Peptide-based introduction of non-anionic polynucleotide analogs for gene expression regulation

Abstract

Described herein are compositions and methods for delivering non-anionic polynucleotide analog cargo to the cytoplasmic / nuclear compartment of eukaryotic cells via synthetic peptide shuttle agents. The non-anionic polynucleotide analog cargo can be charge-neutral or cationic, and the synthetic peptide shuttle agent is a peptide containing an amphipathic alpha-helical motif with both a positively charged hydrophilic exterior and a hydrophobic exterior. The synthetic peptide shuttle agent is not covalently linked to the non-anionic polynucleotide analog cargo, or is cleavably linked under physiological conditions. The non-anionic polynucleotide analog cargo can be a charge-neutral or cationic antisense synthetic oligonucleotide (ASO) that hybridizes to intracellular target RNA for alteration of gene expression.

Description

Technical Field

[0001] This description relates to the intracellular delivery of non - anionic polynucleotide analog cargos. More specifically, this description relates to the use of synthetic peptide shuttle agents for the intracellular delivery of non - anionic polynucleotide analog cargos.

[0002] This description refers to a number of documents whose content is hereby incorporated by reference in its entirety.

Background Art

[0003] Most non - anionic polynucleotide analog cargos are plagued by issues related to their intracellular delivery and often require their covalent conjugation to a delivery moiety, thereby further complicating their synthesis and commercialization. An improved method for increasing the cytoplasmic / nuclear delivery of non - anionic polynucleotide analog cargos is highly desirable.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non - Patent Documents

[0005]

Non - Patent Document 1

Non - Patent Document 2

[0006] Synthetic peptide shuttles represent a recently defined family of peptides that have already been reported to rapidly and efficiently deliver protein cargo into the cytoplasm and / or nucleus of a wide range of target eukaryotic cells. In contrast to traditional intracellular delivery strategies based on cell membrane-permeable peptides, synthetic peptide shuttles are not covalently bound to their polypeptide cargoes. In fact, covalently binding shuttles to their cargoes generally has adverse effects on their transduction activity. The first generation of such peptide shuttles was described in International Publication 2016 / 161516, in which the peptide shuttles contained an endosomal leakage domain (ELD) manipulably linked to a cell membrane-permeable domain (CPD). Subsequently, International Publication 2018 / 068135 further described synthetic peptide shuttles rationally designed based on a set of 15 design parameters solely for the purpose of improving protein cargo transduction while reducing the toxicity of the first-generation peptide shuttles.

[0007] Cell membrane-permeable peptides (CPPs) have been used for decades in transfection strategies for delivering DNA / RNA into cells. However, first-generation synthetic peptide shuttles containing CPP-derived CPDs failed to efficiently transduce plasmid DNA cargo into the nucleus for gene expression, as the DNA cargo remained largely trapped in endosomes. Subsequent experiments revealed that second-generation synthetic peptide shuttles were also unsuitable for efficiently transduction of plasmid DNA into the nucleus for gene expression. Furthermore, strategies involving neutralizing the negatively charged phosphate backbone of DNA / RNA by coating it with positively charged small molecules failed to significantly improve shuttle-mediated cargo delivery, as endosomal capture remained a problem. This suggests that something more than simple charge neutralization was required for shuttle-mediated delivery of polynucleotides.

[0008] This disclosure relates to the remarkable discovery that synthetic peptide shuttle agents have the ability to rapidly and efficiently introduce non-anionic polynucleotide analog cargoes into the cytoplasmic / nuclear compartment in amounts sufficient to influence the modification of gene expression in eukaryotic cells.

[0009] In one embodiment, the Specified Description describes a composition comprising a non-anionic polynucleotide analog cargo for intracellular delivery and a synthetic peptide shuttle agent that is independent of or not covalently bonded to the non-anionic polynucleotide analog cargo, wherein the synthetic peptide shuttle agent is a peptide comprising an amphiphilic alpha-helix motif having both a positively charged hydrophilic outer surface and a hydrophobic outer surface, and the synthetic peptide shuttle agent increases the cytoplasmic / nuclear delivery of the non-anionic polynucleotide analog cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttle agent.

[0010] In a further embodiment, this specification describes a method for modifying gene expression in eukaryotic cells, the method comprising: (a) providing a non-anionic polynucleotide analog cargo for intracellular delivery, wherein the non-anionic polynucleotide analog cargo is designed to hybridize to a target RNA in eukaryotic cells; (b) providing a synthetic peptide shuttle that is independent of or not covalently bonded to the non-anionic polynucleotide analog cargo; and (c) contacting eukaryotic cells with the non-anionic polynucleotide analog cargo in the presence of a synthetic peptide shuttle at a concentration sufficient to increase the delivery efficiency and / or cytoplasmic / nuclear delivery of the charged-neutral polynucleotide analog cargo compared to the absence of the synthetic peptide shuttle, wherein the non-anionic polynucleotide analog cargo hybridizes to the target RNA upon cytoplasmic / nuclear delivery, thereby affecting the modification of gene expression.

[0011] general definition Titles and other identifiers, such as (a), (b), (i), (ii), etc., are provided solely to facilitate the reading of this specification and the claims. The use of titles and other identifiers in this specification and the claims does not necessarily require that the processes or elements be carried out in alphabetical or numerical order, or in the order in which they are presented.

