cells of interest at concentrations up to 50 μM, 45 μM, 40 μM, 35 μM, 30 μM, 25 μM, 20 μM, 15 μM, 10 μM, 9 μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 0.5 μM, 0.1 μM, or 0.05 μM. The cytotoxicity of the shuttle agents described herein may be measured using any suitable method. Furthermore, transduction protocols may be adapted to reduce or minimize shuttle agent toxicity and / or improve / maximize transfection efficiency (e.g., shuttle and / or cargo concentration used, shuttle / cargo exposure time, exposure in the presence or absence of serum).

[0097] In some embodiments, the described shuttle agents are readily metabolizable by the intended target eukaryotic cells. For example, the shuttle agents may consist entirely or essentially of peptides or polypeptides for which the target eukaryotic cells possess the cellular machinery to metabolize / degrade the shuttle agents. Indeed, the intracellular half-lives of the described synthetic peptide- and polypeptide-based shuttle agents are predicted to be significantly shorter than the half-lives of exogenous organic compounds, such as fluorophores. However, fluorophores can be toxic and must be investigated before they can be safely used clinically (Alford et al., 2009). In some embodiments, the described shuttle agents may be suitable for clinical use. In some embodiments, the described shuttle agents may avoid the use of domains or compounds whose toxicity is uncertain or not eliminated.

[0098] cocktail In some embodiments, the present description relates to compositions comprising a cocktail of at least two, at least three, at least four, or at least five different types of synthetic peptide or polypeptide-based shuttle agents, as defined herein. In some embodiments, combining different types of synthetic peptide or peptide shuttle agents (e.g., various shuttle agents comprising different types of domains) may provide increased versatility for delivering various polypeptide cargoes into cells. Furthermore, without being bound by theory, combining low concentrations of various types of shuttle agents may help reduce cytotoxicity associated with using a single type of shuttle agent (e.g., at a higher concentration).

[0099] Methods, kits, uses and cells In some embodiments, the present description relates to methods for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, the method comprising contacting the target eukaryotic cell with the polypeptide cargo in the presence of a shuttle agent at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to the absence of the shuttle agent. In some embodiments, contacting the target eukaryotic cell with the polypeptide cargo in the presence of the shuttle agent increases the transduction efficiency of the polypeptide cargo by at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or even 100-fold compared to the absence of the shuttle agent.

[0100] In some embodiments, the present description relates to methods for increasing the transduction efficiency of a polypeptide cargo into the cytosol of a target eukaryotic cell. As used herein, the phrase "increased transduction efficiency" refers to the ability of the presently described shuttle agents to improve the percentage or proportion of a population of target cells in which a cargo of interest (e.g., a polypeptide cargo) is delivered across the cell membrane and into the cell. Immunofluorescence microscopy, flow cytometry, and other suitable methods can be used to assess cargo transduction efficiency. In some embodiments, the presently described shuttle agents can enable a transduction efficiency 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 presently described shuttle agents may enable one of the above transduction efficiencies along with cell viability of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, e.g., as measured by the assay described in Example 3.3a or by another suitable assay known in the art.

[0101] In addition to increasing the efficiency of target cell transduction, the described shuttle agents can facilitate the delivery of a desired cargo (e.g., a polypeptide cargo) to the cytosol of a target cell. In this regard, efficiently delivering extracellular cargo to the cytosol of a target cell using peptides can be challenging, as the cargo is often trapped in intracellular endosomes after crossing the cell membrane, which limits the cargo's intracellular availability and may lead to its eventual metabolic degradation. For example, the use of a protein transduction domain derived from the HIV-1 Tat protein has been reported to result in the bulk sequestration of the cargo in intracellular vesicles. In some embodiments, the described shuttle agents can facilitate the ability of endosomal-trapped cargo to escape from the endosome and reach the cytoplasmic compartment. In this context, the expression "to the cytosol" in the phrase "increasing the transduction efficiency of an independent polypeptide cargo into the cytosol" refers to the ability of the described shuttle agents to enable the intracellularly delivered cargo of interest to escape endosomal trapping and reach the cytoplasmic compartment. After the cargo of interest reaches the cytosol, it may subsequently be targeted to various intracellular compartments (e.g., nucleus, nucleolus, mitochondria, peroxisomes). In some embodiments, the phrase "to the cytosol" is therefore intended to encompass not only cytosolic delivery, but also delivery to other intracellular compartments where the cargo first needs to enter the cytoplasmic compartment.

[0102] In some embodiments, the described methods are in vitro methods. In some embodiments, the described methods are in vivo methods.

[0103] In some embodiments, the methods described herein may include contacting a target eukaryotic cell with a shuttle agent, or a composition as defined herein, and a polypeptide cargo. In some embodiments, the shuttle agent or composition may be pre-incubated with the polypeptide cargo to form a mixture, and then the target eukaryotic cell is exposed to the mixture. In some embodiments, the type of shuttle agent may be selected based on the amino acid sequence of the polypeptide cargo to be delivered into the cell. In other embodiments, the type of shuttle agent may be selected taking into consideration the amino acid sequence of the polypeptide cargo to be delivered into the cell, the cell type, the tissue type, etc.

[0104] In some embodiments, the method may involve multiple treatments of target cells with the shuttle agent or composition (e.g., 1, 2, 3, 4, or more times per day and / or on a predetermined schedule). In such cases, a low concentration 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, one skilled in the art may adapt the teachings described herein to the particular needs of delivering polypeptide cargo to particular cells using different combinations of shuttle agents, domains, uses, and methods.

[0105] In some embodiments, the methods described herein may be applied to intracellular delivery of polypeptide cargo into cells in vivo, which may be achieved by parenteral administration or direct injection into a tissue, organ, or system.

[0106] In some embodiments, the shuttle agent or composition and polypeptide cargo may be exposed to the target cells in the presence or absence of serum. In some embodiments, the method may be suitable for clinical or therapeutic use.

[0107] In some embodiments, the description relates to a kit for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell. In some embodiments, the description relates to a kit for increasing the transduction efficiency of a polypeptide cargo into the cytosol of a target eukaryotic cell. The kit may include a shuttle agent or composition as defined herein and a suitable container.

[0108] In some embodiments, the target eukaryotic cell may be an animal cell, a mammalian cell, or a human cell. In some embodiments, the target eukaryotic cell may be a stem cell (e.g., embryonic stem cell, pluripotent stem cell, induced pluripotent stem cell, neural stem cell, mesenchymal stem cell, hematopoietic stem cell, peripheral blood stem cell), primary cell (e.g., myoblast, fibroblast), or immune cell (e.g., NK cell, T cell, dendritic cell, antigen-presenting cell). In some embodiments, the present description relates to an isolated cell comprising a synthetic peptide- or polypeptide-based shuttle agent as defined herein. In some embodiments, the cell may be a protein-induced pluripotent stem cell. It will be understood that cells that are often resistant or receptive to protein transduction may be interesting candidates for the synthetic peptide- or polypeptide-based shuttle agents described herein.

[0109] In some embodiments, the present description relates to a method for producing a synthetic peptide shuttle agent that delivers a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, the method comprising synthesizing a peptide comprising: (1) at least 20 amino acids in length; (2) an amphipathic alpha-helical motif with a positively charged hydrophilic outer surface, and (3) comprising a hydrophobic outer surface; A peptide that satisfies at least five of the following parameters (4) to (15): (4) the hydrophobic exterior surface comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids representing 12–50% of the peptide's amino acids, based on an open cylinder representation of an alpha helix with 3.6 residues per turn; (5) have a hydrophobic moment (μ) of 3.5 to 11; (6) have a predicted net charge of at least +4 at physiological pH; (7) have an isoelectric point (pI) of 8 to 13; (8) 35% to 65% amino acids consisting of any combination of: A, C, G, I, L, M, F, P, W, Y, and V; (9) 0% to 30% amino acids: composed of any combination of N, Q, S, and T; (10) 35% to 85% amino acids: composed of any combination of A, L, K, or R; (11) composed of any combination of 15% to 45% amino acids: A and L, provided that at least 5% L is present in the peptide; (12) 20% to 45% of amino acids: composed of any combination of K and R; (13) 0% to 10% of the amino acids: composed of any combination of D and E; (14) The difference between the percentage of A and L in the peptide (%A+L) and the percentage of K and R in the peptide (%K+R) is 10% or less; (15) 10% to 45% of the amino acids are composed of any combination of Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[0110] In some embodiments, the present description relates to a method for identifying a shuttle agent that delivers a polypeptide cargo from the 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 polypeptide cargo in the presence of the peptide; (c) measuring the transduction efficiency of the polypeptide cargo in the target eukaryotic cell; and (d) identifying the peptide as a shuttle agent that transduces the polypeptide cargo if an increase in the transduction efficiency of the polypeptide cargo in the target eukaryotic cell is observed.

[0111] In some embodiments, the present description relates to a genome editing system, the system comprising: (a) a shuttle agent as defined herein; (b) a CRISPR-binding endonuclease; and (c) one or more guide RNAs. In some embodiments, the genome editing system may further comprise a linear DNA template that controls genome editing.

[0112] Genome editing for improved cell therapy In some embodiments, the shuttle agents, synthetic peptides, compositions, and methods described herein can be used to transduce genome editing complexes (e.g., CRISPR-based genome editing complexes) into engineered cells for improved cell therapy compared to native or non-engineered cells. Such improvements can include, for example, reducing the immunogenicity of the engineered cells and / or improving the activity / efficacy of the engineered cells.

[0113] Given their natural ability to recognize and kill tumor cells, natural killer (NK) cells are particularly attractive immune cells for genome engineering. Thus, in some embodiments, the present description relates to the use of shuttle agents, synthetic peptides, compositions, and methods described herein to transduce genome editing complexes (e.g., CRISPR-based genome editing complexes) to genetically engineer NK cells (or other immune cells that may benefit from the same modification) for the purpose of improving cell-based immunotherapy. For example, the present description may relate to the intracellular delivery of one or more CRISPR-based genome editing complexes containing guide RNAs and / or linear DNA templates targeting the CBLB gene, c-CBL gene, GSK3 gene, ILT2 gene, CISH gene, NKG2a gene, B2M gene, or any combination thereof. Such gene targets may enhance NK-mediated cytotoxic activity after knockout, as discussed below.

[0114] 1. NKG2A (KLRC1, CD159A, killer cell lectin-like receptor C1) CD94 / NKG2A functions as an MHC class I-specific NK inhibitory receptor (Braud et al., 1998; Lee et al., 1998). bright CD16 dim NKG2A is expressed by a subset of NK cells known as NKG2A (approximately 10% of peripheral NK cells), which typically have low cytotoxicity (Cooper et al., 2001; Poli et al., 2009). NKG2A ligands are non-classical MHC class I HLA-E molecules, which are expressed on all human cells. Recognition of HLA-E by NKG2A receptors is part of the "self-tolerance" mechanism (which also involves KIR receptors) and results in the negative regulation of NK cytotoxicity (Lee et al., 1998).

[0115] Clinical evidence exists demonstrating the role of non-classical HLA class I, primarily HLA-E and HLA-G (see ILT-2 targets), in escaping immune surveillance and resulting in more aggressive cancer recurrence and reduced overall survival after surgery (de Kruijf et al., 2010; Levy et al., 2008; Ye et al., 2007a; Yie et al., 2007b; Yie et al., 2007c; Yie et al., 2007d; Guo et al., 2015; Ishigami et al., 2015; Zhen et al., 2013). The use of NKG2A-KO NK cells during adoptive cell therapy may attenuate the impact of the presence of HLA-E molecules (membrane-bound or soluble) in the tumor microenvironment. Furthermore, NK cells expanded from IL15- or IL12-expressing K562 feeder cells exhibited NKG2A-KO activity. POS It would be desirable to knock out this inhibitory receptor during the expansion process, resulting in NKG2A-KO cells (Denman et al., 2012). Furthermore, the results in Example G.9 show that NKG2A-KO NK92 cells are highly cytotoxic to IFNγ-treated HeLa cells.

[0116] 2. ILT2 (Ig-like transcript 2 gene) ILT2 is an inhibitory receptor expressed on several immune cells, including NK cells (Kirwan et al., 2005). The ligand for this receptor is the HLA-G molecule, which is naturally expressed only in the thymus and trophoblasts. However, many tumors acquire the ability to express HLA-G to avoid immune cell attack through ILT2 receptor activation-mediated suppression. Indeed, NK cells ILT2- HLA-G-mediated NK cell-mediated cytotoxicity was also significantly reduced in OVCAR-3 cancer cells (Lin et al., 2007). Similar to HLA-E, HLA-G expression on cancer cells is generally associated with poor prognosis.

[0117] 3. c-Cbl and Cbl-B (Casitas B-lineage lymphoma proto-oncogene family). These genes (Cassitas B-lineage lymphoma proto-oncogene, Cbl family members) encode E3 ligases and function in the protein ubiquitination pathway (regulating cytoplasmic protein content). E3 ligases catalyze the formation of covalent bonds between Ub (ubiquitin) and specific lysine residues on target proteins (there are over 1,000 E3 ligases in mammals). Cbl family members are involved in the negative regulation of receptor tyrosine kinase signaling in immune cells by binding and ubiquitinating phosphorylated receptors and adaptors (Liu et al., 2014; Utz-Nicoladoni, 2015). Both c-cbl and Cbl-b have been shown to ubiquitinate phosphorylated LAT adaptors. Phosphorylation of LAT after NK cell activation is required to recruit other mediators, particularly PLC-γ, and siRNA-mediated c-cbl and Cbl-b knockdown enhanced NK cell activity against B-cell lymphoma 721.221-Cw4 ( Matalon et al., 2016 ).

[0118] Other TAM receptors (Tyro3, ​​Axl, Mer) have been identified as targets of Cbl-b ubiquitination (Paolino et al., 2014). However, if TAM receptors negatively regulate NK cells, Cbl-b knockout should instead be associated with decreased NK cell activity. Therefore, TAM receptors may be considered good targets for enhancing NK cells, but this seems unlikely via Cbl-b knockout.

[0119] In vivo studies, Cbl-b - / - Mice were shown to be protected from primary tumor growth (Loeser et al., 2007). Furthermore, NK cells isolated from these mice, upon activation, showed increased proliferation and IFN-γ production (Paolino et al., 2014).

[0120] 4. GSK3B (Glycogen synthase kinase beta) GSK3b is a Ser / Thr kinase involved in several cellular functions, such as proliferation, apoptosis, inflammatory responses, and stress (Patel et al., 2017). Inhibition of GSK3b in NK cells (using small inhibitors) leads to increased cytotoxicity against AML (OCI-AML3), likely through increased IFN-γ and IFN-α production, 2B4 stimulation, and LFA-1 expression (Parameswaran et al., 2016; Aoukaty et al., 2005). We recently showed that the GSK3β inhibitor SB216763 enhances the cytotoxic activity of NK92 against HeLa cells (data not shown). This effect is enhanced by co-incubation with IL-15.

[0121] 5. CISH (cytokine-inducible SH2-containing proteins) CIS proteins are members of the suppressor of cytokine signaling (SOCS) family, which bind to phosphorylated JAKs and inhibit the JAK-STAT signaling pathway. - / - Mice have shown that CIS is a key suppressor of IL15 signaling (Delconte et al., 2016). After IL15 exposure, these cells exhibited prolonged IL15 responses, elevated IFN-γ production, and increased cytotoxicity. Furthermore, there is a clear correlation between IL15 responsiveness and NKG2D-dependent cytotoxicity (Horng et al., 2007).

[0122] In clinical trials, co-infusion of cytokines such as IL2 and IL15 is strongly recommended to sustain NK cell activity during adoptive NK cell therapy. However, such co-infusion induces serious side effects in patients. IL15-hypersensitive NK cells (CISH knockout) would be beneficial for the treatment.

[0123] In some embodiments, disruption of the B2M gene, which encodes beta-2 microglobulin (B2M), a component of MHC class I receptors, can substantially reduce the immunogenicity of all cells expressing MHC class I. In other aspects, the genome of NK cells can be modified after delivery of a genome editing system as described herein. More specifically, the cytotoxicity of NK cells can be improved after delivery of a genome editing system that targets specific putative targets that can enhance NK-mediated cytotoxic activity, such as the NKG2A, ILT2, c-Cbl, Cbl-b, GSK3B, and CISH genes.

[0124] Co-transduction with a polypeptide cargo of interest and a marker protein The ability of domain-based rationally designed peptide shuttle agents to simultaneously transduce two polypeptide cargoes into a target eukaryotic cell population has been demonstrated herein and in WO 2016 / 161516. Example I herein demonstrates that simultaneously transducing a polypeptide cargo of interest (e.g., a CRISPR-endonuclease) and an independent marker protein (e.g., GFP) into a target eukaryotic cell population does not necessarily increase the overall transduction efficiency of the polypeptide cargo of interest. Surprisingly, however, it was discovered that a significantly higher proportion of target eukaryotic cells successfully transduced with the polypeptide cargo of interest also successfully transduced with the marker protein. Conversely, a significantly higher proportion of cells that were not transduced with the polypeptide cargo of interest also did not transduce the marker protein. Isolation of marker protein-positive cells (e.g., by FACS) greatly increased the proportion of cells successfully transduced with the polypeptide cargo of interest, and the correlation was found to be concentration-dependent, in that the cell population exhibiting the highest marker protein fluorescence also exhibited the highest transduction proportion of the polypeptide cargo of interest.

[0125] In some aspects, the present description relates to methods for enriching eukaryotic cells transduced with a polypeptide cargo of interest. The methods may include (a) co-transducing a target eukaryotic cell population with the polypeptide cargo of interest and a marker protein; and (b) isolating or enriching for eukaryotic cells transduced with the marker protein, thereby enriching for eukaryotic cells transduced with the polypeptide cargo of interest.

[0126] In some embodiments, the marker protein may not be covalently linked to the polypeptide cargo of interest (e.g., the marker protein is independent of the polypeptide cargo of interest), the marker protein may be covalently linked to the polypeptide cargo of interest, the marker protein may be non-covalently linked to the polypeptide cargo of interest (e.g., electrostatically and / or conformationally bound via protein domain-protein domain interactions), or the marker protein may be covalently linked to the polypeptide cargo of interest via a cleavable linker (e.g., a linker peptide comprising a cleavage site by an enzyme, such as an enzyme expressed in endosomes, lysosomes, or in the cytosol). In some embodiments, the intracellular concentration of the transduced marker protein may positively correlate with the intracellular concentration of the transduced polypeptide cargo of interest.

[0127] In some embodiments, the marker protein may comprise a detectable label. As used herein, a "detectable label" refers to a molecule or particle that allows one skilled in the art to identify and separate cells containing the label from cells that lack the label. In some embodiments, the marker protein may be a fluorescent protein, a fluorescently labeled protein, a bioluminescent protein, an isotopically labeled protein, a magnetically labeled protein, or another detectable label that allows cells containing the marker protein to be separated from cells that lack the marker protein, or that allows cells to be separated based on the level of the marker protein in the cells.

[0128] In some embodiments, eukaryotic cells transduced with a marker protein can be isolated or enriched using flow cytometry, fluorescence-activated cell sorting (FACS), magnetic cell sorting (MACS), or other known cell / sorting techniques. Successful isolation or enrichment of transduced cells can be particularly advantageous, for example, in the case of polypeptide cargoes such as functional CRISPR-based genome editing complexes, which can result in relatively low transduction efficiency.

[0129] It was also surprisingly disclosed in Example I herein that cells that fail to transduce after a first round of transduction with a polypeptide cargo of interest can be isolated and re-transduced with the polypeptide cargo of interest in a subsequent transduction round. These results suggest that cells that fail to transduce after a first round of transduction with a polypeptide cargo of interest are not necessarily refractory to subsequent transduction.

[0130] In some embodiments, the present description relates to a method for enriching eukaryotic cells transduced with a polypeptide cargo of interest, the method comprising: (a) co-transducing a target eukaryotic cell population with the polypeptide cargo of interest and a marker protein; and (b) isolating the eukaryotic cells transduced with the marker protein from cells lacking the marker protein, thereby producing a marker protein-positive cell population and a marker protein-negative cell population. In some embodiments, steps (a) and (b) may be repeated one or more times on the marker protein-negative cell population, the marker protein-positive cell population, or both the marker protein-negative and marker protein-positive cell populations. In some embodiments, the method for enriching eukaryotic cells transduced with a polypeptide cargo of interest may be automated, for example, by isolating cells negative for marker protein transduction and re-transducing the marker protein-negative cell population.

[0131] In some embodiments, the present description relates to a method for enriching eukaryotic cells transduced with a polypeptide cargo of interest, the method comprising: (a) co-transducing a target eukaryotic cell population with the polypeptide cargo of interest and a marker protein; and (b) isolating eukaryotic cells transduced with the marker protein based on their intracellular concentration of the marker protein.

[0132] In some embodiments, the marker proteins described herein can be proteins that stimulate cell proliferation (e.g., growth factors or transcription factors), proteins that stimulate cell differentiation, proteins that promote cell survival, anti-apoptotic proteins, or proteins with another biological activity.

[0133] In some embodiments, a polypeptide cargo of interest and a marker protein can be co-transduced by contacting a target eukaryotic cell with the polypeptide cargo and the marker protein in the presence of a peptide transduction agent, the peptide transduction agent being present at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo and the marker protein compared to the transduction efficiency in the absence of the peptide transduction agent. In some embodiments, the peptide transduction agent can be an endosomolytic peptide. In some embodiments, the peptide transduction agent is or comprises a domain-based peptide shuttle agent, or a rationally designed peptide shuttle agent of WO 2016 / 161516 and / or U.S. Pat. No. 9,738,687, as defined herein. In some embodiments, the domain-based peptide shuttle agent can be a synthetic peptide comprising an endosomal leakage domain (ELD) operably linked to a plasma membrane permeable domain (CPD), or a histidine-rich domain and an ELD operably linked to a CPD. In some embodiments, the domain-based peptide shuttle agents described above: (a) comprise a minimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, and a maximum length of 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues; (b) have a predicted net charge of at least +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, or +15 at physiological pH; (c) are soluble in aqueous solution; or (d) any combination of (a)-(c). In some embodiments, the ELD, CPD, histidine-rich domain, and linker domain are as defined herein. In some embodiments, the target eukaryotic cell comprises or consists of an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, a NK cell, a dendritic cell, or other cell type or cell subtype.

[0134] In some embodiments, the polypeptide cargo of interest may be (i) a polypeptide cargo as defined herein; and / or (ii) one or more CRISPR-binding endonucleases alone or in conjunction with one or more corresponding guide RNAs and / or linear DNA templates as defined herein.