[0012] The use of the words “a” or “an” in the claims and / or specification may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.”

[0013] When used herein, the term "about" indicates that a value includes the standard deviation of the error of the device or method used to determine the value. Generally, the technical term "about" specifies a possible variation of up to 10%. Therefore, variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of the value are included within the term "about". Unless otherwise indicated, the use of the term "about" prior to a range applies to both ends of the range.

[0014] As used herein and in the claims, the words “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “includes” and “include”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are comprehensive or open-ended and do not preclude any further unlisted elements or process steps.

[0015] As used herein, “protein” or “polypeptide” or “peptide” means any peptide-linked chain of amino acids, which may or may not include any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfation, SUMOylation, prenylation, ubiquitination, etc.). For further clarity, protein / polypeptide / peptide modifications are assumed as long as the modifications do not disrupt the cargo delivery activity of the shuttle agents described herein. For example, the shuttle agents described herein may be linear or cyclic, may be synthesized from one or more D- or L-amino acids, and / or may be conjugated to fatty acids (e.g., their N-terminus). The shuttle agents described herein may also have at least one amino acid which is replaced by a corresponding synthetic amino acid having a side chain with similar physicochemical properties (e.g., structure, hydrophobicity, or charge) to the amino acid being replaced.

[0016] As used herein, “domain” or “protein domain” generally refers to a portion of a protein having a specific functional group or function. Some domains preserve their function when isolated from the rest of the protein and can therefore be used modularly. The modular nature of many protein domains can provide flexibility through their arrangement within the shuttle agent described herein. However, some domains may function better when genetically engineered at specific locations within the shuttle agent (e.g., in or between the N-terminal and C-terminal regions). The location of a domain within its endogenous protein may also serve as an indicator of where the domain should be genetically engineered within the shuttle agent and what type / length of linker should be used. Standard recombinant DNA techniques may be used by those skilled in the art to manipulate the arrangement and / or number of domains within the shuttle agent described herein in consideration of this disclosure. Furthermore, assays disclosed herein and others known in the art may be used to evaluate the functionality of each domain in terms of the shuttle agent (e.g., its ability to facilitate cell penetration across the cell membrane, endosomal escape, and / or access to the cytoplasm). Furthermore, standard methods can be used to evaluate whether the domains of the shuttle agent affect the activity of the cargo to be delivered into the cell. In this context, the expression “operatably linked,” as used herein, refers to the ability of a domain to perform its intended function (e.g., cell penetration, endosomal escape, and / or intracellular targeting) in the context of the shuttle agent described herein. For clarity, the expression “operatably linked” means defining a functional linkage between two or more domains, not limited to a specific order or distance between them.

[0017] As used herein, the term “synthetic” in expressions such as “synthetic peptide,” “synthetic peptide shuttle,” or “synthetic polypeptide” is intended to refer to molecules that do not exist in nature and can be produced in vitro (e.g., chemically synthesized and / or produced using recombinant DNA technology). The purity of various synthetic preparations can be evaluated, for example, by high-performance liquid chromatography analysis and mass spectrometry. Chemical synthesis approaches may be advantageous over cell expression systems (e.g., yeast or bacterial protein expression systems) because they can eliminate the need for large-scale recombinant protein purification steps (e.g., required for clinical use). In contrast, longer synthetic polypeptides may be more complex and / or more expensive to produce by chemical synthesis approaches, and such polypeptides may be more advantageously produced using cell expression systems. In some embodiments, the peptides or shuttles described herein may be chemically synthesized (e.g., solid-phase or liquid-phase peptide synthesis) as opposed to being expressed from recombinant host cells. In some embodiments, the peptides or shuttles described herein may lack an N-terminal methionine residue. Those skilled in the art can adapt the synthetic peptides or shuttle agents described herein to meet specific stability requirements or other needs by using one or more modified amino acids (e.g., amino acids that do not occur naturally) or by chemically modifying the synthetic peptides or shuttle agents described herein.

[0018] As used herein, the term "independent" generally refers to molecules or agents (e.g., shuttle agents and cargos) that are not covalently bonded to each other or that can transiently covalently bond via a cleavable bond such that they separate from each other upon administration (e.g., when exposed to a reducing cellular environment and / or before, simultaneously, or immediately after being delivered into a cell) through cleavage of the bond. For example, the expression "independent cargo" is intended to refer to a cargo that is not covalently bonded (e.g., not fused) to the shuttle agent described herein at the point of introduction across the cell membrane and that is delivered (introduced) into the cell. In some embodiments, having a shuttle agent that is independent (not fused) of the cargo can be advantageous by increasing the versatility of the shuttle agent, e.g., by allowing the ratio of the shuttle agent to the cargo to be readily varied (in contrast to being limited to a fixed ratio in the case of a covalent bond between the shuttle agent and the cargo). In some embodiments, covalently bonding the shuttle agent to its cargo via a cleavable bond such that they separate from each other upon contact with the target cell can be advantageous from a manufacturing and / or control perspective.

[0019] As used herein, the expression "is or is derived from" or "is derived from" includes conservative amino acid substitutions, deletions, modifications, and functional variants such as variants or functional derivatives of a given protein or peptide (e.g., a shuttle agent described herein) or their domains (e.g., CPD or ELD) that do not suppress the activity of the protein domain.