[0135] item In some embodiments, the description may relate to the following: 1. A method for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, said method comprising contacting the target eukaryotic cell with a polypeptide cargo in the presence of a shuttle agent at a concentration sufficient to increase the transduction efficiency of said polypeptide cargo compared to that in the absence of said shuttle agent, wherein said shuttle agent: (1) at least 20 amino acids in length; (2) an amphipathic alpha-helical motif with a positively charged hydrophilic outer surface, and (3) comprising a hydrophobic outer surface; A peptide that satisfies at least five of the following parameters (4) to (15): (4) the hydrophobic exterior surface comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids representing 12–50% of the peptide's amino acids, based on an open cylinder representation of an alpha helix with 3.6 residues per turn; (5) have a hydrophobic moment (μ) of 3.5 to 11; (6) have a predicted net charge of at least +4 at physiological pH; (7) have an isoelectric point (pI) of 8 to 13; (8) 35% to 65% amino acids consisting of any combination of: A, C, G, I, L, M, F, P, W, Y, and V; (9) 0% to 30% amino acids: composed of any combination of N, Q, S, and T; (10) 35% to 85% amino acids: composed of any combination of A, L, K, or R; (11) composed of any combination of 15% to 45% amino acids: A and L, provided that at least 5% L is present in the peptide; (12) 20% to 45% of amino acids: composed of any combination of K and R; (13) 0% to 10% of the amino acids: composed of any combination of D and E; (14) The difference between the percentage of A and L in the peptide (%A+L) and the percentage of K and R in the peptide (%K+R) is 10% or less; (15) 10% to 45% of the amino acids are composed of any combination of Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[0136] 2. The method of claim 1, wherein the shuttle agent satisfies at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all of the parameters (4) through (15) described herein.

[0137] 3. (i) the shuttle agent is a peptide having a minimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids; (ii) the amphipathic alpha helix motif is 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 , 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, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0; (iii) the amphipathic alpha-helical motif comprises a positively charged hydrophilic outer surface, which comprises (a) at least two, three, or four adjacent positively charged K and / or R residues on a helical wheel projection; and / or (b) a segment of six adjacent residues on a helical wheel projection, which includes three to five K and / or R residues, based on an alpha helix having a rotation angle between consecutive amino acids of 100 degrees and / or 3.6 residues per turn; (iv) the amphipathic alpha-helical motif comprises a hydrophobic exterior, the hydrophobic exterior comprising: (a) at least two adjacent L residues on a helical wheel projection; and / or (b) a segment of 10 adjacent residues comprising at least five hydrophobic residues selected from L, I, F, V, W, and M, based on an alpha helix having a rotation angle between consecutive amino acids of 100 degrees and / or 3.6 residues per turn on a helical wheel projection; (v) the hydrophobic exterior surface comprises a highly hydrophobic core composed 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 of the peptide; (vi) the peptide has a hydrophobic moment (μ) between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5; (vii) the peptide has a predicted net charge of +4, +5, +6, +7, +8, +9 to +10, +11, +12, +13, +14, or +15; (viii) the peptide has a predicted pI of 10 to 13; or (ix) The method according to item 1 or 2, wherein the method is any combination of (i) to (viii).

[0138] 4. The method of any one of items 1 to 3, wherein the shuttle agent satisfies at least one, at least two, at least three, at least four, at least five, at least six, or all of the following parameters: (8) the peptide is composed of any combination of 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40% to 60% of the amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (9) the peptide is composed of any combination 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 the amino acids: N, Q, S, and T; (10) The peptide is composed of 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to 60% of the amino acids: A, L, K, or R in any combination; (11) The peptide is composed of any combination of 15% to 40%, 20% to 40%, 20% to 35%, or 20% to 30% of the amino acids: A and L; (12) The peptide is composed of any combination of 20% to 40%, 20% to 35%, or 20% to 30% of the amino acids: K and R; (13) 5% to 10% of amino acids: composed of any combination of 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 / or R residues in the peptide (%K+R) is 9%, 8%, 7%, 6%, or 5% or less; and (15) The peptide is composed of any combination of 15-40%, 20-35%, or 20-30% of the amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[0139] 5. The method according to any one of items 1 to 4, wherein the peptide comprises a histidine-rich domain.

[0140] 6. The histidine-rich domain is (i) located towards the N-terminus and / or C-terminus of the peptide; (ii) a stretch of at least 3, at least 4, at least 5, at least 6 amino acids containing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues, and / or containing 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) The method according to item 5, wherein both (i) and (ii) are employed.

[0141] 7. The method according to any one of items 1 to 6, wherein the peptide comprises a flexible linker domain enriched in serine and / or glycine residues.

[0142] 8. The method according to any one of items 1 to 7, wherein the peptide comprises or consists of the following amino acid sequence: (a) [X1]-[X2]-[linker]-[X3]-[X4] (Formula 1); (b) [X1]-[X2]-[linker]-[X4]-[X3] (Formula 2); (c) [X2]-[X1]-[linker]-[X3]-[X4] (Formula 3); (d) [X2]-[X1]-[linker]-[X4]-[X3] (Formula 4); (e) [X3]-[X4]-[linker]-[X1]-[X2] (Formula 5); (f) [X3]-[X4]-[linker]-[X2]-[X1] (Formula 6); (g) [X4]-[X3]-[linker]-[X1]-[X2] (Formula 7); or (h) [X4]-[X3]-[linker]-[X2]-[X1] (Formula 8); During the ceremony, [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 the following: -4[+]-A-; -3[+]-G-A-; -3[+]-A-A-; -2[+]-1[Φ]-1[+]-A-; -2[+]-1[Φ]-G-A-; -2[+]-1[Φ]-A-A-; or -2[+]-A-1[+]-A; -2[+]-A-G-A; -2[+]-A-A-A-; -1[Φ]-3[+]-A-; -1[Φ]-2[+]-G-A-; -1[Φ]-2[+]-A-A-; -1[Φ]-1[+]-1[Φ]-1[+]-A; -1[Φ]-1[+]-1[Φ]-G-A; -1[Φ]-1[+]-1[Φ]-A-A; -1[Φ]-1[+]-A-1[+]-A; -1[Φ]-1[+]-A-G-A; -1[Φ]-1[+]-A-A-A; -A-1[+]-A-1[+]-A; -A-1[+]-A-G-A; and -A-1[+]-A-A-A; [X4] is selected from the following: -1[ζ]-2A-1[+]-A; -1[ζ]-2A-2[+]; -1[+]-2A-1[+]-A; -1[ζ]-2A-1[+]-1[ζ]-A-1[+]; -1[ζ]-A-1[ζ]-A-1[+]; -2[+]-A-2[+]; -2[+]-A-1[+]-A; -2[+]-A-1[+]-1[ζ]-A-1[+]; -2[+]-1[ζ]-A-1[+]; -1[+]-1[ζ]-A-1[+]-A; -1[+]-1[ζ]-A-2[+]; -1[+]-1[ζ]-A-1[+]-1[ζ]-A-1[+]; -1[+]-2[ζ]-A-1[+]; -1[+]-2[ζ]-2[+]; -1[+]-2[ζ]-1[+]-A; -1[+]-2[ζ]-1[+]-1[ζ]-A-1[+]; -1[+]-2[ζ]-1[ζ]-A-1[+]; -3[ζ]-2[+]; -3[ζ]-1[+]-A; -3[ζ]-1[+]-1[ζ]-A-1[+]; -1[ζ]-2A-1[+]-A; -1[ζ]-2A-2[+]; -1[ζ]-2A-1[+]-1[ζ]-A-1[+]; -2[+]-A-1[+]-A; -2[+]-1[ζ]-1[+]-A; -1[+]-1[ζ]-A-1[+]-A; -1[+]-2A-1[+]-1[ζ]-A-1[+]; and -1[ζ]-A-1[ζ]-A-1[+]; and [Linker] is selected from the following: -Gn-; -Sn-; -(GnSn)n-; -(GnSn)nGn-; -(GnSn)nSn-; -(GnSn)nGn(GnSn)n-; and -(GnSn)nSn(GnSn)n-; During the ceremony, [Φ] is the amino acid: Leu, Phe, Trp, Ile, Met, Tyr, or Val; [+] is the amino acid: Lys or Arg; [ζ] is the amino acid: Gln, Asn, Thr, or Ser; A is the amino acid Ala; G is the amino acid: Gly; S is the amino acid Ser; and n is an integer of 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.

[0143] 9. (i) the peptide 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 NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242; or the peptide comprises or consists of a functional variant of any one of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242; (ii) the peptide is: (a) comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242; (b) comprising the amino acid sequence motif of SEQ ID NO: 158 and / or 159; or (c) comprising the amino acid sequence motif of SEQ ID NO: 158 operably linked to the amino acid sequence motif of SEQ ID NO: 159; or (iii) The method according to any one of items 1 to 8, wherein both (i) and (ii) are employed.

[0144] 10. The method according to any one of items 1 to 9, wherein the peptide comprises an endosomal leakage domain (ELD) and / or a cell-permeable domain (CPD).

[0145] 11. (i) the ELD is or is derived from an endosomolytic peptide, an antimicrobial peptide (AMP), a linear cationic alpha-helical antimicrobial peptide, a cecropin-A / melittin hybrid (CM series) peptide, a pH-dependent membrane-active peptide (PAMP), a peptide amphiphile, a peptide derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA), CM18, diphtheria toxin T domain (DT), GALA, PEA, INF-7, LAH4, HGP, H5WYG, HA2, EB1, VSVG, Pseudomonas toxin, melittin, KALA, 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, penetratin (antennapedia), pVEC, M918, Pep-1, Pep-2, Xentry, an arginine stretch, transportan, SynB1, SynB3, or any combination thereof; or (iii) The method according to item 10, wherein both (i) and (ii) are employed.

[0146] 12. The peptide is (a) an ELD comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 15, 63, or 64, or a variant or fragment thereof having endosomolytic activity; (b) a CPD comprising the amino acid sequence of any one of SEQ ID NOs: 16 to 27 or 65 or a variant or fragment thereof having cell membrane permeability activity; or (c) The method according to item 10 or 11, comprising both (a) and (b).

[0147] 13. (i) the peptide comprises an ELD that is CM18, KALA, or C(LLKK)3C having the amino acid sequence of SEQ ID NO: 1, 14, or 63, or an ELD thereof that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 1, 14, or 63 and has endosomolytic activity; (ii) the peptide comprises a CPD that is TAT ​​or PTD4 having the amino acid sequence of SEQ ID NO: 17 or 65, or a variant thereof that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 17 or 65 and has cell membrane permeabilization activity; or (iii) The method according to any one of items 10 to 12, wherein both (i) and (ii) are employed.

[0148] 14. The method according to any one of items 1 to 9, wherein the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 57 to 59, 66 to 72, or 82 to 102, or a functional variant thereof having at least 85%, 90%, or 95% identity to any one of SEQ ID NOs: 57 to 59, 66 to 72, or 82 to 102.

[0149] 15. (i) the shuttle agent is fully metabolizable by the target eukaryotic cell; and / or (ii) The method of any one of items 1 to 14, wherein contacting the target eukaryotic cell with the polypeptide cargo in the presence of a shuttle agent increases the transduction efficiency of the polypeptide cargo by at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold compared to the transduction efficiency in the absence of the shuttle agent.

[0150] 16. The method according to any one of items 1 to 15, which is an in vitro method.

[0151] 17. A synthetic peptide shuttle agent, which is a peptide as defined in any one of items 1 to 15.

[0152] 18. The synthetic peptide according to item 17, which is a peptide of 20 to 100 amino acids in length and comprises any one of the amino acid sequences of SEQ ID NOs: 104, 105, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 134, 135, 138, 140, 142, 145, 148, 151, 152, 169 to 242, and 243 to 10242, or comprises the amino acid sequence motif of SEQ ID NOs: 158 and / or 159.

[0153] 19. The synthetic peptide shuttle agent of items 17 or 18 for use in in vitro delivery of a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, wherein the synthetic peptide shuttle agent is used at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to the absence of the synthetic peptide shuttle agent.

[0154] 20. The synthetic peptide shuttle agent of items 17 or 18 for use in the in vivo delivery of a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, wherein the synthetic peptide shuttle agent is used at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to the absence of the synthetic peptide shuttle agent.

[0155] 21. A composition comprising a shuttle agent as defined in any one of items 1 to 15, or a cocktail of at least two, at least three, at least four, at least five different types of shuttle agents as defined in any one of items 1 to 15, and a polypeptide cargo to be delivered from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell.

[0156] 22. Use of a shuttle agent as defined in any one of items 1 to 15, or a synthetic peptide as defined in item 18, for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, wherein the shuttle agent or synthetic peptide is used at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to that in the absence of the shuttle agent or synthetic peptide.

[0157] 23. A kit for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, comprising a shuttle agent as defined in any one of items 1 to 15, or a synthetic peptide as defined in item 18, and a suitable container.

[0158] 24. The method according to any one of items 1 to 16, the synthetic peptide shuttle agent according to any one of items 17 to 20, the composition according to item 21, the use according to item 22, or the kit according to item 23, wherein the polypeptide cargo lacks a cell membrane-permeable domain.

[0159] 25. The method according to any one of items 1 to 16, the synthetic peptide shuttle agent according to any one of items 17 to 20, the composition according to item 21, the use according to item 22, or the kit according to item 23, wherein the polypeptide cargo comprises a cell membrane-permeable domain.

[0160] 26. The method of any one of items 1 to 16, 24 or 25, the synthetic peptide shuttle agent of any one of items 17 to 20, 24 or 25, the composition of item 21, 24 or 25, or the use of any one of items 22 to 25, wherein the polypeptide cargo comprises an intracellular targeting domain.

[0161] 27. The intracellular targeting domain is (a) nuclear localization signal (NLS); (b) nucleolar signal sequence; (c) a mitochondrial signal sequence; or 27. The method, composition, use, or kit according to Item 26, wherein (d) is a peroxisomal signal sequence.

[0162] 28. (a) The NLS is derived from E1a, T-Ag, c-myc, T-Ag, op-T-NLS, Vp3, nucleoplasmin, histone 2B, Xenopus N1, PARP, PDX-1, QKI-5, HCDA, H2B, v-Rel, Amida, RanBP3, Pho4p, LEF-1, TCF-1, BDV-P, TR2, SOX9, or Max; (b) the nucleolar signal sequence is derived from BIRC5 or RECQL4; (c) the mitochondrial signal sequence is derived from Tim9 or yeast cytochrome c oxidase subunit IV, or (d) The method, synthetic peptide shuttle agent, composition, use, or kit according to item 27, wherein the peroxisomal signal sequence is derived from PTS1.

[0163] 29. The method, synthetic peptide shuttle agent, composition, use, or kit according to any one of items 24 to 29, wherein the polypeptide cargo is complexed to a DNA and / or RNA molecule.

[0164] 30. The method, synthetic peptide shuttle agent, composition, use, or kit according to any one of items 24 to 29, wherein the polypeptide cargo is a transcription factor, a nuclease, a cytokine, a hormone, a growth factor, an antibody, a peptide cargo, an enzyme, an enzyme inhibitor, or any combination thereof.

[0165] 31. (a) the transcription factor is HOXB4, NUP98-HOXA9, Oct3 / 4, Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1, Ngn3, MafA, Blimp-1, Eomes, T-bet, FOXO3A, NF-YA, SALL4, ISL1, FoxA1, Nanog, Esrrb, Lin28, HIF1-alpha, Hlf, Runx1t1, Pbx1, Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof; (b) the nuclease is catalytically active or catalytically dead: RNA-guided endonuclease, CRISPR endonuclease, Type I CRISPR endonuclease, Type II CRISPR endonuclease, Type III CRISPR endonuclease, Type IV CRISPR endonuclease, Type V CRISPR endonuclease, Type VI CRISPR endonuclease, CRISPR-associated protein 9 (Cas9), Cpf1, CasY, CasX, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), homing endonuclease or meganuclease, DNA-guided nuclease, Natronobacterium gregorii argonaute (NgAgo), or any combination thereof; (c) the antibody recognizes an intracellular antigen; and / or (d) The method, synthetic peptide shuttle agent, composition, use, or kit according to item 30, wherein the peptide cargo recognizes an intracellular molecule.

[0166] 32. The method, synthetic peptide shuttle agent, composition, use, or kit according to any one of items 24 to 31 for use in cell therapy, genome editing, adoptive cell transfer, and / or regenerative medicine.

[0167] 33. The method, synthetic peptide shuttle agent, composition, use, or kit according to any one of items 24 to 32, wherein the target eukaryotic cell is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, an NK cell, or a dendritic cell.

[0168] 34. A eukaryotic cell comprising a shuttle agent as defined in any one of items 1 to 15, a synthetic peptide shuttle agent as defined in item 18, or a composition as defined in item 21.

[0169] 35. The eukaryotic cell according to item 34, which is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, an NK cell, or a dendritic cell.

[0170] 36. A method for delivering one or more CRISPR-bound endonucleases, alone or together with one or more corresponding guide RNAs and / or linear DNA templates, to a target eukaryotic cell, comprising contacting the target eukaryotic cell with the endonuclease in the presence of a shuttle agent at a concentration sufficient to increase the transduction efficiency of the endonuclease compared to the absence of the shuttle agent, wherein the shuttle agent is as defined in any one of items 1 to 15.

[0171] 37. The method according to item 36, which is an in vitro method or an in vivo method.

[0172] 38. The method of item 36 or 37, wherein the one or more nucleases are a Type I CRISPR endonuclease, a Type II CRISPR endonuclease, a Type III CRISPR endonuclease, a Type IV CRISPR endonuclease, a Type V CRISPR endonuclease, a Type VI CRISPR endonuclease, or any combination thereof.

[0173] 39. The method of item 36 or 37, wherein the one or more endonucleases are CRISPR-associated protein 9 (Cas9), Cpf1, CasX, CasY, or any combination thereof; or catalytically-dead CRISPR-associated protein 9 (dCas9), dCpf1, dCasX, dCasY, or any combination thereof.

[0174] 40. The method according to any one of items 36 to 39, wherein the target eukaryotic cell is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, a NK cell, or a dendritic cell.

[0175] 41. The method of item 58, wherein the one or more corresponding guide RNAs and / or linear DNA templates target one or more genes to reduce immunogenicity, improve cytotoxicity, and / or otherwise improve efficacy for cell therapy of the target eukaryotic cells compared to corresponding parent eukaryotic cells that have not been subjected to the method.

[0176] 42. The method of item 41, wherein the cell therapy is a cell-based cancer immunotherapy.

[0177] 43. The method of any one of items 40 to 42, wherein the one or more corresponding guide RNAs and / or linear DNA templates target the CBLB gene, the c-CBL gene, the GSK3 gene, the ILT2 gene, the CISH gene, the NKG2a gene, the B2M gene, or any combination thereof.

[0178] 44. A method for producing a synthetic peptide shuttle agent that delivers a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, the method comprising synthesizing a peptide comprising: (1) at least 20 amino acids in length; (2) an amphipathic alpha-helical motif with a positively charged hydrophilic outer surface, and (3) comprising a hydrophobic outer surface; A peptide that satisfies at least five of the following parameters (4) to (15): (4) the hydrophobic exterior surface comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids representing 12–50% of the peptide's amino acids, based on an open cylinder representation of an alpha helix with 3.6 residues per turn; (5) have a hydrophobic moment (μ) of 3.5 to 11; (6) have a predicted net charge of at least +4 at physiological pH; (7) have an isoelectric point (pI) of 8 to 13; (8) 35% to 65% amino acids consisting of any combination of: A, C, G, I, L, M, F, P, W, Y, and V; (9) 0% to 30% amino acids: composed of any combination of N, Q, S, and T; (10) 35% to 85% amino acids: composed of any combination of A, L, K, or R; (11) composed of any combination of 15% to 45% amino acids: A and L, provided that at least 5% L is present in the peptide; (12) 20% to 45% of amino acids: composed of any combination of K and R; (13) 0% to 10% of the amino acids: composed of any combination of D and E; (14) The difference between the percentage of A and L in the peptide (%A+L) and the percentage of K and R in the peptide (K+R) is 10% or less; (15) 10% to 45% of the amino acids are composed of any combination of Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[0179] 45. The method according to item 44, wherein the peptide is as defined in any one of items 2 to 15.

[0180] 46. ​​A method for identifying a shuttle agent that delivers a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, comprising: (a) synthesizing a peptide, the peptide being a peptide as defined in any one of items 1 to 15 or 18; (b) contacting the target eukaryotic cell with the polypeptide cargo in the presence of said peptide; (c) measuring the transduction efficiency of the polypeptide cargo in the target eukaryotic cells; and (d) Identifying the peptide as a shuttle agent for transducing said polypeptide cargo if an increase in transduction efficiency of said polypeptide cargo in the target eukaryotic cell is observed.

[0181] 47. The method according to item 46, wherein the polypeptide cargo is as defined in any one of items 24 to 31.

[0182] 48. Genome editing systems, including: (a) a shuttle agent as defined in any one of items 1 to 15 or 18; (b) one or more CRISPR-binding endonucleases; and (c) one or more guide RNAs.

[0183] 49. The genome editing system of item 48, further comprising a linear DNA template that controls genome editing.

[0184] 50. The genome editing system of item 48 or 49, wherein the one or more CRISPR-binding endonucleases are Type I CRISPR endonuclease, Type II CRISPR endonuclease, Type III CRISPR endonuclease, Type IV CRISPR endonuclease, Type V CRISPR endonuclease, Type VI CRISPR endonuclease, CRISPR-binding protein 9 (Cas9), Cpf1, CasX, CasY, or any combination thereof.

[0185] 51. A method for enriching eukaryotic cells transduced with a polypeptide cargo of interest, comprising: (a) co-transducing a target eukaryotic cell population with a polypeptide cargo of interest and a marker protein; and (b) isolating or enriching for eukaryotic cells transduced with the marker protein, thereby enriching for eukaryotic cells transduced with the polypeptide cargo of interest.

[0186] 52. (i) the marker protein is not covalently bound to the polypeptide cargo of interest, the marker protein is covalently bound to the polypeptide cargo of interest, the marker protein is noncovalently bound to the polypeptide cargo of interest, or the marker protein is covalently bound to the polypeptide cargo of interest via a cleavable linker; and / or (ii) The method of item 51, wherein the marker protein comprises a detectable label, or the marker protein is a fluorescent protein, a fluorescently labeled protein, a bioluminescent protein, an isotope-labeled protein, or a magnetically labeled protein.

[0187] 53. The method according to item 51 or 52, wherein the intracellular concentration of the transduction marker protein positively correlates with the intracellular concentration of the transduction polypeptide cargo of interest.

[0188] 54. The method according to any one of items 51 to 53, wherein eukaryotic cells transduced with the marker protein are isolated or enriched using flow cytometry, fluorescence-activated cell sorting (FACS), or magnetic cell sorting (MACS).

[0189] 55. The method of any one of items 51 to 54, wherein eukaryotic cells transduced with a marker protein are isolated or sorted from cells lacking the marker protein, thereby producing a marker protein-positive cell population and / or a marker protein-negative cell population.

[0190] 56. The method of item 55, further comprising repeating steps (a) and (b) one or more times on the marker protein-negative cell population, the marker protein-positive cell population, or both the marker protein-negative and marker protein-positive cell populations.