[0020] Other objects, advantages, and features of the present description will become more apparent upon reading the following non-limiting description of its specific embodiments, given by way of example only and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] [Figure 1]Figure showing intracellular delivery of PMO-FITC cargo in HeLa cells via a first-generation "domain-based" and rationally designed synthetic peptide shuttle agent, evaluated by flow cytometry. Results are averages calculated from experiments performed at least twice. [Figure 2] Figure showing the ability of the synthetic peptide shuttle agent FSD10 to introduce antisense PMO into the cytoplasm of HeLa cells, thereby enabling knockdown of GFP gene expression, evaluated by flow cytometry. Results are averages calculated from experiments performed at least twice. [Figure 3] Figure showing the ability of the synthetic peptide shuttle agent FSD250 to introduce antisense PMO targeting Wnt1 and Gli1 for knockdown of Gli1 protein expression in DU145 cells, evaluated by Western blot. Results are averages calculated from experiments performed at least twice. [Figure 4] Figure showing the ability of the synthetic peptide shuttle agent FSD250 to introduce antisense PMO targeting Gli1 for knockdown of Gli1 protein expression in DU145 cells, evaluated by Western blot. Results are averages calculated from experiments performed at least twice. [Figure 5] Figure showing the results of a large-scale screening of candidate peptide shuttle agents for propidium iodide (PI) and GFP-NLS introduction activities. Results are averages calculated from experiments performed at least twice. [Figure 6] Figure showing the results of further screening of candidate peptide shuttle agents for propidium iodide (PI) and / or GFP-NLS introduction activities. Results are averages calculated from experiments performed at least twice. [Figure 7]This figure shows the ability of the synthetic peptide shuttle FSD250 to introduce antisense PMO and PNA into HeLa cells. Figure 7A shows the results of intracellular delivery by flow cytometry. Figure 7B shows the results of cell viability by flow cytometry. The results are averages calculated from experiments performed at least twice. [Figure 8] This figure shows the results of flow cytometry on the inhibitory effects of the synthetic peptide shuttle FSD250 on the intracellular delivery of PMO via naked DNA (Figure 8A) and RNA (sgRNA; Figure 8B). [Figure 9] This figure shows the ability of second-generation synthetic peptide shuttles FSD10 and FSD250 to introduce antisense PMOs into RH-30 cells, compared to His-CM18-PTD4 (first-generation shuttle). Figure 9A shows the results of intracellular delivery by flow cytometry. Figure 9B shows the results of cell viability by flow cytometry. The results are averages calculated from experiments performed at least twice. [Figure 10] This shows the results of Gli1 knockdown in RH-30 cells after introduction of PMO-Gli1 using the synthetic peptide shuttle FSD250, compared to VivoPMO-Gli1. Figure 10A shows the Western blot results for Gli1 and GAPDH (control). Figure 10B shows the results of densitometry scan analysis of the corresponding Western blots in Figure 10A. [Figure 11] This figure shows the ability of the synthetic peptide shuttle FSD396 to introduce antisense PMO into HeLa cells compared to Endoporter®. Representative immunofluorescence microscopy images are shown for untreated cells (Figure 11A), cells treated with PMO-FITC (Figure 11B), cells treated with PMO-FITC + FSD396 (Figure 11C), and cells treated with PMO-FITC + Endoporter (Figure 11D). [Figure 12]This figure shows the ability of four different PMOs targeting Gli1 to knock down Gli1 in human DU145 cells after induction using the synthetic peptide shuttle FSD250. Representative Western blots and corresponding densitometry scan analyses for Gli1 and actinin (control) are shown. [Figure 13] This figure shows the results of introducing PMO-Gli1 into basal cell carcinoma tumor explants using the synthetic peptide shuttle FSD250. Representative fluorescence microscope images of Cy5 (left), Cy5+DAPI (nuclear staining; center), and Cy5+DAPI+Nomarski (i.e., differential interference contrast microscopy [DIC]) (right) after treatment with PMO-Gli1-Cy5 and FSD250, or PMO-Gli1-Cy5 alone are shown. [Modes for carrying out the invention]

[0022] Sequence List This application includes a computer-readable sequence listing prepared on October 15, 2021. The computer-readable format is incorporated herein by reference.

[0023] [Table 1-1]

[0024] [Table 1-2]

[0025] [Table 1-3] [Table 1-4]

[0026] Detailed explanation In some embodiments, this specification describes compositions and methods for introducing non-anionic polynucleotide analog cargoes. The methods generally involve contacting target eukaryotic cells with a composition comprising a non-anionic polynucleotide analog cargo and a synthetic peptide shuttle agent that is independent of or not covalently bonded to the non-anionic polynucleotide analog cargo, wherein the synthetic peptide shuttle agent increases cytoplasmic / nuclear delivery of the non-anionic polynucleotide analog cargo in the eukaryotic cells.

[0027] Non-anionic polynucleotide analog cargo In some embodiments, the non-anionic polynucleotide analog cargo may be a charged-neutral or cationic antisense synthetic oligonucleotide (ASO). In some embodiments, the ASO may be a charged-neutral or cationic splice-switching oligonucleotide (SSO). In some embodiments, the non-anionic polynucleotide analog cargo may be a charged-neutral polynucleotide analog cargo having a phosphorodiamidate skeleton, an amide (e.g., peptide) skeleton, a methylphosphonate skeleton, a neutral phosphotryester skeleton, a sulfone skeleton, or a triazole skeleton. In some embodiments, the non-anionic polynucleotide analog cargo may be a cationic polynucleotide analog cargo having an aminoalkylated phosphoramidate skeleton, a guanidium skeleton, an S-methylthiourea skeleton, or a nucleosyl amino acid (NAA) skeleton. In some embodiments, the non-anionic polynucleotide analog cargo may be a phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid (PNA), a methylphosphonate oligomer, or a short-chain interfering ribonucleic acid neutral oligonucleotide (siRNN). In some embodiments, the non-anionic polynucleotide analog cargo may be 5-50 mers, 5-75 mers, or 5-100 mers. In some embodiments, the non-anionic polynucleotide analog cargo is not covalently bound to a cell membrane-permeable peptide, octaganidine dendrimer, or other intracellular delivery portion. In some embodiments, the non-anionic polynucleotide analog cargo is cell membrane-impermeable or has low membrane permeability (e.g., due to the physicochemical properties of the cargo that prevent it from freely diffusing across the cell membrane), and the peptide shuttle agents described herein facilitate or increase its intracellular delivery and / or access to the cytoplasm / nucleus. In some embodiments, the non-anionic polynucleotide analog cargo may be cell membrane-permeable, and the peptide shuttle agents described herein nevertheless increase its intracellular delivery and / or access to the cytoplasm.In some embodiments, the peptide shuttle agents described herein can reduce the amount or concentration of cargo required to be administered to achieve their intended biological effect compared to the administration of cargo alone.

[0028] In some embodiments, the non-anionic polynucleotide analog cargo may be a drug for treating any disease or condition that modifies the gene expression of a target RNA relevant to the treatment. In some embodiments, the non-anionic polynucleotide analog cargo may be a drug for treating cancer (e.g., skin cancer, basal cell carcinoma, nevus basal cell carcinoma syndrome), inflammation or inflammation-related diseases (e.g., psoriasis, atopic dermatitis, ulcerative colitis, urticaria, dry eye disease, atrophic or exudative age-related macular degeneration, finger ulcers, actinic keratosis, idiopathic pulmonary fibrosis), pain (e.g., chronic or acute), or diseases affecting the lungs (e.g., cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), or idiopathic pulmonary fibrosis).

[0029] In some embodiments, the non-anionic polynucleotide analog cargo described herein may be, for example, a splice-switching oligonucleotide (SSO) for modifying or altering the splicing of a therapeutically relevant target mRNA. In some embodiments, the target mRNA may be a cystic fibrosis membrane conductance regulator (CFTR), and the compositions or methods described herein may be for the treatment of cystic fibrosis (e.g., via administration to the lungs of a cystic fibrosis subject). In this context, synthetic peptide shuttles have been shown to enable efficient delivery of recombinant protein cargoes to refractory airway epithelial cells (Krishnamurthy et al., 2018).

[0030] In some embodiments, the non-anionic polynucleotide analog cargoes described herein are not covalently bound to cell membrane-permeable or cationic peptides, octaganidine dendrimers, or other intracellular delivery moieties. For example, such conventional delivery strategies used in peptide conjugate phosphorodiamidate morpholino oligomers (PPMOs) and Vivo-Morpholino add further complexity to the PMO synthesis process. In contrast, the synthetic peptide shuttle agents described herein can advantageously introduce unmodified or "naked" non-anionic polynucleotide analog cargoes, greatly facilitating manufacturing and formulation.

[0031] Rational design parameters and peptide shuttle agents In some embodiments, the shuttle agents described herein may be peptides having transduction activity to non-anionic polynucleotide analog cargoes, protein cargoes, or both in target eukaryotic cells. In some embodiments, the shuttle agents described herein preferably satisfy one or more of the following 15 rational design parameters or any combination thereof.

[0032] (1) In some embodiments, the shuttle agent is a peptide having at least 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid lengths. For example, the peptide may have a minimum length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues and a maximum length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acid residues. In some embodiments, shorter peptides (e.g., in the range of 17–50 or 20–50 amino acids) may be particularly advantageous because they can be more easily synthesized and purified by chemical synthesis methods and are more suitable for clinical use (in contrast to recombinant proteins that must be purified from cell expression systems). While the numbers and ranges described herein are often listed as multiples of 5, this description is not to be limited in that way. For example, the maximum length described herein includes lengths such as 56, 57, 58…61, 62, etc., and it should be understood that the non-listing of such lengths is simply for the sake of brevity. The same reasoning applies to the percentage of identity described herein.