[0191] 57. The method of any one of items 51 to 56, wherein eukaryotic cells transduced with a marker protein are isolated or selected on the basis of their intracellular concentration of the marker protein.

[0192] 58. The method according to any one of items 51 to 57, wherein the marker protein is a protein that stimulates cell proliferation, a protein that stimulates cell differentiation, a protein that promotes cell survival, an anti-apoptotic protein, or a protein with another biological activity.

[0193] 59. The method of any one of items 51 to 58, wherein the polypeptide cargo of interest and the marker protein are co-transduced by contacting the target eukaryotic cell with the polypeptide cargo and the marker protein in the presence of a peptide transduction agent, wherein the peptide transduction agent is present at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo and the marker protein compared to that in the absence of the peptide transduction agent.

[0194] 60. (a) The peptide transduction agent is an endosomolytic peptide; (b) the peptide transduction agent is or comprises a synthetic peptide shuttle agent as defined in item 17 or 18; (c) the target eukaryotic cell comprises an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, a NK cell, or a dendritic cell; (d) the polypeptide cargo of interest is (i) a polypeptide cargo as defined in any one of items 24 to 31; and / or (ii) one or more CRISPR-binding endonucleases alone or together with one or more corresponding guide RNAs and / or linear DNA templates as defined in any one of items 38 to 43; (e) The method according to Item 59, wherein the method is any combination of (a) to (d).

[0195] Other objects, advantages and features of the present description will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of example only, with reference to the accompanying drawings.

[0196] Example Example 1: material and method 1.1 Materials All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA or Oakville, ON, Canada) or of equivalent grade from BioShop Canada Inc. (Mississauga, ON, Canada) or VWR (Ville Mont-Royal, QC, Canada) unless otherwise noted. 1.2 Reagents [Table 6-1] [Table 6-2]

[0197] 1.3 Cell lines HeLa, HEK293A, HEK293T, THP-1, CHO, NIH3T3, CA46, Balb3T3, and HT2, KMS-12, DOHH2, REC-1, HCC-78, NCI-H196, and HT2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured according to the manufacturer's instructions. Myoblasts are primary human cells donated by Professor J.P. Tremblay (Université Laval, Quebec, Canada). [Table 7-1] [Table 7-2]

[0198] 1.4 Protein purification Fusion proteins were expressed in bacteria (Escherichia coli BL21DE3) under standard conditions using an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector containing a T5 promoter. The medium contained 24 g of yeast extract, 12 g of tryptone, 4 mL of glycerol, 2.3 g of KH2PO4, and 12.5 g of K2HPO4 per liter. Bacterial cultures were incubated with the appropriate antibiotic (e.g., ampicillin) at 37°C under stirring. Expression was induced with a final concentration of 1 mM IPTG at an optical density (600 nm) between 0.5 and 0.6 for 3 hours at 30°C. Bacteria were harvested after centrifugation at 5000 RPM, and the bacterial pellet was stored at -20°C.

[0199] The bacterial pellet was resuspended in Tris buffer (Tris 25 mM pH 7.5, NaCl 100 mM, imidazole 5 mM) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and lysed by passing three times through a homogenizer - Panda 2K™ at 1000 bar. The solution was centrifuged at 15000 RPM and 4°C for 30 minutes. The supernatant was collected and filtered using a 0.22 μM filter device.

[0200] Using an FPLC (AKTA Explorer 100R), the solubilized proteins were loaded onto a HisTrap™ FF column pre-equilibrated with 5 column volumes (CV) of Tris buffer. The column was washed with 30 column volumes (CV) of Tris buffer supplemented with 0.1% Triton™ X-114, followed by 30 CV of Tris buffer containing 40 mM imidazole. The proteins were eluted with 5 CV of Tris buffer containing 350 mM imidazole and collected. The collected fractions corresponding to specific proteins were examined by standard denaturing SDS-PAGE.

[0201] The purified protein was diluted in 20 mM Tris at the desired pH according to the protein's pI and loaded onto an appropriate ion-exchange column (Q Sepharose™ or SP Sepharose™) pre-equilibrated with 5 CV of 20 mM Tris, 30 mM NaCl. The column was washed with 10 CV of 20 mM Tris, 30 mM NaCl, and the protein was eluted with a 15 CV gradient up to 1 M NaCl. The collected fractions corresponding to the specific protein were examined by standard denaturing SDS-PAGE. The purified protein was then washed and concentrated in PBS 1X with an Amicon Ultra™ centrifugal filter 10,000 MWCO. Protein concentration was assessed using a standard Bradford assay.

[0202] 1.5 Synthetic peptides and shuttle agents All peptides used in this study were purchased from GLBiochem (Shanghai, China), and their purity was confirmed by high-performance liquid chromatography and mass spectrometry. In some cases, peptides were synthesized to contain a C-terminal cysteine ​​residue, allowing for the preparation of peptide dimers. These dimeric peptides were synthesized directly with a disulfide bridge between the C-terminal cysteines of the two monomers. The amino acid sequences and characteristics of each of the synthetic peptides and shuttle agents tested in this example are summarized in Tables 1.3, B1, and C1. [Table 8-1] [Table 8-2] [Table 8-3]

[0203] Results were calculated using the ProtParam™ online tool available from the ExPASy™ Bioinformatics Resource Portal (http: / / web.expasy.org / protparam / ). MW: molecular weight pI: isoelectric point Charge: total number of positively (+) and negatively (-) charged residues

[0204] Example 2: Peptide shuttle agents promote the escape of endosome-trapped calcein 2.1 Endosomal escape assay To study endosomal leakage and to determine whether the addition of a shuttle agent promotes endosomal leakage of polypeptide cargo, microscopy- and flow cytometry-based fluorescence assays were developed, and these methods are described in Example 2 of International Application No. PCT / CA2016 / 050403.

[0205] 2.1.1 Visualization of endosomal leakage by microscopy Calcein is a membrane-impermeable fluorescent molecule that is readily internalized by cells when administered into the extracellular medium. Its fluorescence is pH-dependent, and calcein self-quenches at high concentrations. Upon internalization, calcein becomes trapped in cellular endosomes at high concentrations and can be visualized by fluorescence microscopy as a punctate pattern. After endosomal leakage, calcein is released into the cytoplasm, and this release can be visualized by fluorescence microscopy as a diffuse pattern.

[0206] One day before the calcein assay, cells in logarithmic growth phase were harvested and seeded into 24-well plates (80,000 cells per well). Cells were allowed to attach by overnight incubation in the appropriate growth medium, as described in Example 1. The next day, the medium was removed and replaced with 300 μL of fresh medium containing 62.5 μg / mL (100 μM) calcein without FBS (except for HEK293A, which contained 250 μg / mL, 400 μM calcein). At the same time, the shuttle agent to be tested was added at the designated concentration. The plate was incubated at 37°C for 30 minutes. The cells were washed with 1x PBS (37°C), and fresh medium containing FBS was added. The plate was incubated at 37°C for 2.5 hours. The cells were washed three times and visualized using a phase contrast and fluorescence microscope (IX81™, Olympus).

[0207] A typical result is shown in Figure 1A, in which untreated HEK293A cells loaded with calcein (100 μM calcein) exhibit a low-intensity, punctate fluorescence pattern when visualized by fluorescence microscopy (upper left panel). In contrast, HeLa cells treated with a shuttle agent that promotes endosomal escape of calcein (100 μM calcein + CM18-TAT 5 μM) exhibit a high-intensity, more nucleic acid-rich fluorescence pattern in a greater proportion of cells (upper right panel).

[0208] 2.1.2 Quantification of endosomal leakage by flow cytometry In addition to microscopy, flow cytometry allows for more quantitative analysis of endosomal leakage because calcein release into the cytoplasm results in an increased fluorescence intensity signal. Calcein fluorescence is optimal at physiological pH (e.g., in the cytosol) compared to the acidic environment of the endosome.

[0209] One day before the calcein assay, cells in logarithmic growth phase were harvested and seeded into 96-well plates (20,000 cells per well). Cells were allowed to attach by overnight incubation in the appropriate growth medium, as described in Example 1. The next day, the medium was removed and replaced with 50 μL of fresh serum-free medium containing 62.5 μg / mL (100 μM) calcein (except for HEK293A, which contained 250 μg / mL, 400 μM calcein). At the same time, the shuttle agent to be tested was added at the indicated concentration. The plate was incubated at 37°C for 30 minutes. The cells were washed with 1x PBS (37°C), and fresh medium containing 5-10% serum was added. The plate was incubated at 37°C for 2.5 hours. The cells were washed with 1x PBS and detached using trypsinization. Trypsinization was stopped by the addition of the appropriate growth medium, and calcein fluorescence was quenched using flow cytometry (Accuri C6, Becton, Dickinson and Company (BD)).

[0210] To distinguish cells with baseline fluorescence due to endosome-trapped calcein from cells with increased fluorescence due to calcein release from endosomes, cells loaded with untreated calcein were used as a control. The average fluorescence signal ("mean count") was analyzed to quantify endosomal escape. In some cases, the "mean fold" corresponding to the fold increase in mean count relative to the control (untreated calcein-loaded cells) was calculated. The size and granularity of the events scanned by flow cytometry corresponding to the cells were also analyzed. Cell mortality was monitored as the percentage of cells among the total scanned events. If it was lower than the control, the number of necrotic cell debris was considered to be increased due to toxicity, and the assay was discarded.

[0211] Typical results are shown in Figure 1B, where an increase in fluorescence intensity (shift to the right) was observed in calcein-loaded HeLa cells treated with a shuttle agent that promotes endosomal escape ("Calcein 100 μM + CM18-TAT 5 μM," right panel) compared to untreated calcein-loaded HeLa cells ("Calcein 100 μM," left panel). The increase in calcein fluorescence is caused by the increase in pH associated with the release of calcein from endosomes (acidic) into the cytoplasm (physiological).

[0212] 2.2 Results from endosomal escape assay 2.2.1 HeLa cells HeLa cells were cultured and tested in an endosomal escape assay as described in Example 2.1. The results of the flow cytometry analysis are summarized below. In each case, the flow cytometry results were also confirmed by fluorescence microscopy (data not shown). [Table 9] [Table 10]

[0213] The results in Tables 2.1 and 2.2 show that treatment of calcein-loaded HeLa cells with the shuttle agents CM18-Penetratin-Cys and CM18-TAT-Cys (with the domain structure ELD-CPD) resulted in an increase in mean cellular calcein fluorescence intensity compared to untreated control cells or cells treated with the single domain peptides used alone (CM18, TAT-Cys, Penetratin-Cys) or together (CM18 + TAT-Cys, CM18 + Penetratin-Cys). These results suggest that CM18-Penetratin-Cys and CM18-TAT-Cys, but not the single domain peptides (used alone or together), promote the escape of calcein trapped by endosomes. [Table 11] [Table 12] [Table 13]

[0214] The results in Table 2.3 (Figure 2), Table 2.4, and Table 2.5 (Figure 3) suggest that CM18-TAT-Cys and CM18-Penetratin-Cys promote the escape of endosome-trapped calcein in HeLa cells in a dose-dependent manner. In some cases, concentrations of CM18-TAT-Cys or CM18-Penetratin-Cys greater than 10 μM were associated with increased cytotoxicity in HeLa cells. [Table 14] [Table 15]

[0215] The results in Tables 2.6 and 2.7 suggest that shuttle peptide dimers (which are molecules containing two or more ELDs and CPDs) can promote calcein endosomal escape levels that are comparable to the corresponding monomers.

[0216] 2.2.3 HEK293A cells To examine the effect of the shuttle agents on various cell lines, HEK293A cells were cultured and tested in the endosomal escape assay as described in Example 2.1. The results of the flow cytometry analysis are summarized in Table 2.8 below and in Figure 1B. [Table 16]

[0217] The results in Table 2.8 and Figure 1B show that treatment of calcein-loaded HEK293A cells with the shuttle agent CM18-TAT-Cys resulted in an increase in mean cellular calcein fluorescence intensity compared to untreated control cells.

[0218] 2.2.2 Myoblasts To examine the effects of the shuttle agents on primary cells, primary myoblasts were cultured and tested in an endosomal escape assay as described in Example 2.1. The results of the flow cytometry analysis are summarized in Tables 2.9 and 2.10 below, and in Figure 4. In each case, the flow cytometry results were also confirmed by fluorescence microscopy. [Table 17] [Table 18]

[0219] The results in Table 2.9 (shown graphically in Figure 4) and Table 2.10 suggest that CM18-TAT-Cys promotes the escape of endosome-trapped calcein in primary myoblasts in a dose-dependent manner. Concentrations of CM18-TAT-Cys greater than 10 μM were associated with increased cytotoxicity in myoblasts, as was the case for HeLa cells. [Table 19]

[0220] The results in Table 2.11 suggest that the shuttle peptide dimers can promote calcein endosomal escape levels in primary myoblasts that are comparable to the corresponding monomers.

[0221] Example 3: Peptide shuttling agents enhance GFP transduction efficiency 3.1 Protein transduction assay One day before transduction assays were performed, cells in logarithmic growth phase were harvested and seeded into 96-well plates (20,000 cells per well). Cells were incubated overnight in the appropriate growth medium containing FBS (see Example 1). The next day, in separate sterile 1.5 mL tubes, cargo protein at the indicated concentration was premixed (preincubated) with the peptide to be tested as a shuttle agent (0.5–5 μM) in 50 μL of fresh, serum-free medium (unless otherwise noted) at 37°C for 1 or 10 minutes (depending on the protocol). The medium in the wells was removed, and the cells were washed three times with freshly prepared phosphate-buffered saline (PBS) prewarmed to 37°C. The cells were incubated with the cargo protein / shuttle agent mixture at 37°C for the indicated time (e.g., 1, 5, or 60 minutes). After incubation, the cells were rapidly washed three times with freshly prepared PBS and / or heparin (0.5 mg / mL) prewarmed to 37°C. Human THP-1 blood cells required washing with heparin to avoid unwanted cell membrane-associated protein background in subsequent analyses (microscopy and flow cytometry). Cells were finally incubated in 50 μL of fresh medium containing serum at 37°C before analysis.

[0222] 3.1a Protocol A: Protein transduction assay for adherent cells One day before transduction assays were performed, cells in logarithmic growth phase were harvested and seeded into 96-well plates (20,000 cells per well). The cells were incubated overnight in the appropriate growth medium containing serum (see Example 1). The following day, in separate sterile 1.5 mL tubes, the shuttle agent was diluted in sterile distilled water at room temperature (nuclease-free water was used if the cargo was or contained a nucleic acid). Peptide was added to the shuttle agent, and sterile PBS or cell culture medium (serum-free) was added as needed to obtain the desired concentrations of shuttle agent and cargo in a final volume sufficient to cover the cells (e.g., 10–100 μL per well of a 96-well plate). The peptide / cargo mixture was then immediately used for experiments. Each experiment included at least three controls: (1) peptide alone (e.g., at the highest concentration tested), (2) cargo alone, and (3) no cargo or shuttle agent controls. The medium in the wells was removed, and the cells were washed once with PBS pre-warmed to 37°C and incubated with the cargo protein / peptide mixture at 37°C for the desired time. The peptide / cargo mixture in the wells was removed, and the cells were washed once with PBS, and fresh complete medium was added. Prior to analysis, the cells were washed one last time with PBS, and fresh complete medium was added.

[0223] 3.1b Protocol B: Suspension cell protein transduction assay One day before transduction assays were performed, logarithmically growing suspension cells were harvested and seeded into 96-well plates (20,000 cells per well). The cells were incubated overnight in the appropriate growth medium containing serum (see Example 1). The following day, in separate sterile 1.5 mL tubes, the shuttle agent was diluted in sterile distilled water at room temperature (nuclease-free water was used if the cargo was or contained a nucleic acid). Peptide was added to the shuttle agent, and sterile PBS or cell culture medium (serum-free) was added as needed to obtain the desired concentrations of shuttle agent and cargo in a final volume sufficient for cell resuspension (e.g., 10–100 μL per well of a 96-well plate). The shuttle agent / peptide was then immediately used for experiments. Each experiment included at least three controls: (1) peptide alone (e.g., at the highest concentration tested), (2) cargo alone, and (3) no cargo or shuttle agent. The cells were centrifuged at 400g for 2 minutes, then the medium was removed and the cells were resuspended in PBS pre-warmed to 37°C. The cells were centrifuged again at 400g for 2 minutes, the PBS was removed, and the cells were resuspended with the shuttle agent / cargo mixture at 37°C for the desired time. 200μL of complete medium was then added directly to the cells. The cells were centrifuged at 400g for 2 minutes and the medium was removed. The pellet was resuspended in 200μL of PBS pre-warmed to 37°C and washed. After another centrifugation, the PBS was removed and the cells were resuspended in 50μL of trypsin-EDTA solution for 2 minutes. 200μL of complete medium was added directly and the cells were centrifuged at 400g for 2 minutes. The medium was removed and the cells were resuspended in 200μL of complete medium.

[0224] 3.2 Fluorescence microscopy analysis Delivery of fluorescent protein cargo in the cytoplasmic and nuclear compartments was observed using an Olympus IX70™ microscope (Japan) equipped with a fluorescent lamp (model U-LH100HGAPO) and various filters. Fluorescent signals of GFP and FITC-labeled antibodies were observed using an Olympus filter U-MF2™ (C54942-Exc495 / Em510). mCherry™ and GFP antibody fluorescent signals were observed using an Olympus filter HQ-TR™ (V-N41004-Exc555-60 / Em645-75). DAPI or Blue Hoechst fluorescent signals were observed using an Olympus filter U-MWU2™ (Exc330 / Em385). Cells incubated in 50 µL of fresh medium were directly observed under a microscope (bright field and fluorescent) at various magnifications (4x–40x). Cells were observed using a CoolSNAP-PRO™ camera (series A02D874021) and images were acquired using Image-Proplus™ software.

[0225] 3.2a Cell immunolabeling Adherent cells were plated at 1.5x10 per well of a 24-well plate. 5Single cells were seeded onto sterile glass strips and incubated overnight at 37°C. For fixation, cells were incubated in 500 μL of formaldehyde (3.7% v / v) per well at room temperature for 15 minutes and washed three times with PBS for 5 minutes. For permeabilization, cells were incubated in 500 μL of Triton™ X-100 (0.2%) per well at room temperature for 10 minutes and washed three times with PBS for 5 minutes. For blocking, cells were incubated in 500 μL of PBS containing 1% BSA (PBS / BSA) per well at room temperature for 60 minutes. Primary mouse monoclonal antibodies were diluted in PBS / BSA (1%). Cells were incubated overnight in 30 μL of primary antibody at 4°C. Cells were washed three times with PBS for 5 minutes. Secondary antibodies were diluted in PBS / BSA (1%), and cells were incubated in 250 μL of secondary antibody at room temperature in the dark for 30 minutes. Cells were washed three times with PBS for 5 minutes. The glass strips containing the cells were mounted on glass microscope slides using 10 μL of mounting medium, Fluoroshield™ containing DAPI.

[0226] 3.3 Flow cytometry analysis: GFP fluorescence was quantified using flow cytometry (Accuri C6, Becton, Dickinson and Company (BD)). To quantify the increase in fluorescence due to internalization of the fluorescent protein in treated cells, a baseline was established using untreated cells. The percentage of cells with a fluorescent signal above the maximum fluorescence of untreated cells, "mean %" or "Pos cells (%)," was used to identify positively fluorescent cells. "Relative fluorescence intensity (FL1-A)" corresponds to the average of all fluorescent intensities from each cell with a fluorescent signal after fluorescent protein delivery using the shuttle agent. The size and granularity of the events scanned by flow cytometry corresponding to the cells were also analyzed. Cytotoxicity (% cell viability) was monitored by comparing the percentage of cells in the total scanned events of treated cells compared to untreated cells.

[0227] 3.3a ​​Survival analysis Where applicable, cell viability was assessed using the resazurin test. Resazurin is a sodium salt colorant that is converted from blue to pink by mitochondrial enzymes in metabolically active cells. To quantify the percentage of viable cells, this colorimetric conversion, which occurs only in viable cells, can be measured by spectrophotometric analysis. A stock solution of resazurin was prepared in water at 1 mg / 100 mL and stored at 4°C. 25 μL of the stock solution was added to each well of a 96-well plate, and the cells were incubated at 37°C for 1 hour before spectrophotometric analysis. The incubation time used for the resazurin enzyme reaction varied depending on the amount of cells and the volume of medium used in the well.

[0228] 3.4 GFP construction and amino acid sequence The gene encoding GFP was inserted into a T5 bacterial expression vector to express a GFP protein containing a 6x histidine tag and a serine / glycine-rich linker at the N-terminus and a serine / glycine-rich linker and a stop codon (-) at the C-terminus. The recombinant GFP protein was purified as described in Example 1.4. The sequence of the GFP construct was as follows: [ka] (MW=31.46kDa; pI=6.19) The serine / glycine-rich linker is in bold. The GFP sequence is underlined.

[0229] 3.5 GFP transduction with CM18-TAT-Cys in HeLa cells: fluorescence microscopy HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein was co-incubated with 0, 3, or 5 μM CM18-TAT and then exposed to HeLa cells for 1 hour. The cells were observed by bright-field and fluorescence microscopy as described in Example 3.2. The results shown in Figure 5 indicate that GFP was delivered intracellularly to HeLa cells in the presence of the shuttle agent CM18-TAT.

[0230] 3.6 GFP transduction by shuttle agents in HeLa cells: dose response (CM18-TAT-Cys, dCM18-TAT-Cys, GFP) and cell viability HeLa cells were cultured and tested in the protein transduction assay described in Examples 3.1-3.3. Briefly, GFP recombinant protein was co-incubated with various concentrations of CM18-TAT-Cys or dimerized CM18-TAT-Cys (dCM18-TAT-Cys) and then exposed to HeLa cells for 1 hour. The results are shown in Table 3.1 and Figures 6A-6B. [Table 20]

[0231] Table 3.1 and Figure 6A show the fluorescence intensity results of flow cytometry analysis of HeLa cells transduced with GFP (5 μM) without or with 5, 3, 1, and 0.5 μM CM18-TAT-Cys. The corresponding cytotoxicity data are shown in Table 3.1 and Figure 6B. These results suggest that the shuttle agent CM18-TAT-Cys increases the transduction efficiency of GFP in a dose-dependent manner. [Table 21]

[0232] Table 3.2 and Figure 7 show the results of fluorescence intensity from flow cytometry analysis of HeLa cells transduced with various concentrations of GFP (1-10 μM) without or with 5 μM CM18-TAT-Cys (Figure 7A) or 2.5 μM dCM18-TAT-Cys (Figure 7B).

[0233] 3.7 GFP transduction in HeLa cells: dose response of CM18-TAT-Cys and CM18-Penetratin-Cys and their dimers HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein (5 μM) was co-incubated with various concentrations and combinations of CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys, dCM18-Penetratin-Cys), and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 3.3 and Figure 8, and Table 3.4 and Figure 9. [Table 22]

[0234] The results in Table 3.3 and Figure 8 show that the use of shuttle agents CM18-TAT-Cys and dCM18-TAT-Cys enhanced GFP transduction efficiency in HeLa cells (see bars "1" and "2" in Figure 8). While no GFP intracellular delivery was observed using CM18-Penetratin-Cys or dCM18-Penetratin-Cys alone (see bars "3" or "4" in Figure 8), the combination of CM18-TAT-Cys with CM18-Penetratin-Cys (monomer or dimer) improved GFP protein delivery (see the four right-most bars in Figure 8). [Table 23]

[0235] The results in Table 3.4 and Figure 9 show that the use of shuttle agents CM18-TAT-Cys and dCM18-TAT-Cys (see bars "1" and "2" in Figure 9) increased the transduction efficiency of GFP in HeLa cells. While no intracellular delivery of GFP was observed using CM18-Penetratin-Cys or dCM18-Penetratin-Cys alone (see bars "3" or "4" in Figure 9), the combination of CM18-TAT-Cys with CM18-Penetratin-Cys (monomer or dimer) improved GFP protein delivery (see the four right-most bars in Figure 9).

[0236] 3.8 GFP transduction with shuttle agents in HeLa cells: Control HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP recombinant protein (5 μM) was co-incubated with 5 μM each of the following peptides: TAT-Cys, CM18, penetratin-Cys, TAT-Cys + CM18, penetratin-Cys + CM18, and CM18-TAT-Cys, and then exposed to HeLa cells for 1 hour. GFP fluorescence was visualized by bright-field and fluorescence microscopy. Microscopic results (data not shown) indicated that GFP was successfully delivered into cells using CM18-TAT-Cys. However, GFP was not successfully delivered into cells when single-domain peptides were used alone (CM18, TAT-Cys, penetratin-Cys) or together (CM18 + TAT-Cys, CM18 + penetratin-Cys). These results are consistent with those shown in Tables 2.1 and 2.2 for the calcein endosomal escape assay.

[0237] Example 4: Peptide shuttling agents enhance TAT-GFP transduction efficiency The experiments in Example 3 demonstrated the ability of the shuttle agent to deliver GFP into cells. In this example, the experiments shown demonstrate that the shuttle agent can also enhance the intracellular delivery of a GFP cargo protein fused to a CPD (TAT-GFP).

[0238] 4.1 Construction and amino acid sequence of TAT-GFP The construction was performed as described in Example 3.4, except that the TAT sequence was cloned between the 6x histidine tag and the GFP sequence. The 6x histidine tag, TAT, GFP, and stop codon (-) are separated by a serine / glycine-rich linker. The recombinant TAT-GFP protein was purified as described in Example 1.4. The sequence of the TAT-GFP construct was: [ka] [SEQ ID NO: 61] (MW=34.06kDa; pI=8.36) The TAT sequence is underlined. The serine / glycine-rich linker is in bold.

[0239] 4.2 TAT-GFP transduction with CM18-TAT-Cys in HeLa cells: visualization by fluorescence microscopy HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, TAT-GFP recombinant protein (5 μM) was co-incubated with 3 μM CM18-TAT-Cys and then exposed to HeLa cells for 1 hour. Cells and GFP fluorescence were visualized by bright-field and fluorescence microscopy at 10x and 40x magnification (as described in Example 3.2), and example results are shown in Figure 10. Microscopic results revealed that in the absence of CM18-TAT-Cys, TAT-GFP exhibited low-intensity endosomal distribution, as reported in the literature. In contrast, TAT-GFP was delivered to the cytoplasm and nucleus in the presence of the shuttle agent CM18-TAT-Cys. Without wishing to be bound by theory, the TAT peptide itself may act as a nuclear localization signal (NLS), explaining the nuclear localization of TAT-GFP. These results indicate that CM18-TAT-Cys increases TAT-GFP transduction efficiency and allows endosome-trapped TAT-GFP to reach the cytoplasmic and nuclear compartments.

[0240] 4.3 TAT-GFP transduction with CM18-TAT-Cys in HeLa cells: dose response and viability of transduced cells HeLa cells were cultured and tested in a protein transduction assay as described in Example 3.1. Briefly, TAT-GFP recombinant protein (5 μM) was co-incubated with various concentrations of CM18-TAT-Cys (0, 0.5, 1, 3, or 5 μM) and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 4.3 and Figure 11A. The corresponding cytotoxicity data are shown in Figure 11B. [Table 24]

[0241] Example 5: Peptide Shuttling Agents Enhance GFP-NLS Transduction Efficiency and Nuclear Localization The experiments in Examples 3 and 4 demonstrated the ability of the shuttle agents to deliver GFP and TAT-GFP into cells. The experiments presented in this example demonstrate that the shuttle agents can facilitate nuclear delivery of GFP protein cargo fused to a nuclear localization signal (NLS).

[0242] 5.1 Construction and amino acid sequence of GFP-NLS The construction was carried out as described in Example 3.4, except that the optimized NLS sequence was inserted between the GFP sequence and the stop codon (-). The NLS sequence is separated from the GFP sequence and the stop codon by two serine / glycine-rich linkers. The recombinant GFP-NLS protein was purified as described in Example 1.4. The sequence of the GFP-NLS construct was: [ka] (MW=34.85kDa; pI=6.46) The NLS sequence is underlined. The serine / glycine-rich linker is in bold.

[0243] 5.2 Nuclear delivery of GFP-NLS by CM18-TAT-Cys in HeLa cells in 5 min: visualization by fluorescence microscopy HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. Briefly, GFP-NLS recombinant protein (5 μM) was co-incubated with 5 μM CM18-TAT-Cys and then exposed to HeLa cells. After 5 minutes, GFP fluorescence was visualized by bright-field and fluorescence microscopy at 10×, 20×, and 40× magnification (as described in Example 3.2); example results are shown in Figure 12. Microscopic results indicated that GFP-NLS was efficiently delivered to the nucleus in the presence of the shuttle agent CM18-TAT-Cys after only 5 minutes of incubation.

[0244] 5.3 GFP-NLS transduction with CM18-TAT-Cys in HeLa cells: dose response and viability of transduced cells HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. GFP-NLS recombinant protein (5 μM) was co-incubated with 0, 0.5, 1, 3, or 5 μM CM18-TAT-Cys and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 5.1 and Figure 13A. The corresponding cytotoxicity data are shown in Figure 13B. [Table 25]

[0245] These results indicate that CM18-TAT-Cys can enhance GFP-NLS transduction efficiency in HeLa cells in a dose-dependent manner.

[0246] 5.4 GFP-NLS transduction with CM18-TAT-Cys, CM18-Penetratin-Cys, and their dimers in HeLa cells HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. GFP-NLS recombinant protein (5 μM) was co-incubated with various concentrations and combinations of CM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys, dCM18-Penetratin-Cys), and then exposed to HeLa cells for 1 hour. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Tables 5.2 and 5.3 and Figures 14 and 15. [Table 26] [Table 27]

[0247] The results in Tables 5.2 and 5.3 and Figures 14 and 15 show that the use of the shuttle agents CM18-TAT-Cys and dCM18-TAT-Cys increased the transduction efficiency of GFP-NLS into HeLa cells (see bars "1" and "2" in Figures 14 and 15). While no GFP-NLS intracellular delivery was observed using CM18-Penetratin-Cys or dCM18-Penetratin-Cys alone (see bars "3" and "4" in Figures 14 and 15), the combination of CM18-TAT-Cys with CM18-Penetratin-Cys (monomer or dimer) improved GFP-NLS intracellular delivery (see the four right-most bars in Figures 14 and 15).

[0248] 5.5 GFP-NLS transduction with shuttle agents in HeLa cells: 5 min vs. 1 h incubation with or without FBS HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. GFP-NLS recombinant protein (5 μM) was co-incubated with CM18-TAT-Cys (3.5 μM) alone or together with dCM18-Penetratin-Cys (1 μM). Cells were incubated for 5 minutes or 1 hour in plain DMEM medium ("DMEM") or DMEM medium containing 10% FBS ("FBS") and then subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 5.4 and Figure 16. Cells not treated with shuttle agent or GFP-NLS ("ctrl") and cells treated with GFP-NLS but without shuttle agent ("GFP-NLS 5 μM") were used as controls. [Table 28]

[0249] The results in Table 5.4 and Figure 16 show that the addition of even a relatively small amount of dimeric dCM18-Penetratin-Cys (1 μM, "dCM18pen") to the CM18-TAT-Cys monomer improved GFP-NLS transduction efficiency. Interestingly, intracellular GFP-NLS delivery was achieved with only 5 minutes of incubation, and delivery was still achievable (albeit reduced) in the presence of FBS.

[0250] 5.6 GFP-NLS transduction by shuttle agents in THP-1 suspension cells The ability of the shuttle agent to deliver GFP-NLS into cells was tested in THP-1 cells, an acute monocytic leukemia cell line grown in suspension. THP-1 cells were cultured (see Example 1) and tested in the protein transduction assay described in Example 3.1. GFP-NLS recombinant protein (5 μM) was co-incubated with or without 1 μM CM18-TAT-Cys, exposed to THP-1 cells for 5 minutes, and then subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 5.5 and Figure 17A. The corresponding cytotoxicity data are shown in Figure 17B. [Table 29]

[0251] The results in Table 5.5 and Figure 17 demonstrate the ability of the shuttle agents to intracellularly deliver protein cargo to a human monocytic cell line grown in suspension.

[0252] Example 6: Peptide shuttling agents increase the transduction efficiency of FITC-labeled anti-tubulin antibodies The experiments in Examples 3-5 demonstrated the ability of the shuttle agent to increase the transduction efficiency of GFP, TAT-GFP, and GFP-NLS. The experiments provided in this example demonstrate that the shuttle agent can also deliver a larger protein cargo: a FITC-labeled anti-tubulin antibody. The FITC-labeled anti-tubulin antibody was purchased from Abcam (ab64503) and has an estimated molecular mass of 150 KDa. The delivery and microscopy protocols are described in Example 3.

[0253] 6.1 Transduction of functional antibodies by CM18-TAT-Cys in HeLa cells: visualization by microscopy FITC-labeled anti-tubulin antibody (0.5 μM) was co-incubated with 5 μM CM18-TAT-Cys and exposed to HeLa cells for 1 hour. Antibody delivery was visualized by bright-field (20x) and fluorescence microscopy (20x and 40x). As shown in Figure 18, fluorescent tubulin fibers were visualized in the cytoplasm, demonstrating the functionality of the antibody inside the cells.

[0254] 6.2 Transduction of Functional Antibodies by CM18-TAT-Cys, CM18-Penetratin-Cys, and Dimers in HeLa Cells: Flow Cytometry HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. FITC-labeled anti-tubulin antibody (0.5 μM) was co-incubated with 3.5 μM CM18-TAT-Cys, CM18-Penetratin-Cys, or dCM18-Penetratin-Cys, or a combination of 3.5 μM CM18-TAT-Cys and 0.5 μM dCM18-Penetratin-Cys, and exposed to HeLa cells for 1 hour. Cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 6.1 and Figure 19A. The corresponding cytotoxicity data are shown in Figure 19B. [Table 30]

[0255] The results in Table 6.1 and Figures 18 and 19 show that both CM18-TAT-Cys and CM18-Penetratin-Cys promote the intracellular delivery of FITC-labeled anti-tubulin antibodies. In contrast to the results using GFP, TAT-GFP, and GFP-NLS in Examples 3-5, CM18-Penetratin-Cys was able to deliver the antibody cargo intracellularly when used alone (without CM18-TAT-Cys). However, the combination of CM18-TAT-Cys and dCM18-Penetratin-Cys enabled higher intracellular delivery compared to CM18-TAT-Cys alone and had less cytotoxicity compared to CM18-Penetratin-Cys and dCM18-Penetratin-Cys (see Figures 19A and 19B).

[0256] Example 7: CM18-TAT-Cys enables intracellular plasmid DNA delivery but poor plasmid expression In this example, the ability of the CM18-TAT-Cys shuttle agent to deliver plasmid DNA intracellularly was tested in HEK293A cells using a plasmid encoding GFP.

[0257] 7.1 Gene transfer assay in HEK293A cells One day before the transfection assay, mammalian cells (HEK293A) in logarithmic growth phase were harvested and seeded into 24-well plates (50,000 cells per well). The cells were incubated overnight in the appropriate growth medium containing FBS. The next day, in separate sterile 1.5 mL tubes, Cy5™ fluorescently labeled pEGFP was mixed with CM18-TAT-Cys (0.05, 0.5, or 5 μM) in fresh PBS in a final volume of 100 μL for 10 minutes at 37°C. The medium in the wells was removed, the cells were quickly washed three times with PBS, and 500 μL of warm medium without FBS was added. The pEGFP and CM18-TAT-Cys solution was added to the cells and incubated for 4 hours at 37°C. After incubation, the cells were washed with PBS and fresh medium containing FBS was added. The cells were incubated at 37° C. and then subjected to flow cytometry analysis as described in Example 3.

[0258] 7.2 Plasmid DNA delivery using CM18-TAT-Cys Plasmid DNA (pEGFP) was labeled with Cy5™ dye according to the manufacturer's instructions (Mirus Bio LLC). The Cy5™ moiety did not affect transfection efficiency when compared with unlabeled plasmid using standard transfection protocols (data not shown). Flow cytometry analysis allowed for quantification of Cy5™ emission, corresponding to DNA intracellular delivery, and GFP emission, corresponding to successful nuclear delivery, DNA transcription, and protein expression. The results are shown in Table 7.1 and Figure 20. [Table 31]

[0259] The results shown in Table 7.1 and Figure 20 indicate that CM18-TAT-Cys was able to increase intracellular delivery of plasmid DNA when used at concentrations of 0.05, 0.5, and 5 μM compared to cells incubated with DNA alone ("pEGFP-Cy5"). However, GFP expression was not detected in the cells, suggesting that very little plasmid DNA reached the cytoplasmic compartment, allowing nuclear localization. Without wishing to be bound by theory, it is possible that the plasmid DNA was largely sequestered in endosomes, preventing its escape into the cytoplasmic compartment. Salomone et al. (2013) reported the use of the CM18-TAT11 hybrid peptide to intracellularly deliver plasmid DNA. They used a luciferase enzyme reporter assay to evaluate gene transfer efficiency, but this may not be ideal for quantifying the efficiency of cytoplasmic / nuclear delivery, as the percentage of plasmid DNA successfully released from endosomes and delivered to the nucleus may be overestimated due to the strong activity of the luciferase enzyme. In this regard, the authors of Salomone et al., 2013 further note that luciferase expression occurs along with extensive entrapment of (naked) DNA molecules into vesicles, which is consistent with the results shown in Table 7.1 and Figure 20.

[0260] 7.3 Peptide-mediated plasmid DNA delivery in HeLa cells Following poor transfection efficiency of peptide CM18-TAT-Cys (0.1%, see Table 7.1) observed in HEK293A cells, the experiment was repeated in another cell line (HeLa) with CM18-TAT-Cys, along with other peptides listed in Tables 1.3, B1, and C1.

[0261] One day before the transfection assay, HeLa cells in logarithmic growth phase were harvested and seeded into 96-well plates (10,000 cells per well). The cells were incubated overnight in the appropriate growth medium containing FBS. The next day, in a separate sterile 1.5 mL tube, the peptide to be tested and the polynucleotide cargo (pEGFP-C1) were mixed in serum-free medium in a final volume of 50 μL at 37°C for 10 minutes. The medium in the wells was removed, and the cells were quickly washed once with PBS at 37°C. The mixture containing the peptide to be tested and the polynucleotide cargo was added to the cells and incubated at 37°C for the indicated time (e.g., 1 minute, 1 hour, or 4 hours). After incubation, the cells were washed once with PBS at 37°C, and fresh medium containing FBS was added. The cells were incubated at 37°C and then subjected to flow cytometry analysis as described in Example 3.2 to determine the transfection efficiency (i.e., cellular EGFP expression) and viability. The results are shown in Table 7.2. [Table 32-1] [Table 32-2]

[0262] All tested peptides in Table 7.2 showed transfection efficiencies of less than 1%. Furthermore, the low transfection efficiency of CM18-TAT-Cys was confirmed in HeLa cells (0.08%). These results indicate that peptides suitable for delivering polypeptide cargo may not necessarily be suitable for delivering plasmid DNA. For example, although the shuttle agent His-CM18-PTD4-His herein effectively transduces polypeptide cargo (see, e.g., Example 10), this peptide showed a DNA plasmid transfection efficiency of only 0.34% (Table 7.2).

[0263] Example 8: Addition of a histidine-rich domain to the shuttle agent further improves GFP-NLS transduction efficiency 8.1 GFP-NLS transduction with His-CM18-TAT-Cys in HeLa cells: visualization by microscopy GFP-NLS (5 μM, see Example 5) was co-incubated with 5 μM CM18-TAT-Cys or His-CM18-TAT and exposed to HeLa cells for 1 hour. Nuclear fluorescence of intracellularly delivered GFP-NLS was confirmed by fluorescence microscopy (data not shown), indicating successful delivery of GFP-NLS to the nucleus.

[0264] 8.2 GFP-NLS Transduction with His-CM18-TAT in HeLa Cells: Flow Cytometry HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1. GFP-NLS (5 μM) was co-incubated with 0, 1, 3, or 5 μM of CM18-TAT-Cys or His-CM18-TAT and exposed to HeLa cells for 1 hour. Cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 8.1 and Figure 21A. The corresponding cytotoxicity data are shown in Figure 21B. [Table 33]

[0265] Strikingly, the results in Table 8.1 and Figure 21 show that His-CM18-TAT was able to increase GFP-NLS protein transduction efficiency approximately twofold at 3 μM and 5 μM concentrations compared to CM18-TAT-Cys. These results suggest that adding a histidine-rich domain to a shuttle agent containing an ELD and CPD can significantly increase its polypeptide cargo transduction efficiency. Alternatively, or simultaneously, combining the shuttle agent with an additional, independently synthesized peptide containing a histidine-rich domain fused to a CPD (but lacking an ELD) may provide similar benefits for protein transduction, with the added advantage of allowing the concentration of the histidine-rich domain to be varied or controlled independently of the concentration of the shuttle agent. Without being bound by theory, the histidine-rich domain may act as a proton sponge in endosomes, providing another mechanism for endosomal membrane destabilization.

[0266] Example 9: His-CM18-PTD4 increases the transduction efficiency and nuclear delivery of GFP-NLS, mCherry™-NLS, and FITC-labeled anti-tubulin antibody 9.1 Protein transduction protocols Protocol A: Protein transduction assay for delivery in cell culture media One day before the transduction assay, cells in logarithmic growth phase were harvested and seeded into 96-well plates (20,000 cells per well). The cells were incubated overnight in the appropriate growth medium containing FBS (see Example 1). The next day, in separate sterile 1.5 mL tubes, the desired concentration of cargo protein was premixed (preincubated) with the desired concentration of shuttle agent in 50 μL of fresh serum-free medium (unless otherwise noted) at 37°C for 10 minutes. The medium in the wells was removed, and the cells were washed 1 to 3 times (depending on the cell type used) with PBS prewarmed to 37°C. The cells were incubated with the cargo protein / shuttle agent mixture at 37°C for the desired time. After incubation, the cells were washed three times with PBS and / or heparin (0.5 mg / mL) prewarmed to 37°C. For human THP-1 blood cells, washing with heparin was used to avoid unwanted cell membrane-associated protein background in subsequent analyses (microscopy and flow cytometry). Cells were finally incubated in 50 μL of fresh medium containing serum at 37°C before analysis.

[0267] Protocol B: Protein transduction assay of adherent cells in PBS One day before transduction assays were performed, cells in logarithmic growth phase were harvested and seeded into 96-well plates (20,000 cells per well). The cells were incubated overnight in the appropriate growth medium containing serum (see Example 1). The following day, in separate sterile 1.5 mL tubes, the shuttle agent was diluted in sterile distilled water at room temperature (nuclease-free water was used if the cargo was a nucleic acid or contained a nucleic acid). The cargo protein was added to the shuttle agent, and sterile PBS was added, if necessary, to obtain the desired concentrations of shuttle agent and cargo in a final volume sufficient to cover the cells (e.g., 10–100 μL per well of a 96-well plate). The shuttle agent / cargo mixture was then immediately used for experiments. Each experiment included at least three controls: (1) a control of shuttle agent alone (e.g., at the highest concentration tested), (2) a control of cargo alone, and (3) a control containing neither cargo nor shuttle agent. The medium in the wells was removed, the cells were washed once with PBS pre-warmed to 37°C, and then the shuttle agent / cargo mixture was added to cover all cells for the desired time. The shuttle agent / cargo mixture in the wells was removed, the cells were washed once with PBS, and fresh complete medium was added. Prior to analysis, the cells were washed once with PBS, and fresh complete medium was added.

[0268] Protocol C: Protein transduction assay for suspension cells in PBS One day before transduction assays were performed, logarithmically growing suspension cells were harvested and seeded into 96-well plates (20,000 cells per well). The cells were incubated overnight in the appropriate growth medium containing serum (see Example 1). The following day, in separate sterile 1.5 mL tubes, the shuttle agent was diluted in sterile distilled water at room temperature (nuclease-free water was used if the cargo was a nucleic acid or contained a nucleic acid). The cargo protein was added to the shuttle agent, and sterile PBS or cell culture medium (serum-free) was added as needed to obtain the desired concentrations of shuttle agent and cargo in a final volume sufficient to resuspend the cells (e.g., 10–100 μL per well of a 96-well plate). The shuttle agent / cargo mixture was then immediately used for experiments. Each experiment included at least three controls: (1) a control of shuttle agent alone (e.g., at the highest concentration tested), (2) a control of cargo alone, and (3) a control without either cargo or shuttle agent. The cells were centrifuged at 400g for 2 minutes, then the medium was removed and the cells were resuspended in PBS pre-warmed to 37°C. The cells were centrifuged again at 400g for 2 minutes, the PBS was removed and the cells were resuspended in the shuttle agent / cargo mixture. After the desired incubation time, 100µL of complete medium was added directly to the cells. The cells were centrifuged at 400g for 2 minutes and the medium was removed. The pellet was resuspended and washed in 200µL of PBS pre-warmed to 37°C. After another centrifugation, the PBS was removed and the cells were resuspended in 100µL of complete medium. The last two steps were repeated once before analysis.

[0269] 9.2 GFP-NLS Transduction with His-CM18-PTD4 in HeLa Cells Using Protocol A or B: Flow Cytometry To compare the effects of different protocols on shuttle agent transduction efficiency, HeLa cells were cultured and tested in a protein transduction assay using Protocol A or B as described in Example 9.1. Briefly, using Protocol A, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 10 μM His-CM18-PTD4 and exposed to HeLa cells for 1 hour, or using Protocol B, it was co-incubated with 35 μM His-CM18-PTD4 and exposed to HeLa cells for 10 seconds. Cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 9.1 and Figure 22A. ("Pos cells (%)" is the percentage of cells emitting GFP signal.) [Table 34]

[0270] The above results show that, compared to protocol A, higher protein transduction efficiency of the cargo GFP-NLS using the shuttle agent His-CM18-PTD4 was obtained using protocol B.