[0033] (2) In some embodiments, the peptide shuttle agent comprises an amphiphilic alpha-helix motif at neutral pH. As used herein, the expression “alpha-helix motif” or “alpha-helix” means an alpha-helix having a right-handed coiled or spiral structure (helix) with a rotation angle of 100 degrees between consecutive amino acids and / or 3.6 residues per turn, unless otherwise specified. As used herein, the expression “contains an alpha-helix motif” or “amphiphilic alpha-helix motif,” etc., means the three-dimensional structure that the peptide (or peptide segment) described herein is expected to adopt in a biological setting based on the peptide primary amino acid sequence, regardless of whether the peptide actually adopts it when used in cells as a shuttle agent. Furthermore, the peptide described herein may contain one or more alpha-helix motifs at various peptide positions. For example, the shuttle agent FSD5 in International Publication No. 2018 / 068135 is expected to take the form of an alpha helix over its entire length (see Figure 49C in International Publication No. 2018 / 068135), while the shuttle agent FSD18 in International Publication No. 2018 / 068135 is expected to contain two separate alpha helices directed toward the N and C-terminal regions of the peptide (see Figure 49D in International Publication No. 2018 / 068135). In some embodiments, the shuttle agents described herein are not expected to contain beta sheet motifs, such as those shown in Figures 49E and 49F of International Publication No. 2018 / 068135. Methods for predicting the presence of alpha helices and beta sheets in proteins and peptides are well known in the art. For example, one such method is based on 3D modeling using PEP-FOLD®, an online resource for predicting novel peptide structures (http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD / ) (Lamiable et al., 2016; Shen et al., 2014; Thevenet et al., 2012).Other methods for predicting the presence of alpha helices in peptides and proteins are known and readily available to those skilled in the art.

[0034] As used herein, the expression “amphiphilic” means a peptide having both hydrophobic and hydrophilic elements (for example, based on the side chains of the amino acids that make up the peptide). For example, the expression “amphiphilic alpha-helix” or “amphiphilic alpha-helix motif” means a peptide that is expected to take an alpha-helix motif having a nonpolar hydrophobic surface and a polar hydrophilic surface, based on the properties of the side chains of the amino acids that form the helix.

[0035] (3) In some embodiments, the peptide shuttle agents described herein include an amphiphilic alpha-helix motif having a positively charged hydrophilic outer surface, such as one rich in R and / or K residues. As used herein, the expression “positively charged hydrophilic outer surface” means the presence of at least three lysine (K) and / or arginine (R) residues clustered on one side of the amphiphilic alpha-helix motif, based on an alpha-helix wheel projection (see, for example, the left panel of Figure 49A in International Publication No. 2018 / 068135). Such helical wheel projections can be generated using various programs, such as the online helical wheel projection tool created by Don Armstrong and Raphael Zidovetzki (e.g., available at https: / / www.donarmstrong.com / cgi-bin / wheel.pl) or the online tool developed by Mol et al., 2018 (e.g., available at http: / / lbqp.unb.br / NetWheels / ). In some embodiments, the amphiphilic alpha-helix motif includes a positively charged hydrophilic outer surface, which includes (a) at least two, three, or four adjacent positively charged K and / or R residues on a helical wheel projection, 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 of 100 degrees between consecutive amino acids and / or an alpha-helix having 3.6 residues per turn.

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

[0037] (4) In some embodiments, the peptide shuttle agents described herein include an amphiphilic alpha-helix motif having a highly hydrophobic core composed of spatially adjacent highly hydrophobic residues (e.g., L, I, F, V, W, and / or M). In some embodiments, for example, as shown in Figure 49A, right panel of International Publication No. 2018 / 068135, the highly hydrophobic core may consist of L, I, F, V, W, and / or M amino acids that are spatially adjacent and, after calculation excluding histidine-rich domains (see below), correspond to 12-50% of the amino acids of the peptide. In some embodiments, the highly hydrophobic core may consist of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% to 25%, 30%, 35%, 40%, or 45% of the peptide's amino acids. More specifically, the highly hydrophobic core parameter may be calculated by first arranging the peptide's amino acids in an open cylinder representation, and then depicting regions of consecutive highly hydrophobic residues (L, I, F, V, W, M) as shown in Figure 49A, right panel, of International Publication No. 2018 / 068135. The number of highly hydrophobic residues in this depicted highly hydrophobic core is then divided by the total amino acid length of the peptide excluding histidine-rich domains (e.g., N and / or C-terminal histidine-rich domains). For example, in the case of the peptide shown in Figure 49A of International Publication No. 2018 / 068135, there are 8 residues in the depicted highly hydrophobic core and a total of 25 residues in the peptide (excluding the 12 terminal histidines). Therefore, the highly hydrophobic core is 32% (8 / 25).

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

[0039] (6) In some embodiments, the peptide shuttle agents described herein may have a predicted effective charge of at least +3 or +4 at physiological pH, calculated from the side chains of the K, R, D, and E residues. For example, the effective charge of the peptide may be at least +5, +6, +7, at least +8, at least +9, at least +10, at least +11, at least +12, at least +13, at least +14, or at least +15 at physiological pH. These positive charges are generally conferred by the presence of more positively charged lysine and / or arginine residues, in contrast to negatively charged aspartic acid and / or glutamic acid residues.

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

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

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

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

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

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

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

[0048] (15) In some embodiments, the peptide shuttle agent described herein may consist of 10% to 45% of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, or H (i.e., other than A, L, K, or R). In certain embodiments, the peptide shuttle agent may consist of 15% to 40%, 20% to 35%, or 20% to 30% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

[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 the parameters (1) to (15) described herein. In certain embodiments, the peptide shuttle agents described herein satisfy all of the parameters (1) to (3) and also satisfy at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all of the parameters (4) to (15) described herein.