[0271] 9.3 GFP-NLS Transduction with His-CM18-PTD4 in HeLa Cells Using Protocol B: Flow Cytometry A dose-response experiment was performed to evaluate the effect of His-CM18-PTD4 concentration on protein transduction efficiency. HeLa cells were cultured and tested in the protein transduction assay described in Protocol B of Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 0, 50, 35, 25, or 10 μM His-CM18-PTD4 and then exposed to HeLa cells for 10 seconds. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 9.2 and Figure 22B. [Table 35]

[0272] The above results indicate that His-CM18-PTD4 can increase GFP-NLS transduction efficiency in HeLa cells in a dose-dependent manner.

[0273] 9.4 GFP-NLS Transduction with His-CM18-PTD4 in HeLa Cells Using Protocol B: Visualization by Microscopy GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 35 μM His-CM18-PTD4 using Protocol B as described in Example 9.1 and then exposed to HeLa cells for 10 seconds. The cells were then subjected to fluorescence microscopy analysis as described in Examples 3.2 and 3.2a.

[0274] For the example results shown in Figures 23 and 24, GFP fluorescence of HeLa cells was immediately visualized after the final washing step by bright field and fluorescence microscopy at 4x, 20x and 40x magnification.

[0275] In Figure 23, the top panels of Figures 23A, 23B, and 23C show nuclear labeling (DAPI) at 4x, 20x, and 40x magnification, respectively, and the bottom panels show the corresponding GFP-NLS fluorescence. In Figure 23C, the white triangular window indicates an example of a co-labeled area between the nuclear (DAPI) and GFP-NLS signals. In Figure 23D, the top and bottom panels show example bright-field images of HeLa cells, and the middle panel shows the results of the corresponding FACS analysis (performed as described in Example 3.3), which shows the percentage of cells with GFP signal in a 96-well plate. No significant GFP fluorescence was observed in the negative control sample (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0276] Figure 24 shows bright-field (Figure 24A) and fluorescent images (Figure 24B). The inset in Figure 24B shows the results of the corresponding FACS analysis (performed as described in Example 3.3), which shows the percentage of cells with GFP signal in a 96-plate well. No significant GFP fluorescence was observed in the negative control sample (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0277] For the example results shown in Figure 25, HeLa cells were fixed, permeabilized, and subjected to immunolabeling as described in Example 3.2a, and then visualized by fluorescence microscopy as described in Example 3.2. GFP-NLS was labeled using a primary mouse monoclonal anti-GFP antibody (Feldan, #A017) and a secondary goat anti-mouse Alexa™-594 antibody (Abcam #150116). The top panels of Figures 25A and 25B show nuclear labeling (DAPI), and the bottom panels show the corresponding labeling of GFP-NLS. Figures 25A and 25B show example images at 20x and 40x magnification, respectively. The white triangular window indicates an example area of ​​co-labeling between the nucleus and GFP-NLS. No significant GFP-NLS labeling was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0278] Figure 26 shows example images acquired using a confocal microscope at 63x magnification of live cells. Figure 26A shows a bright-field image, and Figure 26B shows the corresponding fluorescent GFP-NLS. Figure 26C is an overlay between the images in Figure 26A and Figure 26B. No significant GFP-NLS fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0279] 9.4a FTIC-labeled anti-tubulin antibody transduction with His-CM18-PTD4 in HeLa cells using protocol B: Visualization by microscopy FITC-labeled anti-tubulin antibody (0.5 μM, Abcam, ab64503) was co-incubated with 50 μM His-CM18-PTD4 using Protocol B as described in Example 9.1 and then exposed to HeLa cells for 10 seconds. The cells were then subjected to fluorescence microscopy analysis as described in Examples 3.2 and 3.2a, and the FITC fluorescence of the anti-tubulin antibody in HeLa cells was immediately visualized by bright-field and fluorescence microscopy at 20x magnification after the final washing step. Example results are shown in Figures 24C and 24D. No significant FITC fluorescence was observed in the negative control sample (i.e., cells exposed to FITC-labeled anti-tubulin antibody without any shuttle agent; data not shown).

[0280] Overall, the results in Examples 9.4 and 9.4a demonstrate that GFP-NLS and FITC-labeled anti-tubulin antibody cargo are successfully transduced and delivered to the nucleus and / or cytosol of HeLa cells in the presence of the shuttle agent His-CM18-PTD4.

[0281] 9.5 GFP-NLS kinetic transduction by His-CM18-PTD4 in HeLa cells: visualization by microscopy GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 50 μM His-CM18-PTD4 using Protocol B as described in Example 9.1, and then exposed to HeLa cells for 10 seconds. After a washing step, GFP fluorescence of HeLa cells was immediately visualized by fluorescence microscopy (Example 3.2) at 20x magnification after various time intervals. Typical results are shown in Figure 27, where fluorescence microscope images were acquired after 45, 75, 100, and 120 seconds (see Figures 27A, 27B, 27C, and 27D, respectively).

[0282] As shown in Figure 27A, diffuse cellular GFP fluorescence was typically observed after 45 seconds, with many cells exhibiting areas of lower GFP fluorescence in the nucleus. These results suggest a primarily cytoplasmic and low nuclear distribution of GFP-NLS delivered into cells via the shuttle agent after 45 seconds. Figures 27B–27D show the gradual redistribution of GFP fluorescence to the cell nucleus after 75 seconds (Figure 27B), 100 seconds (Figure 27C), and 120 seconds (Figure 27D) of exposure to the His-CM18-PTD4 shuttle agent and GFP-NLS cargo. No significant GFP fluorescence was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0283] The results in Example 9.5 show that GFP-NLS is successfully delivered to the nucleus of HeLa cells by 2 minutes in the presence of the shuttle agent His-CM18-PTD4.

[0284] 9.6 GFP-NLS and mCherry™-NLS Co-Transduction with His-CM18-PTD4 in HeLa Cells: Visualization by Microscopy The mCherry™-NLS recombinant protein was constructed, expressed and purified from a bacterial expression system as described in Example 1.4. The sequence of the mCherry™-NLS recombinant protein was: [ka] (MW=34.71kDa; pI=6.68) The NLS sequence is underlined. The serine / glycine-rich linker is in bold.

[0285] GFP-NLS recombinant protein (5 μM, see Example 5.1) and mCherry™-NLS recombinant protein (5 μM) were co-incubated with 35 μM His-CM18-PTD4 using Protocol B as described in Example 9.1 and then exposed to HeLa cells for 10 seconds. After a washing step, cells were immediately visualized by brightfield and fluorescence microscopy at 20x magnification as described in Example 3.2. Example results are shown in Figure 28, which shows corresponding images showing brightfield (Figure 28A), DAPI fluorescence (Figure 28B), GFP-NLS fluorescence (Figure 28C), and mCherry™-NLS fluorescence (Figure 28D). The open triangle window indicates an example area of ​​co-labeling between GFP-NLS and mCherry™ fluorescent signals in the cell nucleus. No significant GFP or mCherry™ fluorescence was observed in negative control samples (ie, cells exposed to GFP-NLS or mCherry™ without either shuttle agent; data not shown).

[0286] These results demonstrate that GFP-NLS and mCherry™-NLS are successfully delivered together to the nucleus in HeLa cells in the presence of the shuttle agent His-CM18-PTD4.

[0287] 9.7 GFP-NLS Transduction with His-CM18-PTD4 in THP-1 Suspension Cells: Flow Cytometry The ability of His-CM18-PTD4 to deliver GFP-NLS to the nuclei of suspension cells was tested using THP-1 cells. THP-1 cells were cultured and tested in a protein transduction assay using protocols A and C as described in Example 9.1. GFP-NLS (5 μM, see Example 5.1) was co-incubated with 1 μM His-CM18-PTD4 and exposed to THP-1 cells for 1 hour (protocol A) or with 5 μM His-CM18-PTD4 and exposed to THP-1 cells for 15 seconds (protocol C). Cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 9.3 and Figure 31. [Table 36]

[0288] 9.8 GFP-NLS transduction with His-CM18-PTD4 in THP-1 cells: visualization by microscopy GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 5 μM His-CM18-PTD4 using Protocol C as described in Example 9.1 and then exposed to THP-1 cells for 15 seconds. Cells were subjected to microscopic visualization as described in Example 3.2.

[0289] For the example results shown in Figure 32, GFP fluorescence of HeLa cells was visualized immediately after the final washing step by bright-field (top panel) and fluorescence (bottom panel) microscopy at 4x, 10x, and 40x magnification (Figures 32A-32C, respectively). The open triangular window in Figure 32C indicates an example of the area of ​​co-labeling between the bright-field and fluorescence images. Figure 32D shows a typical result of the corresponding FACS analysis (performed as described in Example 3.3), showing the percentage of cells in a 96-well plate with GFP signal. Further results are shown in Figure 33, in which Figures 33A and 33B show bright-field images and Figures 33C and 33D show the corresponding fluorescence images. The open triangular window indicates an example of the area of ​​co-labeling between Figures 33A and 33C, and between Figures 33B and 33D. The right-most panel shows typical results of the corresponding FACS analysis (performed as described in Example 3.3), showing the percentage of cells with a GFP signal in a 96-plate well.

[0290] No significant GFP fluorescence was observed in negative control samples (ie, cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0291] The results in this example demonstrate that GFP-NLS is successfully delivered intracellularly in THP-1 cells in the presence of the shuttle agent His-CM18-PTD4.

[0292] Example 10: Various multi-domain shuttling agents, but not single-domain peptides, successfully transduce GFP-NLS in HeLa and THP-1 cells. 10.1 GFP-NLS transduction by various shuttle agents in HeLa cells: flow cytometry HeLa cells were cultured and tested in a protein transduction assay using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 50 μM of various shuttle agents and exposed to HeLa cells for 10 seconds. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 10.1 and Figure 29A. "Pos cells (%)" is the average percentage of all cells emitting a GFP signal. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 37]

[0293] 10.2 GFP-NLS transduction in HeLa cells with different incubation times and different shuttle agents: Flow cytometry HeLa cells were cultured and tested in a protein transduction assay using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 10 μM TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 1, 2, or 5 minutes. After a final washing step, cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 10.2 and Figure 29B. "Pos cells (%)" is the average percentage of all cells emitting a GFP signal. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 38]

[0294] 10.3 GFP-NLS transduction with various incubation times and TAT-KALA, His-CM18-PTD4, and His-C(LLKK)3C-PTD4 in HeLa cells: flow cytometry HeLa cells were cultured and tested in a protein transduction assay using Protocol C as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 5 μM TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 1, 2, or 5 minutes. After a final washing step, cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 10.3 and Figure 29C. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 39]

[0295] 10.4 GFP-NLS transduction by various shuttle agents in HeLa cells: flow cytometry HeLa cells were cultured and tested in a protein transduction assay using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 50 μM of various shuttle agents (see Table 1.3 for amino acid sequence and characteristics) and exposed to HeLa cells for 10 seconds. Cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Tables 10.3a and 10.3b and Figures 29E and 29F. "Pos cells (%)" is the average percentage of all cells emitting a GFP signal. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 40] [Table 41]

[0296] HeLa cells were cultured and tested in a protein transduction assay using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 10 μM TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 1, 2, or 5 minutes. After a final washing step, cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Tables 10.3c and 10.3b and Figures 29G and 29H. "Pos cells (%)" is the average percentage of all cells emitting a GFP signal. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 42] [Table 43]

[0297] The shuttle agent CM18-PTD4 was used as a model to demonstrate the modularity of individual protein domains and their ability to be modified. More specifically, the presence or absence of an N-terminal cysteine ​​residue ("Cys"), various flexible linkers between the ELD and CPD domains ("L1":GGS, "L2":GGSGGGS, and "L3":GGSGGGSGGGS), and various lengths, positions, and variants of the histidine-rich domain were investigated.

[0298] HeLa cells were cultured and tested in a protein transduction assay using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 20 μM of various shuttle peptide variants of the shuttle agent His-CM18-PTD4 (see Table 1.3 for amino acid sequence and characteristics) for 1 minute. After a final washing step, cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 10.3e and Figure 29I. "Pos cells (%)" is the average percentage of all cells emitting a GFP signal. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 44]

[0299] These results demonstrate that variations in a given shuttle (e.g., CM18-PTD4) can be used to modulate the degree of transduction efficiency and cell viability of a given shuttle. More specifically, the addition of an N-terminal cysteine ​​residue to CM18-PTD4 (see Cys-CM18-PTD4) reduced GFP-NLS transduction efficiency by 11% (from 47.6% to 36.6%) but increased cell viability from 33.9% to 78.7%. Introduction of flexible linker domains of various lengths (L1, L2, and L3) between the CM18 and PTD4 domains did not result in a significant loss of transduction efficiency but did increase cell viability (see CM18-L1-PTD4, CM18-L2-PTD4, and CM18-L3-PTD4). Finally, variation in the amino acid sequence and / or position of the histidine-rich domain did not result in a complete loss of transduction efficiency and cell viability of His-CM18-PTD4 (see 3His-CM18-PTD4, 12His-CM18-PTD4, HA-CM18-PTD4, 3HA-CM18-PTD4, CM18-His-PTD4, and His-CM18-PTD4-His). Of note, adding a second histidine-rich domain to the C-terminus of His-CM18-PTD4 (i.e., His-CM18-PTD4-His) increased transduction efficiency from 60% to 68%, with similar cell viability.

[0300] 10.5 Lack of GFP-NLS transduction by single domain peptides or His-CPD peptide in HeLa cells: flow cytometry HeLa cells were cultured and tested in a protein transduction assay using Protocol B as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 50 μM of various single-domain peptides (TAT, PTD4, penetratin, CM18, C(LLKK)3C, KALA) or the two-domain peptide His-PTD4 (lacking ELD) and exposed to HeLa cells for 10 seconds. After a final washing step, cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 10.4 and Figure 29D. "Pos cells (%)" is the average percentage of all cells emitting a GFP signal. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any single-domain peptide or shuttle agent. [Table 45]

[0301] These results show that the single domain peptides TAT, PTD4, penetratin, CM18, C(LLKK)3C, KALA or the two domain peptide His-PTD4 (lacking the ELD) cannot successfully transduce GFP-NLS in HeLa cells.

[0302] 10.6 GFP-NLS transduction with TAT-KALA, His-CM18-PTD4, His-C(LLKK)3C-PTD4, PTD4-KALA, EB1-PTD4, and His-CM18-PTD4-His in HeLa cells: visualization by microscopy GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 50 μM of shuttle agent using Protocol B as described in Example 9.1 and then exposed to HeLa cells for 10 seconds. After a 2-minute incubation, cells were visualized by microscopy as described in Example 3.2.

[0303] For the example results in Figure 30, GFP fluorescence of HeLa cells was immediately visualized by bright-field (bottom panel) and fluorescence (top and middle panels) microscopy at 20x or 40x magnification after the final washing step. Results using the shuttle agents TAT-KALA, His-CM18-PTD4, and His-C(LLKK)3C-PTD4 are shown in Figures 30A, 30B, and 30C, respectively. Results using the shuttle agents PTD4-KALA, EB1-PTD4, and His-CM18-PTD4-His are shown in Figures 30D, 30E, and 30F, respectively. The inset in the bottom panel shows the results of the corresponding FACS analysis (performed as described in Example 3.3), which indicates the percentage of cells in a 96-well plate with a GFP signal. No significant GFP fluorescence was observed in the negative control sample (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0304] 10.7 GFP-NLS transduction with various incubation times and TAT-KALA, His-CM18-PTD4, and His-C(LLKK)3C-PTD4 in THP-1 cells: Flow cytometry THP-1 cells were cultured and tested in a protein transduction assay using Protocol C as described in Example 9.1. Briefly, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 1 μM TAT-KALA, His-CM18-PTD4, or His-C(LLKK)3C-PTD4 for 15, 30, 60, or 120 seconds. After a final washing step, cells were subjected to flow cytometry analysis as described in Example 3.3. The average percentage of all cells emitting a GFP signal ("Pos cells (%)") is shown in Table 10.4a and Figure 34A. The average fluorescence intensity is shown in Table 10.5 and Figure 34B. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 46] [Table 47]

[0305] Example 11: Repeated daily treatment with low concentrations of shuttle agents in the presence of serum results in GFP-NLS transduction in THP-1 cells 11.1 GFP-NLS Transduction with His-CM18-PTD4 or His-C(LLKK)3C-PTD4 in THP-1 Cells: Flow Cytometry THP-1 cells were cultured and tested in a protein transduction assay using Protocol A described in Example 9.1, with the following modifications: GFP-NLS recombinant protein (5, 2.5, or 1 μM, see Example 5.1) was co-incubated with 0.5 or 0.8 μM His-CM18-PTD4 or 0.8 μM His-C(LLKK)3C-PTD4, and then exposed to THP-1 cells for 150 minutes each day in the presence of serum-containing cell culture medium. After repeated exposure to the shuttle agent / cargo for 1 or 3 days, the cells were washed and subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table 11.1 and Figures 35A, 35B, 35C, and 35F. The negative control ("Ctrl") corresponds to cells incubated with GFP-NLS recombinant protein (5 μM) without any shuttle agent. [Table 48]

[0306] The viability of THP-1 cells repeatedly exposed to His-CM18-PTD4 and GFP-NLS was examined as described in Example 3.3a. The results are shown in Tables 11.2 and 11.3 and in Figures 35D and 35E. The results in Table 11.2 and Figure 35D show the metabolic activity index of THP-1 cells after 1, 2, 4, and 24 hours, while the results in Table 11.3 and Figure 35E show the metabolic activity index of THP-1 cells after 1 to 4 days. [Table 49] [Table 50]

[0307] The results in Example 11 show that repeated daily (or chronic) treatment with relatively low concentrations of His-CM18-PTD4 or His-C(LLKK)3C-PTD4 in the presence of serum results in intracellular delivery of GFP-NLS in THP-1 cells. The results also suggest that the dosage of shuttle agent and cargo can be independently adjusted to improve cargo transduction efficiency and / or cell viability.

[0308] Example 12: His-CM18-PTD4 increases transduction efficiency and nuclear delivery of GFP-NLS in multiple cell lines 12.1 GFP-NLS Transduction by His-CM18-PTD4 in Various Adherent and Suspension Cells: Flow Cytometry The ability of the shuttle agent His-CM18-PTD4 to deliver GFP-NLS to the nuclei of various adherent and suspension cells was examined using Protocol B (adherent cells) or Protocol C (suspension cells) as described in Example 9.1. Cell lines tested included HeLa, Balb3T3, HEK293T, CHO, NIH3T3, myoblast, Jurkat, THP-1, CA46, and HT2 cells, which were cultured as described in Example 1. GFP-NLS (5 μM, see Example 5.1) was co-incubated with 35 μM His-CM18-PTD4 and exposed for 10 seconds to adherent cells (Protocol B) or with 5 μM His-CM18-PTD4 and exposed for 15 seconds to suspension cells (Protocol C). Cells were washed and subjected to flow cytometry analysis as described in Example 3.3. Results are shown in Table 12.1 and Figure 36. "Pos cells (%)" is the average percentage of all cells emitting a GFP signal. [Table 51]

[0309] 12.2 GFP-NLS transduction with His-CM18-PTD4 in several adherent and suspension cells: visualization by microscopy As described in Example 9.1, GFP-NLS recombinant protein (5 μM, see Example 5.1) was co-incubated with 35 μM His-CM18-PTD4 using Protocol A and exposed for 10 seconds to adherent cells, or with 5 μM His-CM18-PTD4 using Protocol B and exposed for 15 seconds to suspension cells. After washing the cells, GFP fluorescence was visualized by bright-field and fluorescence microscopy. Example images of GFP fluorescence acquired at 10x magnification are shown for 293T (Figure 37A), Balb3T3 (Figure 37B), CHO (Figure 37C), myoblast (Figure 37D), Jurkat (Figure 37E), CA46 (Figure 37F), HT2 (Figure 37G), and NIH3T3 (Figure 37H) cells. The inset shows the percentage of GFP-NLS-positive cells with corresponding flow cytometry results performed as described in Example 3.3. No significant GFP fluorescence was observed in negative control samples (ie, cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0310] The nuclear localization of GFP-NLS in fixed and permeabilized myoblasts was further confirmed using cell immunolabeling as described in Example 3.2a. GFP-NLS was labeled using a primary mouse monoclonal anti-GFP antibody (Feldan, #A017) and a secondary goat anti-mouse Alexa™-594 antibody (Abcam #150116). Nuclei were labeled with DAPI. Example results for primary human myoblasts are shown in Figure 38, where GFP immunolabeling is shown in Figure 38A and an overlay of GFP immunolabeling and DAPI labeling is shown in Figure 38B. No significant GFP labeling was observed in negative control samples (i.e., cells exposed to GFP-NLS without any shuttle agent; data not shown).

[0311] Microscopic results showed that GFP-NLS was successfully delivered to the nuclei of all tested cells using the shuttle agent His-CM18-PTD4.

[0312] Example 13: His-CM18-PTD4 enables CRISPR / Cas9-NLS system transduction and genome editing in HeLa cells 13.1 Cas9-NLS Recombinant Protein The Cas9-NLS recombinant protein was constructed, expressed from a bacterial expression system, and purified as described in Example 1.4. The sequence of the produced Cas9-NLS recombinant protein was as follows: [ka] (MW=162.9kDa; pI=9.05) The NLS sequence is underlined. The serine / glycine-rich linker is in bold.

[0313] 13.2 Gene Transfer Plasmid Replacement Assay This assay allows for visual identification of successfully delivered active CRISPR / Cas9 complexes. As shown in Figure 39A, the assay involves transfecting cells with expression plasmid DNA encoding the fluorescent proteins mCherry™ and GFP, with a stop codon separating the two open reading frames. Transfection of cells with the expression plasmid results in mCherry™ expression but not GFP expression (Figure 39B). A CRISPR / Cas9 complex designed / programmed to cleave the plasmid DNA at the stop codon is then delivered intracellularly to the transfected cells expressing mCherry™ (Figure 39D). Successful transduction of the active CRISPR / Cas9 complex results in the CRISPR / Cas9 complex cleaving the plasmid DNA at the stop codon (Figure 39C). In some cells, random non-homologous DNA repair of the cleaved plasmid occurs, resulting in removal of the stop codon and thus GFP expression and fluorescence (Figure 39E).

[0314] On day 1 of the transfection plasmid replacement assay, DNA plasmids (250 ng) for various experimental conditions were diluted in 50 μL of DMEM in separate sterile 1.5 mL tubes, vortexed, and briefly centrifuged. In separate sterile 1.5 mL tubes, Fastfect™ transfection reagent was diluted 3:1 (1 μg of DNA for 3 μL of Fastfect™ transfection reagent) in 50 μL of serum-free, antibiotic-free DMEM, vortexed briefly, and briefly centrifuged. The Fastfect™ / DMEM mixture was then added to the DNA mixture, vortexed briefly, and briefly centrifuged. The Fastfect™ / DMEM / DNA mixture was then incubated at room temperature for 15–20 minutes before being added to the cells (100 μL per well). The cells were then incubated at 37°C and 5% CO2 for 5 hours. The medium was then replaced with complete medium (containing serum) and further incubated for 24-48 hours at 37°C and 5% CO. The cells were then visualized under a fluorescent microscope to view the mCherry™ signal.

[0315] 13.3 His-CM18-PTD4-mediated CRISPR / Cas9-NLS system delivery and cleavage of plasmid DNA RNAs (crRNA and tracrRNA) were designed to target the nucleotide sequence of the EMX1 gene containing a stop codon between the mCherry™ and GFP coding sequences in the plasmid of Example 13.2. The sequences of the crRNA and tracrRNA used were as follows: [ka]

[0316] HeLa cells were cultured and subjected to a transfection plasmid replacement assay as described in Example 13.2. On day 1, HeLa cells were transfected with a plasmid replacement encoding mCherry™ protein, as shown in Figure 39A. On day 2, using Protocol B as described in Example 9.1, a mixture of Cas9-NLS recombinant protein (2 μM, see Example 13.1) and RNA (crRNA and tracrRNA; 2 μM, see above) was co-incubated with 50 μM His-CM18-PTD4, and the mixture (CRISPR / Cas9 complex) was exposed to HeLa cells for 10 seconds. Double-stranded plasmid DNA cleavage by the CRISPR / Cas9 complex at the stop codon between the mCherry™ and GFP coding sequences (Figure 39B) and subsequent non-homologous repair by the cell in some cases results in stop codon removal (Figure 39C), thereby allowing expression of both mCherry™ and GFP fluorescent proteins in the same cell on day 3 (Figures 39D-E). The open triangular windows in Figures 39D and 39E show examples of areas of co-labeling between mCherry™ and GFP.

[0317] As a positive control for the CRISPR / Cas9-NLS system, HeLa cells were cultured and co-transfected with three plasmids: a plasmid substitute (as described in Example 13.2) and another expression plasmid encoding the Cas9-NLS protein (Example 13.1) and crRNA / tracrRNA (Example 13.3). Typical fluorescence microscopy results are shown in Figures 40A-D. Panels A and B show cells 24 hours after transfection, and panels C and D show cells 72 hours after transfection.

[0318] Figures 40E-40H show the results of a parallel gene transfer plasmid replacement assay performed using 35 μM of the shuttle His-CM18-PTD4, as described in Figure 39. Figures 40E and 40F show cells 24 hours after transduction, and panels G and H show cells 48 hours after transduction. Figures 40E and 40G show mCherry™ fluorescence, and Figures 40F and 40H show GFP fluorescence, the latter resulting from the transduced CRISPR / Cas9-NLS complex and subsequent removal of the stop codon by nonhomologous repair by the cells. No significant cellular GFP fluorescence was observed in negative control samples (i.e., cells exposed to CRISPR / Cas9-NLS complexes without any shuttle agent; data not shown).

[0319] 13.4 T7E1 Assay On-target CRISPR / Cas genome editing events can be detected in cultured cells using T7 endonuclease I (T7E1). Briefly, genomic DNA from target cells is amplified by PCR. The PCR products are then denatured and reannealed to allow heteroduplex formation between wild-type and CRISPR / Cas-mutated DNA. The heteroduplex is digested using T7E1, which recognizes and cleaves mismatched DNA. The resulting cleaved and full-length PCR products are visualized by gel electrophoresis.

[0320] The T7E1 assay was performed using Edit-R™ synthetic crRNA positive control (Dharmacon #U-007000-05) and T7 endonuclease I (NEB, catalog #M0302S). After delivery of the CRISPR / Cas complex, cells were lysed in 100 μL of Phusion™ High-Fidelity DNA Polymerase (NEB #M0530S) with additives. Cells were incubated at 56°C for 15–30 minutes, followed by inactivation at 96°C for 5 minutes. The plate was briefly centrifuged to collect the liquid at the bottom of the wells. For each sample analyzed, a 50 μL PCR sample was prepared. The PCR sample was heated to 95°C for 10 minutes and then slowly cooled (>15 minutes) to room temperature. PCR products (approximately 5 μL) were then separated on a 2% agarose gel to confirm amplification. 15 μL of each reaction was incubated with T7E1 nuclease for 25 minutes at 37° C. The entire reaction volume was immediately run on an agarose gel (2%) with the appropriate gel loading buffer.

[0321] 13.5 His-CM18-PTD4 and His-C(LLKK)3C-PTD4-mediated CRISPR / Cas9-NLS system delivery and cleavage of genomic PPIB sequences Using protocol A as described in Example 9.1, a mixture composed of Cas9-NLS recombinant protein (25 nM, Example 13.1) and crRNA / tracrRNA (50 nM, see below) targeting the nucleotide sequence of the PPIB gene was co-incubated with 10 μM His-CM18-PTD4 or His-C(LLKK)3C-PTD4 and incubated with HeLa cells in serum-free medium for 16 hours. The sequences of the constructed crRNA and tracrRNA and their targets were as follows: [ka] After 16 hours, HeLa cells were washed with PBS and incubated in serum-containing medium for 48 hours. HeLa cells were harvested and subjected to the T7E1 protocol assay as described in Example 13.4.

[0322] Figure 41A shows an agarose gel with the PPIB DNA sequence after PCR amplification. Lane A shows the amplified PPIB DNA sequence in HeLa cells without any treatment (i.e., without the shuttle or Cas9 / RNA complex). Lane B: The two bands boxed in white box number 1 are the cleavage products of the PPIB DNA sequence by the CRIPR / Cas9 complex after delivery of the complex using the shuttle His-C(LLKK)3C-PTD4. Lane C: These bands represent the PPIB DNA sequence amplified after incubation of HeLa cells with the Cas9 / RNA complex without the shuttle (negative control). Lane D: The band boxed in white box number 2 represents the amplified PPIB DNA sequence after incubation of HeLa cells with the Cas9 / RNA complex in the presence of a lipid transfection agent (DharmaFect™ Transfection Reagent #T-20XX-01) (positive control). Similar results were obtained using the shuttle His-CM18-PTD4 (data not shown).

[0323] Figure 41B shows an agarose gel with the PPIB DNA sequence after PCR amplification. The left panel shows the cleavage product of the amplified PPIB DNA sequence by the CRIPR / Cas9 complex after delivery of the complex using the shuttle agent His-CM18-PTD4 in HeLa cells. The right panel shows the amplified DNA sequence before the T7E1 digestion procedure as a negative control.

[0324] Figure 41C shows an agarose gel with the PPIB DNA sequence after PCR amplification. The left panel shows the PPIB DNA sequence amplified after incubating HeLa cells with Cas9 / RNA complexes in the presence of a lipid transfection agent (DharmaFect™ Transfection Reagent #T-20XX-01) (positive control). The right panel shows the amplified DNA sequence before the T7E1 digestion procedure as a negative control.

[0325] These results demonstrate that the shuttle agents His-CM18-PTD4 and His-C(LLKK)3C-PTD4 successfully deliver functional CRISPR / Cas9 complexes to the nuclei of HeLa cells and that this delivery results in CRISPR / Cas9-mediated cleavage of genomic DNA.

[0326] 13.6 CRISPR / Cas9-NLS system delivery and cleavage of genomic HPTR sequences by various shuttle agents in HeLa and Jurkat cells Using protocol B as described in Example 9.1, a mixture composed of Cas9-NLS recombinant protein (2.5 μM, Example 13.1) and crRNA / tracrRNA (2 μM, see below) targeting the nucleotide sequence of the HPTR gene was co-incubated with 35 μM His-CM18-PTD4, His-CM18-PTD4-His, His-C(LLKK)3C-PTD4 or EB1-PTD4 and incubated with HeLa or Jurkat cells in PBS for 2 minutes. The sequences of the constructed crRNA and tracrRNA and their targets were as follows: [ka]

[0327] After 2 minutes, the cells were washed with PBS and incubated in serum-containing medium for 48 hours. The cells were harvested and subjected to the T7E1 protocol assay as described in Example 13.4. Figure 46 shows an agarose gel with the PCR-amplified HPTR DNA sequence and the cleavage products of the amplified HPTR DNA sequence by the CRISPR / Cas9 complex after delivery of the complex using various shuttle agents. Figure 46A shows the results using the following shuttle agents in HeLa cells: His-CM18-PTD4, His-CM18-PTD4-His, and His-C(LLKK)3C-PTD4. Figure 46B shows the results using His-CM18-PTD4 and His-CM18-L2-PTD4 in Jurkat cells. The negative control (lane 4) shows the amplified HPTR DNA sequence after incubation of cells with the CRISPR / Cas9 complex without the shuttle agent. The positive control (lane 5 in Figures 46A and 46B) shows the HPTR DNA sequence amplified after incubating cells with Cas9 / RNA complexes in the presence of a lipid transfection agent (Lipofectamine® RNAiMAX™ Transfection Reagent ThermoFisher product number 13778100).

[0328] These results demonstrate that the various polypeptide shuttle agents described herein can successfully deliver functional CRISPR / Cas9 complexes into the nuclei of HeLa and Jurkat cells, and that this delivery results in CRISPR / Cas9-mediated cleavage of genomic DNA.

[0329] Example 14: His-CM18-PTD4 enables transduction of the transcription factor HOXB4 in THP-1 cells 14.1 HOXB4-WT recombinant protein Human HOXB4 recombinant protein was constructed, expressed from a bacterial expression system, and purified as described in Example 1.4. The sequence of the produced HOXB4-WT recombinant protein was as follows: [ka] (MW=28.54kDa; pI=9.89) The initiator methionine and 6x histidine tag are in bold.

[0330] 14.2 Real-time polymerase chain reaction (rt-PCR) Control and treated cells were transferred to separate sterile 1.5 mL tubes and centrifuged at 300 g for 5 minutes. The cell pellet was resuspended in the appropriate buffer to lyse the cells. RNAse-free 70% ethanol was then added, followed by mixing by pipetting. The lysate was transferred to an RNeasy™ Mini spin column and centrifuged at 13,000 RPM for 30 seconds. After several washing and centrifugation steps with the appropriate buffer, the eluate was collected in a sterile 1.5 mL tube on ice, and the amount of RNA in each tube was then quantified using a spectrophotometer. For DNase treatment, 2 μg of RNA was diluted with 15 μL of RNase-free water. Then, 1.75 μL of 10X DNase buffer and 0.75 μL of DNase were added, followed by incubation at 37°C for 15 minutes. For reverse transcriptase treatment, 0.88 μL of EDTA (50 nM) was added, followed by incubation at 75°C for 5 minutes. In a PCR tube, 0.5 μg of DNase-treated RNA is mixed with 4 μL of iScript™ Reverse Transcription Supermix (5X) and 20 μL of nuclease-free water. The mixture is incubated in a PCR machine using the following program: 25°C for 5 minutes, 42°C for 30 minutes, and 85°C for 5 minutes. The newly synthesized cDNA is transferred into a sterile 1.5 mL tube and diluted with 2 μL of nuclease-free water. 18 μL of the mixture per well of the qPCR machine (CFX-96™) is added to a PCR plate for analysis.

[0331] 14.3 HOXB4-WT Transduction with His-CM18-PTD4 in THP-1 Cells: Dose Response and Viability THP-1 cells were cultured and tested in a protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were seeded at 30,000 cells / well one day before transduction. HOXB4-WT recombinant protein (0.3, 0.9, or 1.5 μM, Example 14.1) was co-incubated with various concentrations of His-CM18-PTD4 (0, 0.5, 7.5, 0.8, or 1 μM) and then exposed to THP-1 cells in the presence of serum for 2.5 hours. To measure target gene mRNA levels as a marker of HOXB4 activity, cells were subjected to real-time PCR analysis as described in Example 14.2, and then normalized to the target gene mRNA levels detected in negative control cells (untreated) to obtain "fold over control" values. Total RNA levels (ng / μL) were also measured as a marker of cell viability. The results are shown in Table 14.1 and Figure 42. [Table 52]

[0332] These results show that exposure of THP-1 cells to a mixture of the shuttle agent His-CM18-PTD4 and the transcription factor HOXB4-WT for 2.5 hours in the presence of serum resulted in a dose-dependent increase in target gene mRNA transcription. These results suggest that HOXB4-WT can be successfully delivered to the nucleus of THP-1 cells in an active form and mediate transcriptional activation.

[0333] 14.4 HOXB4-WT Transduction with His-CM18-PTD4 in THP-1 Cells: Time Course and Viability (0-48 hours) THP-1 cells were cultured and tested in a protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were seeded at 30,000 cells / well one day before the first time course experiment. HOXB4-WT recombinant protein (1.5 μM, Example 14.1) was co-incubated with His-CM18-PTD4 (0.8 μM) and then exposed to THP-1 cells in the presence of serum for 0, 2.5, 4, 24, or 48 hours. To measure target gene mRNA levels as a marker of HOXB4 activity, cells were subjected to real-time PCR analysis as described in Example 14.2 and then normalized to the target gene mRNA levels detected in negative control cells (untreated) to obtain "fold over control" values. Total RNA levels (ng / μL) were also measured as a marker of cell viability. The results are shown in Table 14.2 and Figure 43. [Table 53]

[0334] 14.5 Transduction of HOXB4-WT with His-CM18-PTD4 in THP-1 cells: Time course and viability (0-4 hours) THP-1 cells were cultured and tested in a protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were seeded at 30,000 cells / well one day before the first time course experiment. HOXB4-WT recombinant protein (0.3 μM, Example 14.1) was co-incubated with His-CM18-PTD4 (0.8 μM) and then exposed to THP-1 cells in the presence of serum for 0, 0.5, 1, 2, 2.5, 3, or 4 hours. To measure target gene mRNA levels as a marker of HOXB4 activity, cells were subjected to real-time PCR analysis as described in Example 14.2 and then normalized to the target gene mRNA levels detected in negative control cells (untreated) to obtain "fold over control" values. Total RNA levels (ng / μL) were also measured as a marker of cell viability. The results are shown in Table 14.3 and Figure 44. [Table 54]

[0335] 14.6 HOXB4-WT transduction with His-CM18-PTD4 in HeLa cells: Immunolabeling and microscopic visualization Using Protocol B as described in Example 9.1, recombinant HOXB4-WT transcription factor (25 μM, Example 14.1) was co-incubated with 35 μM His-CM18-PTD4 and exposed to HeLa cells for 10 seconds. After a 30-minute incubation to allow transduced HOXB4-WT to accumulate in the nucleus, cells were fixed, permeabilized, and immunolabeled as described in Example 3.2a. HOXB4-WT was labeled using a primary mouse anti-HOXB4 monoclonal antibody (Novus Bio wt% NBP2-37257) diluted 1 / 500 and a secondary anti-mouse antibody Alexa™-594 (Abcam #150116) diluted 1 / 1000. Nuclei were labeled with DAPI. Cells were visualized by brightfield and fluorescence microscopy at 20x and 40x magnification as described in Example 3.2, and example results are shown in Figure 45. Colocalization was observed between nuclear labeling (Figures 45A and 45C) and HOXB4-WT labeling (Figures 45B and 45D), indicating that HOXB4-WT was successfully delivered to the nucleus after 30 minutes in the presence of the shuttle agent His-CM18-PTD4. The open triangle window shows an example of an area of ​​colocalization between nuclear (DAPI) and HOXB4-WT immunolabeling.

[0336] 14.7 HOXB4-WT transduction with various shuttling agents in THP-1 cells: dose response and viability THP-1 cells were cultured and tested in a protein transduction assay using Protocol A as described in Example 9.1. Briefly, THP-1 cells were seeded at 30,000 cells / well one day before the first time course experiment. HOXB4-WT recombinant protein (1.5 μM, Example 14.1) was co-incubated with 0.8 μM of the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4, His-C(LLKK)3C-PTD4, and His-CM18-PTD4-His, and then exposed to THP-1 cells in the presence of serum for 2.5 hours. To measure target gene mRNA levels as a marker of HOXB4 activity, cells were subjected to real-time PCR analysis as described in Example 14.2, and then normalized to the target gene mRNA levels detected in negative control cells (untreated) to obtain "fold over control" values. Total RNA levels (ng / μL) were also measured as a marker of cell viability. The results are shown in Table 14.4 and Figure 47. [Table 55]

[0337] Example 15: In vivo GFP-NLS delivery in rat parietal cortex by His-CM18-PTD4 The ability of the shuttle agent His-CM18-PTD4 to deliver GFP-NLS in vivo in the nuclei of rat brain cells was tested. In a separate sterile 1.5 mL tube, the shuttle agent His-CM18-PTD4 was diluted with sterile distilled water at room temperature. GFP-NLS, used as a cargo protein, was then added to the shuttle agent, and sterile PBS was added, if necessary, to obtain the desired concentration of shuttle agent and cargo in a final volume sufficient for injection into the rat brain (e.g., 5 μL per injection brain site). The shuttle agent / cargo mixture was then immediately used for experiments. One negative control corresponding to the injection of GFP-NLS alone was included in the experiment.

[0338] Bilateral injections were performed into the parietal cortex of three rats. The left parietal cortex (ipsilateral) was injected with a mixture consisting of the shuttle agent (20 μM) and GFP-NLS (20 μM). The right parietal cortex (contralateral) was injected with GFP-NLS (20 μM) alone as a negative control. For surgical procedures, mice were anesthetized with isoflurane. The animals were then placed in a stereotaxic frame, and the skull surface was exposed. Two holes were drilled at the appropriate sites using a 5 μL Hamilton syringe to allow for bilateral injection of the shuttle / cargo mixture or GFP-NLS alone (20 μM). The anterior-posterior (AP), lateral (L), and dorsoventral (DV) coordinates relative to the bregma were: (a) AP +0.48 mm, L ±3 mm, V -5 mm; (b) AP -2 mm, L ±1.3 mm, V -1.5 mm; (c) AP -2.6 mm, L ±1.5 mm, V -1.5 mm. The injection volume of the shuttle / cargo mixture or cargo alone was 5 μL per injection site, and the injection was performed over 10 minutes. The experimenter then waited 1 minute before removing the needle from the brain. All measurements were performed before, during, and after surgery to minimize pain and discomfort to the animals. After surgery, animals were sacrificed by perfusion with paraformaldehyde (4%) for 2 hours, and the brains were collected and prepared for microscopic analysis. The study procedures conformed to guidelines from the Canadian Council on Animal Care and were approved by the Institutional Animal Care Committee.

[0339] Dorsoventral rat brain sections were collected and analyzed by fluorescence microscopy at 4x (Figure 48A), 10x (Figure 48C), and 20x (Figure 48D) magnifications. The injection site was located in the deepest layer of the parietal cortex (PCx). In the presence of the His-CM18-PTD4 shuttle agent, GFP-NLS diffused into the nuclei of the PCx, corpus callosum (Cc), and striatum (Str) (white curves mark the boundaries between brain structures). Figure 48B shows the stereotaxic coordinates of the injection site (black arrow) from the Franklin and Paxinos rat brain atlas. Injections of GFP-NLS in the presence of His-CM18-PTD4 were performed on the left side of the brain, and negative controls (injections of GFP-NLS alone) were performed on the contralateral side. The black circles and black connecting lines in Figure 48B indicate the observed areas in the fluorescent photographs (Figures 48A, 48C, and 48D).

[0340] This experiment demonstrated cellular delivery of the cargo GFP-NLS after stereotaxic injection in the rat parietal cortex in the presence of the shuttle agent His-CM18-PTD4. The results show delivery of GFP-NLS in the nuclei of cells from the deeper layers of the parietal cortex (injection site) to the corpus callus and dorsal levels of the striatum (putamen). In contrast, in the negative control, GFP-NLS was only detectable locally around the injection site. This experiment demonstrates that the shuttle agent induced nuclear delivery of the cargo at the injection site (parietal cortex) and its diffusion through adjacent brain regions (corpus callus and striatum) on both sides of the rat brain.

[0341] Example A: Physiochemical properties of domain-based peptide shuttle agents Several different peptides were initially screened with the aim of identifying polypeptide-based shuttle agents capable of intracellularly delivering independent polypeptide cargos to the cytosol / nucleus of eukaryotic cells. On the one hand, large-scale screening efforts led to the discovery that domain-based peptide shuttle agents comprising an endosomal leakage domain (ELD) operably linked to a cell-permeable domain (CPD) and, optionally, one or more histidine-rich domains, increase the transduction efficiency of independent polypeptide cargos in eukaryotic cells, thereby allowing the cargos to reach the cytosol / nuclear compartment (see Examples 1-15). Conversely, these screening efforts revealed several peptides with no or poor polypeptide cargo transduction ability, excessive toxicity, and / or other undesirable properties (e.g., poor solubility and / or stability).

[0342] Based on these empirical data, we compared the physiochemical properties of successful, less successful, and unsuccessful peptides to better understand the commonalities of the more successful shuttle agents. This approach included, for example, manual stratification of various peptides according to their transduction potential, taking into account: (2) their ability to facilitate endosomal escape of calcein (see, e.g., Example 2); (3) their ability to deliver one or more types of independent polypeptide cargo into cells, as assessed by flow cytometry, in various cell types and cell lines (e.g., primary, immortalized, adherent, suspension, etc.) and under various transduction protocols (see, e.g., Examples 3-6 and 8-15); and (4) their ability to deliver polypeptide cargo to the cytosol and / or nucleus, as assessed by fluorescence microscopy (e.g., for fluorescently labeled cargo), increased transcriptional activity (e.g., for transcription factor cargo), or genome editing capability (e.g., for nuclease cargo such as CRISPR / Cas9 or CRISPR / Cpf1) (see, e.g., Examples 3-6 and 8-15), as well as toxicity to various cell types and cell lines (e.g., primary, immortalized, adherent, suspension, etc.) under various transduction protocols.

[0343] Concurrent with the manual curation described above, we integrated the transduction ability and cytotoxicity of each peptide for a given fluorescently labeled cargo (GFP, GFP-NLS, or fluorescently labeled antibody) and cell line into a single "transduction score" as an additional screening tool. This score was calculated as follows: [(maximum transduction efficiency observed by flow cytometry for a given peptide in a given cell type%) x (% viability of the peptide in the tested cell line)] / 1000, giving an overall transduction score of 0 to 10 for a given cell type and polypeptide cargo. These analyses identified domain-based peptides with transduction scores ranging from approximately 8 (e.g., for successful domain-based peptide shuttle agents) to as low as 0.067 (e.g., for single-domain negative control peptides).