[0050] In some embodiments, the peptide shuttle agent described herein contains only one histidine-rich domain, and the residues of the single histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein. In some embodiments, if the peptide shuttle agent described herein contains two or more histidine-rich domains, only the residues of the single histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein. For example, if the peptide shuttle agent described herein contains two histidine-rich domains, a first histidine-rich domain facing the N terminus and a second histidine-rich domain facing the C terminus, only the first histidine-rich domain may be included in the calculation / evaluation of parameters (1) to (15) described herein.

[0051] In some embodiments, machine learning or computer-aided design techniques may be used to generate peptides that satisfy one or more of the parameters (1) to (15) described herein. Some parameters, such as parameters (1) and (5) to (15), are more readily implemented by computer-aided design techniques, while structural parameters, such as parameters (2), (3), and (4), are more readily applicable by manual design techniques. Therefore, in some embodiments, peptides that satisfy one or more parameters (1) to (15) may be generated by combining computer-aided and manual design techniques. For example, multiple sequence alignment analyses of several peptides shown herein (or elsewhere) to function as effective shuttle agents have revealed the presence of several consensus sequences—i.e., patterns of commonly recognized alternations of hydrophobic, cationic, and hydrophilic alanine and glycine amino acids. The presence of these consensus sequences may induce the satisfying of structural parameters (2), (3), and (4) (i.e., amphiphilic alpha-helix formation, positively charged surfaces, and a highly hydrophobic core of 12% to 50%). Therefore, these and other consensus sequences can be employed in machine learning and / or computer-aided design methods to generate peptides that satisfy one or more parameters (1) to (15).

[0052] Therefore, in some embodiments, the peptide shuttle agents described herein may include or consist of the following amino acid sequences: (a)[X1]-[X2]-[Linker]-[X3]-[X4](Equation 1); (b)[X1]-[X2]-[Linker]-[X4]-[X3](Equation 2); (c)[X2]-[X1]-[Linker]-[X3]-[X4](Equation 3); (d)[X2]-[X1]-[Linker]-[X4]-[X3](Equation 4); (e)[X3]-[X4]-[Linker]-[X1]-[X2](Equation 5); (f)[X3]-[X4]-[Linker]-[X2]-[X1](Equation 6); (g)[X4]-[X3]-[linker]-[X1]-[X2](Equation 7); (h)[X4]-[X3]-[linker]-[X2]-[X1](Equation 8); (i) [linker]-[X1]-[X2]-[linker](Equation 9); (j)[linker]-[X2]-[X1]-[linker](Equation 10); (k)[X1]-[X2]-[linker](formula 11); (l)[X2]-[X1]-[linker](Equation 12); (m)[linker]-[X1]-[X2](Equation 13); (n)[linker]-[X2]-[X1](Equation 14); (o)[X1]-[X2](Equation 15); or (p)[X2]-[X1] (Equation 16), During the ceremony, [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:-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-!+]-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]-!+]-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 I[ζ]-A-1[ζ]-A-1[+]; and The linker is selected from the following: -Gn-; -Sn-; -(GnSn)n-; -(GnSn)nGn-; -(GnSn)nSn-; -(GnSn)nGn(GnSn)n-; and (GnSn)nSn(GnSn)n-; In the formula, [Φ] is an amino acid: Leu, Phe, Trp, Ile, Met, Tyr, or Val, preferably Leu, Phe, Trp, or Ile; [+] is an amino acid: Lys or Arg; [ζ] is an amino acid: Gln, Asn, Thr, or Ser; A is an amino acid: Ala; G is an amino acid: Gly; S is an amino acid: Ser; n is an integer between 1 and 20, 1 and 19, 1 and 18, 1 and 17, 1 and 16, 1 and 15, 1 and 14, or 1 and 3.

[0053] In some embodiments, the peptide shuttle agents described herein are any of the amino acid sequences of SEQ ID NOs: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, 344 or 353-364 disclosed in International Publication No. 2018 / 068135, or SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 1 A peptide that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one amino acid sequence of 52, 169-242, or 243-10242, or a functional variant thereof. In some embodiments, the peptide shuttle agents described herein may include amino acid sequence motifs of SEQ ID NO: 158 and / or 159 of International Publication No. 2018 / 068135, as found in the peptides FSD5, FSD16, FSD18, FSD19, FSD20, FSD22, and FSD23. In some embodiments, the peptide shuttle agents described herein may include the amino acid sequence motif of SEQ ID NO: 158 of International Publication No. 2018 / 068135, ligated to the amino acid sequence motif of SEQ ID NO: 159 of International Publication No. 2018 / 068135 to function. As used herein, “functional variant” means a peptide having cargo delivery activity that differs from the reference peptide by one or more conserved amino acid substitutions. As used herein in the context of functional variant, “conserved amino acid substitution” means that one amino acid residue is replaced by another amino acid residue having a similar side chain.The family of amino acid residues having similar side chains is well defined in the art and includes basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and possibly proline), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0054] In some embodiments, the peptide shuttle agents described herein do not include one or more of the amino acid sequences of SEQ ID NOs. 57-59, 66-72, or 82-102 of International Publication No. 2018 / 068135. In some embodiments, the peptide shuttle agents described herein do not include one or more of the amino acid sequences of SEQ ID NOs. 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10242 disclosed in International Publication No. 2018 / 068135. Rather, in some embodiments, the peptide shuttle agents described herein may relate to variants of such previously described shuttle agent peptides, which are further manipulated for improved delivery activity (i.e., more reliably able to deliver non-anionic polynucleotide analog cargoes).

[0055] In some embodiments, the peptide shuttle agents described herein may have a minimum threshold for the delivery efficiency and / or cargo delivery score for a “surrogate” cargo when measured in a eukaryotic cell model system (e.g., an immortalized eukaryotic cell line) or model organism. The term “delivery efficiency” refers to the percentage or proportion of the target cell population to which the cargo of interest is delivered intracellularly, which can be determined, for example, by flow cytometry, immunofluorescence microscopy, and other suitable methods that can be used to assess cargo delivery efficiency (e.g., described in International Publication No. 2018 / 068135). In some embodiments, delivery efficiency may be expressed as a percentage of cargo-positive cells. In some embodiments, delivery efficiency may be expressed as a doubling (or doubling) against a suitable negative control evaluated under the same conditions, except in the absence of cargo and shuttle agent (“untreated”; NT) or in the absence of shuttle agent (“cargo alone”).

[0056] In some embodiments, the shuttle agent described herein is: (i) Any one amino acid sequence of sequence numbers 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, 344, or 353-364; (ii) Any amino acid sequence that differs from any one of sequence numbers 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, 344, or 353-364 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acids (for example, excluding any linker domain); (iii) At least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57% of any one of the following sequence numbers: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, 344, or 353-364 , 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences (calculated, for example, excluding any linker domain); (iv) One of the following sequence numbers: 1-50, 58-78, 80-107, 109-139, 141-146, 149-161, 163-169, 171, 174-234, 236-240, 242-260, 262-285, 287-294, 296-300, 302-308, 310, 311, 313-324, 326-332, 338-342, 344 or 353-364, and only conservative amino acid substitutions (for example, 1, 2, 3, 4, 5, 6, 7, An amino acid sequence having 8, 9, or 10 or fewer conservative amino acid substitutions (preferably excluding any linker domain), wherein each conservative amino acid substitution is selected from amino acids within the same amino acid class, where the amino acid classes are: aliphatic: G, A, V, L, and I; hydroxyl or sulfur / selenium-containing: S, C, U, T, and M; aromatic: F, Y, and W; basic: H, K, and R; acidic and their amides: D, E, N, and Q; or (v) Any combination of (i) to (iv), including or consisting of them.

[0057] In some embodiments, the shuttle agents described herein for the delivery of non-anionic polynucleotide analog cargoes are preferably second-generation shuttle agents lacking a cell membrane permeable domain or lacking a cell membrane permeable domain fused to an endosomal leakage domain. In some embodiments, the shuttle agents described herein that are particularly suitable for the delivery of non-anionic polynucleotide analog cargoes are preferably those having a relatively high delivery score, meaning the shuttle agent delivers a larger total number of cargo molecules per cell. Since the synthetic polynucleotide analogs described herein function sterically by hybridizing to their target intracellular RNA molecules (i.e., one cargo molecule binds to one intracellular RNA molecule), shuttle agents with a higher delivery score are expected to be particularly advantageous for such applications. In some embodiments, the shuttle agents described herein (and / or the sequence numbers listed above in the preceding paragraphs) are those listed in Figure 5, having a standardized average delivery score for the delivery of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 PI and / or GFP cargoes.

[0058] In some embodiments, the shuttle agents described herein include or consist of a variant of a synthetic peptide shuttle agent, the variant being identical to the synthetic peptide shuttle agent defined herein except that at least one amino acid is replaced by a corresponding synthetic amino acid having a side chain with similar physicochemical properties (e.g., structure, hydrophobicity, or charge) to the amino acid being replaced, the variant increasing the cytoplasmic / nuclear delivery of the non-anionic polynucleotide analog cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttle agent.

[0059] Chemically modified and synthetic amino acids In some embodiments, the shuttle agent described herein may comprise an oligomer (e.g., a dimer, trimer, etc.) of the peptide described herein. Such oligomers can be constructed by covalently bonding the same or different types of shuttle agent monomers (e.g., by linking cysteine ​​residues introduced into the monomer sequence using disulfide crosslinks). In some embodiments, the shuttle agent described herein may comprise N-terminal and / or C-terminal cysteine ​​residues.

[0060] In some embodiments, the shuttle agent described herein may include or consist of a cyclic peptide. In some embodiments, the cyclic peptide may be formed via a covalent bond between a first residue positioned toward the N-terminus of the shuttle agent and a second residue positioned toward the C-terminus of the shuttle agent. In some embodiments, the first and second residues are adjacent residues located at the N-terminus and C-terminus of the shuttle agent. In some embodiments, the first and second residues may be linked via an amide bond to form a cyclic peptide. In some embodiments, the cyclic peptide may be formed by a disulfide bond between two cysteine ​​residues in the shuttle agent, with the two cysteine ​​residues positioned toward the N-terminus and C-terminus of the shuttle agent. In some embodiments, the shuttle agent may include, or be manipulated to include, cysteine ​​residues adjacent to the N-terminus and C-terminus, linked via a disulfide bond to form a cyclic peptide. In some embodiments, the cyclic shuttle agents described herein may be more resistant to degradation (e.g., by proteases) and / or may have a longer half-life than the corresponding linear peptide.