[0344] The above manual curation and "transduction score"-based analysis revealed several parameters common to many successful domain-based shuttle agents. Some of these parameters are listed in Table A1. An example of a "transduction score"-based analysis in HeLa cells using GFP as the polypeptide cargo is shown in Table A2. Other transduction score-based analyses using cell lines other than HeLa and polypeptide cargoes other than GFP were also performed but are not shown here for brevity.

[0345] Unsuccessful shuttlers were found to have a length of less than 20 amino acids (see parameter 1 in Tables A1 and A2). Four amino acids: alanine, leucine, lysine, and arginine, were the major, most frequent residues in most successful shuttlers (35–85% of the peptide residues; see parameter 10). These residues are responsible for the alpha-helical structure and amphipathic nature of these peptide sequences (parameters 2–5). A balance often existed between the percentage of A / L residues (15–45%) and the percentage of K / R residues (20–45%) in shuttlers (parameters 11, 12, and 14), and the percentage of negatively charged residues was often found to be less than 10% (parameter 14). Conversely, the 16 other amino acid residues (other than A, L, K, and R) typically accounted for 10–45% of shuttlers (parameter 15). Successful shuttle agents typically have a predicted isoelectric point (pI) of 8–13 (parameter 7) and a predicted net charge of +4 or higher (parameter 6), with DCM18-TAT-Cys having a predicted net charge as high as +26. Hydrophobic residues (A, C, G, I, L, M, F, P, W, Y, V) typically comprise 35–65% of the shuttle agent, while neutral hydrophilic residues (N, Q, S, T) typically comprise 0–30% (parameters 8 and 9).

[0346] As shown in Table A2, the most successful shuttle agents (e.g., those with transduction scores above 5.0) typically had few parameters outside the ranges shown in Table A1. However, large increases in transduction efficiency were also observed with shuttle agents in which some parameters were not met, depending, for example, on the extent to which the unmet parameters fell outside the recommended range and / or whether other parameters fell near the middle of the recommended range. Thus, shuttle agents with some parameters falling within the "optimal" range may compensate for other parameters falling outside the recommended range. As previously mentioned, peptides shorter than 20 amino acids did not demonstrate any significant transduction ability (e.g., transduction scores below 0.4), regardless of how many other parameters were met. Among peptides longer than 20 amino acids with transduction scores below 0.4, VSVG-PTD4 (score of 0.35) failed to meet six parameters, and JST-PTD4 (score of 0.083) failed to meet 10 parameters. KALA (score of 0.12) failed to meet four parameters, with parameters 11 and 14 far exceeding the recommended range, reflecting an excess of A / L residues and a large imbalance between the A / L and L / R percentages. It should be understood that the transduction score ranges in Table A2 are chosen arbitrarily, and other ranges may be selected and are within the scope of this description. [Table 56-1] [Table 56-2] [Table 57-1] [Table 57-2]

[0347] Y = Yes; N = No; open cells = values ​​falling within the parameter ranges shown in Table A1; closed cells = values ​​not falling within the parameter ranges shown in Table A1. Although His-LAH4-PTD4 obtained a transduction score greater than 5.0, it was excluded from this analysis because the intracellular GFP fluorescence pattern was observed by fluorescence microscopy as punctate, suggesting that the GFP cargo remained trapped in endosomes. Nevertheless, it is noteworthy that His-LAH4-PTD4 had several parameters that did not fall within the ranges shown in Table A1 for parameters 2, 3, 11, 12, 14, and 15.

[0348] Example B: Rational Design of Synthetic Peptide Shuttle Agents Using the parameters shown in Table A1 and empirical knowledge gained (e.g., Examples 1-15), the peptides listed in Table B1 were manually designed to assess whether the parameters could be used to design successful peptide shuttle agents.

[0349] The peptides listed in Table B1 were tested for their ability to transduce GFP-NLS cargo into HeLa cells (see Example 3.4) using the protein transduction assay generally described in Example 3.1a. GFP-NLS recombinant protein (10 μM) was co-incubated with 10 μM of peptide before exposure to HeLa cells for 1 minute. Cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Tables B2 and B3. The "transduction score"-based analysis discussed in Example A was also performed, and the results are shown in Table B4. Successful nuclear delivery of transduced GFP-NLS (typically after only 1 minute of peptide exposure) was confirmed by fluorescence microscopy as described in Example 3.2 (data not shown).

[0350] Peptides FSD1-FSD5 were initially designed based on the successful domain-based shuttle agent His-CM18-PTD4-His. Peptides FSD1-FSD4 were intentionally designed to not satisfy one or more parameters shown in Table A1, while FSD5 was designed to satisfy all 15 parameters. As can be seen in Table B2, peptides FSD1-FSD4 exhibited transduction efficiencies ranging from 2.45% to 37.6%. In contrast, peptide FSD5 exhibited high transduction efficiency (70.5%) and low toxicity (86% cell viability).

[0351] Three-dimensional modeling using PEP-FOLD, an online resource for de novo peptide structure prediction, predicted an alpha-helical structure for FSD5 (Figure 49C; see http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD / ). In contrast, peptide VSVG-PTD4, which showed only a 3.5% transduction efficiency (see Table 10.3a), was predicted to adopt a different structure, including a shorter alpha helix, a short beta sheet (white arrow), and a random coil (white shape with fuzzy boundaries).

[0352] The helical wheel projection and lateral open cylinder representations of FSD5 and VSVG-PTD4 shown in Figures 49A and 49B (source: http: / / rzlab.ucr.edu / scripts / wheel / wheel.cgi) illustrate the amphipathic nature of FSD5 compared to VSVG-PTD4. The geometric shape of each amino acid residue corresponds to its biochemical properties (i.e., hydrophobicity, charge, or hydrophilicity) based on the residue's side chain. One of the major differences between the two open cylinder representations of FSD5 and VSVG-PTD4 is the presence of a highly hydrophobic core in FSD5 (outlined in Figure 49A, left and right panels), which is absent in VSVG-PTD4. The cylinder in the bottom center panel of Figures 49A and 49B is a simplified version of the open cylinder representation in the right panel, where "H" represents a highly hydrophobic surface area; "h" represents a hydrophobic surface area; "+" represents a positively charged residue; and "h" represents a hydrophilic residue.

[0353] Given the high transduction efficiency of FSD5, we used this shuttle agent as a model for designing peptides FSD6–FSD26. As shown in Table B2, a relatively high degree of amino acid substitution is possible without complete loss of transduction ability, provided that most of the design parameters listed in Table A1 are met. Among FSD6–FSD26, the only peptide that showed a near-complete loss of transduction efficiency was FSD6, which was not predicted to adopt an amphipathic alpha-helical structure. Interestingly, peptide FSD18 exhibited high toxicity in HeLa cells when used at 10 μM, but showed high transduction efficiency and relatively low toxicity when used in other cell types (see Examples E and G), suggesting that peptide toxicity may vary depending on the cell type. Three-dimensional modeling using PEP-FOLD predicted two alternative alpha-helices for FSD18 (see Figure 49D).

[0354] Peptides FSN1-FSN8 were designed to investigate the effect on transduction efficiency when one or more of the design parameters listed in Table A1 are not met. For example, FSN7 showed a transduction efficiency of only 3.56%, indicating that PEP-FOLD predicts the formation of two beta sheets and no alpha helix (Figure 49F). [Table 58-1] [Table 58-2] [Table 58-3] Results were calculated using the ProtParam™ online tool available from the ExPASy™ Bioinformatics Resource Portal (http: / / web.expasy.org / protparam / ). pI: isoelectric point; charge: total number of positively (+) and negatively (-) charged residues [Table 59-1] [Table 59-2] [Table 60] [Table 61-1] [Table 61-2]

[0355] The primary amino acid sequences of peptides FSD5, FSD16, FSD18, FSD19, FSD20, FSD22, and FSD23 are related as shown in the alignment below. [ka]

[0356] Example C: Computer-aided design of synthetic peptide shuttle agents C.1 Machine learning-aided design methods The peptides listed in Table C1 were designed using the algorithm described in the paper by Sebastien Giguere et al. entitled "Machine Learning Assisted Design of Highly Active Peptides for Drug Discovery" (Giguere et al., 2014). This computational prediction method is based on the use of algorithms based on kernel learning and machine learning methods (Shawe-Taylor J. and Cristianini N., 2004). These algorithms aim to select peptides with maximal biological activity according to the desired biological effect. Here, we considered all peptides tested to date in protein transduction assays and divided them into three separate groups. The composition of the groups was based on the "transduction score," calculated as described in Example A. Group 1 consisted of peptides that showed effective cellular delivery with low toxicity; group 2 consisted of peptides that showed effective cellular delivery but were highly toxic; and group 3 consisted of peptides that did not demonstrate any ability to transduce large polypeptide cargoes.

[0357] The scores of peptides from each group were used as starting data points for generating additional peptide variants. The algorithm used the sequences and scores of peptides from group 1 as positive criteria for predicting peptide variants with effective transduction ability. The sequences and scores from groups 2 and 3 were included as negative controls for the algorithm, representing the search field. Peptide variants generated by the algorithm were limited to those with a length of 35 amino acids. After execution, the prediction method generated 16 sequences (FSD27–FSD42). After analyzing these 16 sequences with respect to the design parameters listed in Table A1, only peptides FSD27, FSD34, and FSD40 met all design parameters (see Table C2). The other peptide variants had one or more parameters that fell outside the parameters listed in Table A1. [Table 62] Results were calculated using the ProtParam™ online tool available from the ExPASy™ Bioinformatics Resource Portal (http: / / web.expasy.org / protparam / ). pI: isoelectric point; charge: total number of positively (+) and negatively (-) charged residues [Table 63]

[0358] HeLa cells were cultured and tested in the protein transduction assay described in Example 3.1a. GFP-NLS recombinant protein (10 μM) was co-incubated with 10 μM of peptide and then exposed to HeLa cells for 1 minute. The cells were subjected to flow cytometry analysis as described in Example 3.3. The results are shown in Table C3. [Table 64]

[0359] Interestingly, three peptides (i.e., FSD27, FSD34, and FSD40) generated using the algorithm that satisfied all design parameters shown in Table A1 exhibited transduction efficiencies of 25–33%, with cell viabilities ranging from 83.9% to 98%, respectively. The other peptides, with the exception of FSD41, typically exhibited transduction efficiencies below 12%. FSD41 exhibited a transduction efficiency of 37% (although its toxicity was higher than that of FSD27, FSD34, and FSD40). Although only a single parameter (e.g., efficiency score) was used to program the algorithm, the results for FSD27, FSD34, and FSD40 validate the usefulness of the design parameters shown in Table A1.

[0360] C2. Computer-assisted generation of peptide variants A computer-aided design approach was employed to demonstrate the feasibility of designing and generating peptide variants that satisfy most or all of the design parameters listed in Table A1. First, the approach involved manual review and comparison of the primary amino acid sequences of structurally distinct but still successful peptide shuttle agents to identify a general consensus sequence that led to the satisfaction of structural parameters (2), (3), and (4) (i.e., amphipathic alpha-helix formation, a positively charged face, and a highly hydrophobic core of 12%–50%). Second, the approach involved computer-aided random peptide sequence generation, followed by descriptor filtering, the incorporation of consensus sequences, and one or more of the design parameters (1) and (5)–(15) to generate a list of peptide variants that satisfied most or all of the design parameters. This is discussed in more detail below.

[0361] First, the primary amino acid sequences of peptides shown herein to have relatively high transduction efficiency scores were compared using online multiple sequence alignment tools, including CLUSTALW 2.1 (http: / / www.genome.jp / tools-bin / clustalw); multiple sequence comparison by Log-Expectation (MUSCLE) (https: / / www.ebi.ac.uk / Tools / msa / muscle / ); and PRALINE (http: / / www.ibi.vu.nl / programs / pralinewww / ). The peptides selected for comparison included the following 11: His-CM18-PTD4; EB1-PTD4; His-C(LLKK)3C-PTD4; FSD5; FSD10; FSD19; FSD20; Peptides from FSD21, FSD44, FSD46, and FSD63 were included, but the analysis was not limited to these 11 peptides. Figure 49G shows the alignment of 11 representative peptides using PRALINE, with the "match" score below each aligned residue position indicating the degree of conservation at that residue position (zero being least conserved and 10 being most conserved). For example, alanine (A) at relative position 29 is conserved in all 11 peptides shown in Figure 49G, and was assigned a "match" score of 10. Such multiple sequence analysis of the library of peptides described herein (or elsewhere) revealed the following general structure: (a) [X1]-[X2]-[linker]-[X3]-[X4] (Formula 1); (b) [X1]-[X2]-[linker]-[X4]-[X3] (Formula 2); (c) [X2]-[X1]-[linker]-[X3]-[X4] (Formula 3); (d) [X2]-[X1]-[linker]-[X4]-[X3] (Formula 4); (e) [X3]-[X4]-[linker]-[X1]-[X2] (Formula 5); (f) [X3]-[X4]-[linker]-[X2]-[X1] (Formula 6); (g) [X4]-[X3]-[linker]-[X1]-[X2] (Formula 7); or (h) [X4]-[X3]-[linker]-[X2]-[X1] (Formula 8); wherein [X1], [X2], [X3], [X4], and [Linker] are as defined in the table below: [Table 65-1] [Table 65-2]

[0362] Second, a script was designed and implemented in the programming language, Python, to randomly generate and filter sequences that satisfied all parameters (except for the parameter for predicted isoelectric point (pI, parameter 7), since the source code for calculating this parameter was not available at the time of writing this example). Structural parameters 2, 3, and 4 listed in Table A1 (amphipathic alpha helix, positively charged surface, and highly hydrophobic core) were satisfied by inputting the consensus sequences shown in Formulas 1-8 and Table C4 (appropriate alternation of hydrophobicity, cationicity, hydrophilicity, and Ala and Gly amino acids in the sequence) into the code. Biochemical parameters (1), (5), (6), and (8)-(15) listed in Table A1 were all individually input into the code to generate 10,000 variant peptide sequences. These variant peptide sequences correspond to SEQ ID NOs: 243-10242.

[0363] Example D: Rationally designed peptides promote the escape of endosome-trapped calcein Calcein endosomal escape assays were performed as generally described in Example 2 and characterized by fluorescence microscopy (data not shown) and flow cytometry (results for FSD5 are shown below). FSD18 showed similar results to FSD5 (data not shown). [Table 66]

[0364] Results from fluorescence microscopy and flow cytometry experiments showed that the rationally designed peptide shuttle agent, similar to the domain-based peptide shuttle agent, promoted the escape of endosome-trapped calcein in a dose-dependent manner.

[0365] Example E: Rationally designed peptides enhance transduction efficiency in various cell types Protein transduction assays in various cell types were performed using the rationally designed peptides at the indicated concentrations, with 10 μM GFP-NLS as cargo, for the indicated times, as generally described in Example 3.1a (adherent cells) or Example 3.1b (suspension cells), and then characterized by flow cytometry (Example 3.3) and fluorescence microscopy (Example 3.2). The results of the flow cytometry analysis are shown in the table below. Fluorescence microscopy demonstrated successful delivery of GFP-NLS to the nuclei of the cells (data not shown). [Table 67] [Table 68] [Table 69] [Table 70] [Table 71]

[0366] Example F: Rationally designed peptide shuttle agents enable antibody transduction F.1 Transduction of fluorescently labeled antibodies with FSD5 in HeLa cells Protein transduction assays were performed using peptide FSD5 and antibodies as cargo after a 1-minute incubation period, as generally described in Example 3.1, followed by characterization by fluorescence microscopy (Example 3.2). Figure 50 shows the cytoplasmic transduction of goat anti-mouse IgG H&L (Alexa Fluor® 488) and anti-rabbit IgG H&L (Alexa Fluor® 594) antibodies delivered by peptide FSD5 (8 μM) for 1 minute in HeLa cells, visualized by fluorescence microscopy with Alexa Fluor 594 Ab at 20x magnification (Figure 50A) and Alexa Fluor 488 Ab at 10x and 20x magnification (Figures 50B and 50C, respectively). Bright-field and fluorescent images of live cells are shown in the upper and lower panels, respectively.

[0367] The following experiments demonstrate that other peptides, such as the FSD peptide, can similarly deliver functional antibodies: an anti-NUP98 antibody that can label the nuclear membrane, and two anti-active caspase-3 antibodies that bind to and inactivate the pro-apoptotic caspase-3 protein. Delivery, microscopy, and cell immunolabeling protocols are described in Example 3.

[0368] F.2 Transduction of anti-NUP98 antibody by FSD19 in HeLa cells Anti-NUP98 antibody (10 μg) was co-incubated with 7.5 μM FSD19 and exposed to HeLa cells for 4 hours. The cells were washed, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton™, and labeled with fluorescently labeled (Alexa™ Fluor 488) goat anti-rat antibody. Antibody binding to the perinuclear membrane and cell nuclei was visualized by fluorescence microscopy at 20x (upper panel) and 40x (lower panel). As shown in Figure 50D, green fluorescent signals (left panel) emitted from the nuclear membrane and were overlapped with Hoechst staining (right panel), indicating that the anti-NUP98 antibody retained its function inside the cells after transduction.

[0369] Transduction of two functional anti-active caspase 3 antibodies with FSD23 in F3.THP-1 and Jurkat cells. Quantification by ELISA cleaved PARP assay. Monoclonal (mAb) and polyclonal (pAb) anti-activated caspase 3 antibodies (2 μg) were independently co-incubated with THP-1 and Jurkat cells in the presence of 7.5 μM FSD23 for 5 min. The anti-apoptotic effect of each antibody was assessed by the level of caspase 3-activated apoptosis using an ELISA cleaved PARP assay and quantified by spectroscopy as described below.

[0370] On the day of the experiment, cells in logarithmic growth phase were harvested, centrifuged (400 g, 3 min), and resuspended in serum-free RPMI in 96-well plates (500,000 cells in 150 μL per well). Cells were centrifuged and incubated for 5 min with a mixture of the test peptide (7.5 μM) and 2 μg of the transduced antibody. Cells were centrifuged and resuspended in serum-containing RPMI in 24-well plates at 37°C for 1 h. Actinomycin D (2 μg / mL), a cytotoxic apoptosis inducer, was incubated with the cells for 4 h. Cells were washed with cold PBS and tested using a PARP (cleaved) [214 / 215] human ELISA kit (ThermoFisher) according to the manufacturer's instructions, followed by spectroscopic analysis. The results are shown in Table F1. [Table 72]

[0371] Results from THP-1 and Jurkat cells demonstrate that FSD23 successfully transduced functional anti-active caspase 3 antibodies. Anti-TNF antibody was used as a nonspecific negative control, and actinomycin D was used as a cytotoxic apoptosis inducer. In the absence of actinomycin D ([-]), delivery of each anti-active caspase 3 mAb and pAb reduced basal levels of apoptosis compared to the "anti-TNF" control, in which delivery of anti-TNF antibody had no discernible effect on cell viability. In the presence of actinomycin D ("+"), delivery of both anti-active caspase 3 antibodies with FSD23 reduced the resulting apoptosis compared to the "anti-TNF" control.

[0372] Example G: Rationally designed peptide shuttle agents enable transduction of CRISPR-based genome editing complexes We tested the ability of rationally designed peptide shuttle agents to deliver functional CRISPR-based genome editing complexes into the nuclei of eukaryotic cells using standard DNA cleavage assays. Using these assays, we measured CRISPR / Cas9- and CRISPR / Cpf1-mediated cleavage of HPRT (hypoxanthine phosphoribosyltransferase 1) and DNMT1 (DNA (cytosine-5-)-methyltransferase 1) cellular genomic DNA sequences. After intracellular delivery of genome editing complexes with various shuttle agents, homologous recombination (HDR) of short (72 bp) and long (1631 bp) DNA templates was performed and measured at the HPRT genomic cleavage site.

[0373] G1. CRISPR / Cas9-NLS complex transduction with rationally designed peptide shuttle agents, cleavage of genomic target sequences, and homologous recombination in various cell lines G.1.1 Transduction of functional CRISPR / Cas9-NLS complexes Cas9-NLS recombinant protein was prepared as described in Example 13.1. Using the transduction protocol generally described in Example 3.1a, a mixture composed of Cas9-NLS recombinant protein and crRNA / tracrRNA (see below) targeting the nucleotide sequence of the HPTR gene was co-incubated with various concentrations of FSD5, FSD8, FSD10, or FSD18 and further incubated with HeLa, HCC-78, NIC-H196, or REC-1 cells for 2 minutes in PBS or for 48 hours in serum-containing medium. Cells were washed with PBS, harvested, and proceeded with the T7E1 protocol assay as described in Example 13.4. The sequences of the constructed crRNA and tracrRNA and their targets were as follows: [ka]

[0374] Figures 51A-51F show the results of cleavage of a target genomic HPRT DNA sequence after separation by agarose gel electrophoresis using CRISPR / Cas9 (2.5 μM) and crRNA / tracrRNA (2 μM) in the absence ("-ctrl") or presence of shuttle agents FSD5, FSD8, FSD10, or FSD18 at various concentrations and exposure times in various cell types: HeLa (Figures 51A and 51B); NK (Figure 51C); NIC-196H (Figure 51D); HCC-78 (Figure 51E); and REC-1 cells (Figure 51F). In some cases, gel lanes were loaded in duplicate. The light dotted arrow indicates the band corresponding to the target gene, and the darker solid arrow indicates the band corresponding to the cleavage product of this target gene, indicating successful transduction of a functional CRISPR / Cas9-NLS genome editing comple...

Claims

1. 1. A method of delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, said method comprising contacting said target eukaryotic cell with said polypeptide cargo in the presence of a shuttle agent at a concentration sufficient to increase transduction efficiency of said polypeptide cargo compared to that in the absence of said shuttle agent, said shuttle agent comprising: (1) at least 20 amino acids in length; (2) an amphipathic alpha-helical motif with a positively charged hydrophilic exterior, and (3) comprising a hydrophobic outer surface; A peptide that satisfies at least five of the following parameters (4) to (15): (4) the hydrophobic exterior surface comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids representing 12-50% of the amino acids of the peptide, based on an open cylinder representation of an alpha helix with 3.6 residues per turn; (5) having a hydrophobic moment (μ) of 3.5 to 11; (6) having a predicted net charge of at least +4 at physiological pH; (7) having an isoelectric point (pI) of 8 to 13; (8) 35% to 65% of the amino acids are composed of any combination of the following: A, C, G, I, L, M, F, P, W, Y, and V; (9) 0%-30% of the amino acids are composed of any combination of the following: N, Q, S, and T; (10) 35% to 85% of the amino acids are composed of any combination of: A, L, K, or R; (11) Composed of 15%-45% of the amino acids: any combination of A and L, provided that at least 5% L is present in the peptide; (12) 20% to 45% of the amino acids are composed of any combination of K and R; (13) 0% to 10% of the amino acids: D and E in any combination; (14) The difference between the percentage of A and L in the peptide (%A+L) and the percentage of K and R in the peptide (K+R) is 10% or less; (15) A method, wherein 10% to 45% of the amino acids are any combination of Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

2. 2. The method of claim 1, wherein the shuttle agent satisfies at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all of parameters (4) through (15) described herein.

3. (i) the shuttle agent is a peptide having a minimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids; (ii) the amphipathic alpha helix motif is 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 , 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, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0; (iii) the amphipathic alpha-helical motif comprises a positively charged hydrophilic exterior, which exterior comprises: (a) at least 2, 3 or 4 adjacent positively charged K and / or R residues on a helical wheel projection; and / or (b) a segment of 6 adjacent residues, based on an alpha-helix having a rotation angle between consecutive amino acids of 100 degrees and / or 3.6 residues per turn on a helical wheel projection, comprising 3 to 5 K and / or R residues; (iv) the amphipathic alpha-helical motif comprises a hydrophobic exterior comprising: (a) at least two adjacent L residues on a helical wheel projection; and / or (b) a segment of 10 adjacent residues comprising at least five hydrophobic residues selected from L, I, F, V, W, and M, based on an alpha-helix having a rotation angle between consecutive amino acids of 100 degrees and / or 3.6 residues per turn on a helical wheel projection; (v) the hydrophobic exterior surface comprises a highly hydrophobic core composed 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 of the peptide; (vi) the peptide has a hydrophobic moment (μ) between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5; (vii) the peptide has a predicted net charge of +4, +5, +6, +7, +8, +9 to +10, +11, +12, +13, +14, or +15; (viii) the peptide has a predicted pI of 10-13; or The method of claim 1 or 2, wherein (ix) is any combination of (i) to (viii).

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

5. The method of any one of claims 1 to 4, wherein the peptide comprises a histidine-rich domain.

6. The histidine-rich domain is (i) located towards the N-terminus and / or C-terminus of the peptide; (ii) a stretch of at least 3, at least 4, at least 5, at least 6 amino acids containing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% histidine residues, and / or containing 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) The method of claim 5, wherein (i) and (ii) are both.

7. The method of any one of claims 1 to 6, wherein the peptide comprises a flexible linker domain enriched in serine and / or glycine residues.

8. The method according to any one of claims 1 to 7, wherein the peptide comprises or consists of the amino acid sequence: (a) [X1]-[X2]-[linker]-[X3]-[X4] (Formula 1); (b) [X1]-[X2]-[linker]-[X4]-[X3] (Formula 2); (c) [X2]-[X1]-[linker]-[X3]-[X4] (Formula 3); (d) [X2] - [X1] - [Linker] - [X4] - [X3] (Formula 4); (e) [X3] - [X4] - [Linker] - [X1] - [X2] (Formula 5); (f) [X3] - [X4] - [Linker] - [X2] - [X1] (Formula 6); (g) [X4] - [X3] - [Linker] - [X1] - [X2] (Formula 7); or (h) [X4] - [X3] - [Linker] - [X2] - [X1] (Formula 8); wherein, [X1] is selected from the following: 2[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; 2[Φ] - 1[+] - 2[Φ] - 2[+] -; 1[+] - 1[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; and 1[+] - 1[Φ] - 1[+] - 2[Φ] - 2[+] -; [X2] is selected from the following: -2[Φ] - 1[+] - 2[Φ] - 2[ζ] -; -2[Φ] - 1[+] - 2[Φ] - 2[+] -; -2[Φ] - 1[+] - 2[Φ] - 1[+] - 1[ζ] -; -2[Φ] - 1[+] - 2[Φ] - 1[ζ] - 1[+] -; -2[Φ] - 2[+] - 1[Φ] - 2[+] -; -2[Φ] - 2[+] - 1[Φ] - 2[ζ] -; -2[Φ] - 2[+] - 1[Φ] - 1[+] - 1[ζ] -; and -2[Φ] - 2[+] - 1[Φ] - 1[ζ] - 1[+] -; [X3] is selected from the following: -4[+] - A -; -3[+] - G - A -; -3[+] - A - A -; -2[+] - 1[Φ] - 1[+] - A -; -2[+] - 1[Φ] - G - A -; -2[+] - 1[Φ] - A - A -; or -2[+] - A - 1[+] - A; -2[+] - A - G - A; -2[+] - A - A - A -; -1[Φ] - 3[+] - A -; -1[Φ] - 2[+] - G - A -; -1[Φ] - 2[+] - A - A -; -1[Φ] - 1[+] - 1[Φ] - 1[+] - A; -1[Φ] - 1[+] - 1[Φ] - G - A; -1[Φ] - 1[+] - 1[Φ] - A - A; -1[Φ] - 1[+] - A - 1[+] - A; -1[Φ] - 1[+] - A - G - A; -1[Φ] - 1[+] - A - A - A; -A - 1[+] - A - 1[+] - A; -A - 1[+] - A - G - A; and -A - 1[+] - A - A - A; [X4] is selected from the following: -1[ζ]-2A-1[+]-A; -1[ζ]-2A-2[+]; -1[+]-2A-1[+]-A; -1[ζ]-2A-1[+]-1[ζ]-A-1[+]; -1[ζ]-A-1[ζ]-A-1[+]; -2[+]-A-2[+]; -2[+]-A-1[+]-A; -2[+] -A-1[+]-1[ζ]-A-1[+];-2[+]-1[ζ]-A-1[+];-1[+]-1[ζ]-A-1[+]-A;-1[+]-1[ζ]- A-2[+];-1[+]-1[ζ]-A-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-A-1[+];-1[+]-2[ζ]-2[+] ;-1[+]-2[ζ]-1[+]-A;-1[+]-2[ζ]-1[+]-1[ζ]-A-1[+];-1[+]-2[ζ]-1[ζ]-A-1[+] ;-3[ζ]-2[+];-3[ζ]-1[+]-A;-3[ζ]-1[+]-1[ζ]-A-1[+];-1[ζ]-2A-1[+]-A;-1[ζ]- 2A-2[+];-1[ζ]-2A-1[+]-1[ζ]-A-1[+];-2[+]-A-1[+]-A;-2[+]-1[ζ]-1[+]-A;-1 [+]-1[ζ]-A-1[+]-A;-1[+]-2A-1[+]-1[ζ]-A-1[+]; and-1[ζ]-A-1[ζ]-A-1[+]; and [Linker] is selected from the following: -Gn-; -Sn-; -(GnSn)n-; -(GnSn)nGn-; -(GnSn)nSn-; -(GnSn)nGn(GnSn)n-; and -(GnSn)nSn(GnSn)n-; During the ceremony, [Φ] is an amino acid: Leu, Phe, Trp, Ile, Met, Tyr, or Val; [+] is the amino acid: Lys or Arg; [ζ] is the amino acid Gln, Asn, Thr, or Ser; A is the amino acid Ala; G is the amino acid GIy; 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.

9. (i) the peptide 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 NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242; or the peptide comprises or consists of a functional variant of any one of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242; (ii) the peptide is: (a) comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242; (b) comprises the amino acid sequence motif of SEQ ID NO: 158 and / or 159; or (c) comprising the amino acid sequence motif of SEQ ID NO: 158 operably linked to the amino acid sequence motif of SEQ ID NO: 159; or (iii) both (i) and (ii).

10. The method according to any one of claims 1 to 9, wherein the peptide comprises an endosomal leakage domain (ELD) and / or a cell permeability domain (CPD).

11. (i) the ELD is selected from the group consisting of endosomolytic peptides, antimicrobial peptides (AMPs), linear cationic alpha-helical antimicrobial peptides, cecropin-A / melittin hybrid (CM series) peptides, pH-dependent membrane active peptides (PAMPs), peptide amphiphiles, peptides derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA), CM18, diphtheria toxin T domain (DT), GALA, PEA, INF-7, LAH4, HGP, H5WYG, HA2, EB1, VSVG, Pseudomonas toxin, melittin, KALA, JST-1, C(LLKK). ３ C, G (LLKK) ３ G or any combination thereof, (ii) the CPD is or is derived from a cell membrane penetrating peptide or a protein transduction domain derived from a cell membrane penetrating peptide, TAT, PTD4, penetratin (antennapedia), pVEC, M918, Pep-1, Pep-2, Xentry, an arginine stretch, transportan, SynB1, SynB3 or any combination thereof; or The method of claim 10, wherein (iii) both (i) and (ii).

12. The peptide is (a) an ELD comprising the amino acid sequence of any one of SEQ ID NOs: 1-15, 63 or 64, or a variant or fragment thereof that has endosomolytic activity; (b) a CPD comprising the amino acid sequence of any one of SEQ ID NOs: 16-27 or 65, or a variant or fragment thereof having cell membrane permeability activity; or 12. The method of claim 10 or 11, comprising both (c) (a) and (b).

13. (i) the peptide has the amino acid sequence of SEQ ID NO: 1, 14, or 63; ３ C, or an ELD having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 1, 14, or 63, and having endosomolytic activity; (ii) the peptide comprises a CPD that is TAT ​​or PTD4 having the amino acid sequence of SEQ ID NO: 17 or 65, or a variant thereof having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 17 or 65 and having cell membrane permeabilizing activity; or (iii) The method of any one of claims 10 to 12, wherein (i) and (ii) are both.

14. 10. The method of any one of claims 1 to 9, wherein the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 57-59, 66-72 or 82-102, or a functional variant thereof having at least 85%, 90%, or 95% identity to any one of SEQ ID NOs: 57-59, 66-72 or 82-102.

15. (i) the shuttle agent is fully metabolizable by the target eukaryotic cell; and / or (ii) contacting the target eukaryotic cell with the polypeptide cargo in the presence of the shuttle agent increases the transduction efficiency of the polypeptide cargo by at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or even 100-fold compared to the absence of the shuttle agent.

16. The method according to any one of claims 1 to 15, which is an in vitro method.

17. A synthetic peptide shuttle agent which is a peptide as defined in any one of claims 1 to 15.

18. 18. The synthetic peptide of claim 17, which is a peptide of 20 to 100 amino acids in length comprising the amino acid sequence of any one of SEQ ID NOs: 104, 105, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 134, 135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242, or comprising the amino acid sequence motif of SEQ ID NOs: 158 and / or 159.

19. A synthetic peptide shuttle agent as described in claim 17 or 18 for use in in vitro delivery of a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, wherein the synthetic peptide shuttle agent is used at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to the absence of the synthetic peptide shuttle agent.

20. A synthetic peptide shuttle agent as described in claim 17 or 18 for use in in vivo delivery of a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, said synthetic peptide shuttle agent being used at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to the absence of the synthetic peptide shuttle agent.

21. A composition comprising a shuttle agent as defined in any one of claims 1 to 15, or a cocktail of at least two, at least three, at least four, at least five different types of shuttle agents as defined in any one of claims 1 to 15, and a polypeptide cargo to be delivered from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell.

22. Use of a shuttle agent as defined in any one of claims 1 to 15, or a synthetic peptide as defined in claim 18, for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, wherein the shuttle agent or synthetic peptide is used at a concentration sufficient to increase the transduction efficiency of the polypeptide cargo compared to that in the absence of the shuttle agent or synthetic peptide.

23. A kit for delivering a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, comprising a shuttle agent as defined in any one of claims 1 to 15, or a synthetic peptide as defined in claim 18, and a suitable container.

24. The method of any one of claims 1 to 16, the synthetic peptide shuttle agent of any one of claims 17 to 20, the composition of claim 21, the use of claim 22, or the kit of claim 23, wherein the polypeptide cargo lacks a cell membrane permeable domain.

25. The method of any one of claims 1 to 16, the synthetic peptide shuttle agent of any one of claims 17 to 20, the composition of claim 21, the use of claim 22, or the kit of claim 23, wherein the polypeptide cargo comprises a cell membrane permeable domain.

26. A method according to any one of claims 1 to 16, 24 or 25, a synthetic peptide shuttle agent according to any one of claims 17 to 20, 24 or 25, a composition according to claim 21, 24 or 25, a use according to any one of claims 22 to 25, wherein the polypeptide cargo comprises an intracellular targeting domain.

27. The intracellular targeting domain comprises: (a) nuclear localization signal (NLS); (b) a nucleolar signal sequence; (c) a mitochondrial signal sequence; or 27. The method, synthetic peptide shuttle agent, composition, use, or kit of claim 26, wherein (d) a peroxisomal signal sequence.

28. (a) the NLS is derived from E1a, T-Ag, c-myc, T-Ag, op-T-NLS, Vp3, nucleoplasmin, histone 2B, Xenopus N1, PARP, PDX-1, QKI-5, HCDA, H2B, v-Rel, Amida, RanBP3, Pho4p, LEF-1, TCF-1, BDV-P, TR2, SOX9 or Max; (b) the nucleolar signal sequence is derived from BIRC5 or RECQL4; (c) the mitochondrial signal sequence is derived from Tim9 or yeast cytochrome c oxidase subunit IV, or 28. The method, synthetic peptide shuttle agent, composition, use, or kit of claim 27, wherein (d) the peroxisomal signal sequence is derived from PTS1.

29. 30. The method, synthetic peptide shuttle agent, composition, use or kit of any one of claims 24 to 29, wherein said polypeptide cargo is complexed to a DNA and / or RNA molecule.

30. 30. The method, synthetic peptide shuttle agent, composition, use or kit of any one of claims 24 to 29, wherein said polypeptide cargo is a transcription factor, a nuclease, a cytokine, a hormone, a growth factor, an antibody, a peptide cargo, an enzyme, an enzyme inhibitor, or any combination thereof.

31. (a) the transcription factor is HOXB4, NUP98-HOXA9, Oct3 / 4, Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1, Ngn3, MafA, Blimp-1, Eomes, T-bet, FOXO3A, NF-YA, SALL4, ISL1, FoxA1, Nanog, Esrrb, Lin28, HIF1-alpha, Hlf, Runx1t1, Pbx1, Lmo2, Zfp37, Prdm5, Bcl-6, or any combination thereof; (b) the nuclease is catalytically active or catalytically dead: RNA-guided endonuclease, CRISPR endonuclease, Type I CRISPR endonuclease, Type II CRISPR endonuclease, Type III CRISPR endonuclease, Type IV CRISPR endonuclease, Type V CRISPR endonuclease, Type VI CRISPR endonuclease, CRISPR-associated protein 9 (Cas9), Cpf1, CasY, CasX, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), homing endonuclease or meganuclease, DNA-guided nuclease, Natronobacterium gregorii argonaute (NgAgo), or any combination thereof; (c) the antibody recognizes an intracellular antigen; and / or 31. The method, synthetic peptide shuttle agent, composition, use or kit of claim 30, wherein (d) the peptide cargo recognizes an intracellular molecule.

32. 32. The method, synthetic peptide shuttle agent, composition, use or kit of any one of claims 24 to 31 for use in cell therapy, genome editing, adoptive cell transfer and / or regenerative medicine.

33. 33. The method, synthetic peptide shuttle agent, composition, use or kit of any one of claims 24 to 32, wherein the target eukaryotic cell is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, a NK cell or a dendritic cell.

34. 22. A eukaryotic cell comprising a shuttle agent as defined in any one of claims 1 to 15, a synthetic peptide shuttle agent as defined in claim 18, or a composition as defined in claim 21.

35. 35. The eukaryotic cell of claim 34, which is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, a NK cell, or a dendritic cell.

36. 16. A method for delivering one or more CRISPR-binding endonucleases, alone or together with one or more corresponding guide RNAs and / or linear DNA templates, to a target eukaryotic cell, comprising contacting said target eukaryotic cell with said endonuclease in the presence of a shuttle agent at a concentration sufficient to increase transduction efficiency of said endonuclease compared to that in the absence of said shuttle agent, wherein said shuttle agent is as defined in any one of claims 1 to 15.

37. 37. The method of claim 36, which is an in vitro method or an in vivo method.

38. 38. The method of claim 36 or 37, wherein the one or more nucleases are a type I CRISPR endonuclease, a type II CRISPR endonuclease, a type III CRISPR endonuclease, a type IV CRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPR endonuclease, or any combination thereof.

39. 38. The method of claim 36 or 37, wherein the one or more endonucleases are CRISPR-associated protein 9 (Cas9), Cpf1, CasX, CasY, or any combination thereof; or catalytically-dead CRISPR-associated protein 9 (dCas9), dCpf1, dCasX, dCasY, or any combination thereof.

40. 40. The method of any one of claims 36 to 39, wherein the target eukaryotic cell is an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, a NK cell, or a dendritic cell.

41. 59. The method of claim 58, wherein said one or more corresponding guide RNAs and / or linear DNA templates target one or more genes to reduce immunogenicity, improve cytotoxicity, and / or otherwise improve efficacy for cell therapy of said target eukaryotic cells compared to a corresponding parent eukaryotic cell not subjected to said method.

42. 42. The method of claim 41, wherein the cell therapy is a cell-based cancer immunotherapy.

43. 43. The method of any one of claims 40-42, wherein the one or more corresponding guide RNAs and / or linear DNA templates target the CBLB gene, the c-CBL gene, the GSK3 gene, the ILT2 gene, the CISH gene, the NKG2a gene, the B2M gene, or any combination thereof.

44. 1. A method for producing a synthetic peptide shuttle agent that delivers a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, the method comprising synthesizing a peptide comprising: (1) at least 20 amino acids in length; (2) an amphipathic alpha-helical motif with a positively charged hydrophilic exterior, and (3) comprising a hydrophobic outer surface; A peptide that satisfies at least five of the following parameters (4) to (15): (4) the hydrophobic exterior surface comprises a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids representing 12-50% of the amino acids of the peptide, based on an open cylinder representation of an alpha helix with 3.6 residues per turn; (5) having a hydrophobic moment (μ) of 3.5 to 11; (6) having a predicted net charge of at least +4 at physiological pH; (7) having an isoelectric point (pI) of 8 to 13; (8) 35% to 65% of the amino acids are composed of any combination of the following: A, C, G, I, L, M, F, P, W, Y, and V; (9) 0%-30% of the amino acids are composed of any combination of the following: N, Q, S, and T; (10) 35% to 85% of the amino acids are composed of any combination of: A, L, K, or R; (11) Composed of 15%-45% of the amino acids: any combination of A and L, provided that at least 5% L is present in the peptide; (12) 20% to 45% of the amino acids are composed of any combination of K and R; (13) 0% to 10% of the amino acids: D and E in any combination; (14) The difference between the percentage of A and L in the peptide (%A+L) and the percentage of K and R in the peptide (K+R) is 10% or less; (15) 10% to 45% of the amino acids are composed of any combination of Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

45. The method according to claim 44, wherein said peptide is as defined in any one of claims 2 to 15.

46. 1. A method for identifying a shuttle agent that delivers a polypeptide cargo from the extracellular space to the cytosol and / or nucleus of a target eukaryotic cell, comprising: (a) synthesizing a peptide, which is a peptide as defined in any one of claims 1 to 15 or 18; (b) contacting said target eukaryotic cell with said polypeptide cargo in the presence of said peptide; (c) measuring the transduction efficiency of said polypeptide cargo in said target eukaryotic cells; and (d) if an increase in transduction efficiency of said polypeptide cargo in said target eukaryotic cell is observed, identifying said peptide as a shuttle agent for transducing said polypeptide cargo.

47. The method of claim 46, wherein said polypeptide cargo is as defined in any one of claims 24 to 31.

48. Genome editing systems, including: (a) a shuttle agent as defined in any one of claims 1 to 15 or 18; (b) one or more CRISPR-binding endonucleases; and (c) one or more guide RNAs.

49. 49. The genome editing system of claim 48, further comprising a linear DNA template that controls genome editing.

50. The genome editing system of claim 48 or 49, wherein the one or more CRISPR-binding endonucleases are type I CRISPR endonuclease, type II CRISPR endonuclease, type III CRISPR endonuclease, type IV CRISPR endonuclease, type V CRISPR endonuclease, type VI CRISPR endonuclease, CRISPR binding protein 9 (Cas9), Cpf1, CasX, CasY, or any combination thereof.

51. 1. A method for enriching for eukaryotic cells transduced with a polypeptide cargo of interest, comprising: (a) co-transducing a target eukaryotic cell population with a polypeptide cargo of interest and a marker protein; and (b) isolating or enriching for eukaryotic cells transduced with said marker protein, thereby enriching for eukaryotic cells transduced with said polypeptide cargo of interest.

52. (i) the marker protein is not covalently bound to the polypeptide cargo of interest, the marker protein is covalently bound to the polypeptide cargo of interest, the marker protein is non-covalently bound to the polypeptide cargo of interest, or the marker protein is covalently bound to the polypeptide cargo of interest via a cleavable linker; and / or 52. The method of claim 51 , wherein (ii) the marker protein comprises a detectable label or the marker protein is a fluorescent protein, a fluorescently labeled protein, a bioluminescent protein, an isotopically labeled protein, or a magnetically labeled protein.

53. 53. The method of claim 51 or 52, wherein the intracellular concentration of the transduction marker protein positively correlates with the intracellular concentration of the transduction polypeptide cargo of interest.

54. The method according to any one of claims 51 to 53, wherein the eukaryotic cells transduced with the marker protein are isolated or enriched using flow cytometry, fluorescence activated cell sorting (FACS) or magnetic cell sorting (MACS).

55. 55. The method of any one of claims 51 to 54, wherein eukaryotic cells transduced with the marker protein are isolated or sorted from cells lacking the marker protein, thereby producing a marker protein positive cell population and / or a marker protein negative cell population.

56. 56. The method of claim 55, further comprising repeating steps (a) and (b) one or more times on the marker protein negative cell population, the marker protein positive cell population, or both the marker protein negative and the marker protein positive cell populations.

57. 57. The method of any one of claims 51 to 56, wherein eukaryotic cells transduced with said marker protein are isolated or selected on the basis of their intracellular concentration of said marker protein.

58. 58. The method of any one of claims 51 to 57, wherein the marker protein is a protein that stimulates cell proliferation, a protein that stimulates cell differentiation, a protein that promotes cell survival, an anti-apoptotic protein, or a protein with another biological activity.

59. 59. The method of any one of claims 51 to 58, wherein said polypeptide cargo of interest and said marker protein are co-transduced by contacting said target eukaryotic cell with said polypeptide cargo and said marker protein in the presence of a peptide transduction agent, said peptide transduction agent being present in a concentration sufficient to increase the transduction efficiency of said polypeptide cargo and said marker protein compared to in the absence of said peptide transduction agent.

60. (a) the peptide transduction agent is an endosomolytic peptide; (b) the peptide transduction agent is or comprises the synthetic peptide shuttle agent defined in claim 17 or 18; (c) the target eukaryotic cell comprises an animal cell, a mammalian cell, a human cell, a stem cell, a primary cell, an immune cell, a T cell, a NK cell, or a dendritic cell; (d) said polypeptide cargo of interest is (i) a polypeptide cargo as defined in any one of claims 24 to 31; and / or (ii) one or more CRISPR binding endonucleases alone or together with one or more corresponding guide RNAs and / or linear DNA templates as defined in any one of claims 38 to 43; 60. The method of claim 59, wherein (e) is any combination of (a) through (d).

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