[0061] In some embodiments, the shuttle agent described herein may comprise one or more D-amino acids. In some embodiments, the shuttle agent described herein may comprise D-amino acids at the N-terminus and / or C-terminus of the shuttle agent. In some embodiments, the shuttle agent may consist solely of D-amino acids. In some embodiments, the shuttle agent described herein having one or more D-amino acids may be resistant to degradation (e.g., by proteases) and / or may have a longer half-life than the corresponding peptide composed solely of L-amino acids.

[0062] In some embodiments, the shuttle agents described herein may include chemical modifications to one or more amino acids, and the chemical modifications do not destroy the cargo delivery activity of the synthetic peptide shuttle agent. As used herein, the term “destruction” means that the chemical modification irreversibly destroys the cargo delivery activity of the peptide shuttle agent described herein. Chemical modifications that can temporarily inhibit, attenuate, or delay the cargo delivery activity of the peptide shuttle agent described herein may be included in the chemical modifications to the shuttle agents described herein. In some embodiments, the chemical modification to any one of the shuttle agents described herein may be at the N-terminus and / or C-terminus of the shuttle agent. Examples of chemical modifications include the addition of an acetyl group (e.g., an N-terminal acetyl group), a cysteamide group (e.g., a C-terminal cysteamide group), or a fatty acid (e.g., a C4-C16, C6-C14, C6-C12, C6-C8, or C8 fatty acid, preferably at the N-terminus).

[0063] In some embodiments, the shuttle agent described herein comprises a shuttle agent variant having transduction activity to non-anionic polynucleotide analog cargo in target eukaryotic cells, the variant being identical to any of the shuttle agents described herein except that at least one amino acid is replaced with a corresponding synthetic amino acid or amino acid analog having a side chain with similar physicochemical properties (e.g., structure, hydrophobicity, or charge) to the amino acid being replaced. In some embodiments, the substitution of the synthetic amino acid is (a) Replace a basic amino acid with any one of the following: α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid, 2,6-diamino-4-hexic acid, β-(1-piperazinyl)-alanine, 4,5-dehydroxylysine, δ-hydroxylysine, ω,ω-dimethylarginine, homoarginine, ω,ω'-dimethylarginine, ω-methylarginine, β-(2-quinolyl)-alanine, 4-aminopiperidine-4-carboxylic acid, α-methylhistidine, 2,5-diiodohistidine, 1-methylhistidine, 3-methylhistidine, spinacine, 4-aminophenylalanine, 3-aminotyrosine, β-(2-pyridyl)-alanine, or β-(3-pyridyl)-alanine; (b) Dehydroalanine, β-Fluoroalanine, β-Chloroalanine, β-Iodoalanine, α-Aminobutyric Acid, α-Aminisobutyric Acid, β-Cyclopropylalanine, Azethidine-2-carboxylic Acid, α-Allylglycine, Propargylglycine, tert-Butylalanine, β-(2-Thiazolyl)-Alanine, Thiaproline, 3,4-Dehydroproline, tert-Butylglycine, β-Cyclopentylalanine, β-Cyclohexylalanine, α-Me Tylproline, norvaline, α-methylvaline, penicillamine, β,β-dicyclohexylalanine, 4-fluoroproline, 1-aminocyclopentanecarboxylic acid, pipecolic acid, 4,5-dehydroleucine, alloisoleucine, norleucine, α-methylleucine, cyclohexylglycine, cis-octahydroindole-2-carboxylic acid, β-(2-thienyl)-alanine, phenylglycine, α-methylphenylalanine, homophenylalanine Replace a nonpolar (hydrophobic) amino acid with one of the following: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-(3-benzothienyl)-alanine, 4-nitrophenylalanine, 4-bromophenylalanine, 4-tert-butylphenylalanine, α-methyltryptophan, β-(2-naphthyl)-alanine, β-(1-naphthyl)-alanine, 4-iodophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, 4-methyltryptophan, 4-chlorophenylalanine, 3,4-dichlorophenylalanine, 2,6-difluorophenylalanine, n-in-methyltryptophan, 1,2,3,4-tetrahydronorharmann-3-carboxylic acid, β,β-diphenylalanine, 4-methylphenylalanine, 4-phenylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, or 4-benzoylphenylalanine; (c)β-Cyanolalanine, β-Ureidoalanine, Homocysteine, Allothreonine, Pyroglutamic Acid, 2-Oxothiazolidine-4-carboxylic Acid, Citrulline, Thiocitrulline, Homocitrulline, Hydroxyproline, 3,4-Dihydroxyphenylalanine, β-(1,2,4-Triazole-1-yl)alanine, 2-Mercaptohistidine, β-(3,4-Dihydroxyphenyl)-serine, β-(2-Thienyl)-serine, 4-Azidophenylalanine, 4-Cyanofenyl Replace a polar uncharged amino acid with any one of the following: rualanine, 3-hydroxymethyltyrosine, 3-iodotyrosine, 3-nitrotyrosine, 3,5-dinitrotyrosine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 5-hydroxytryptophan, tyronine, β-(7-methoxycoumarin-4-yl)alanine, or 4-(7-hydroxy-4-coumarinyl)-aminobutyric acid; and / or (d) Replace the acidic amino acid with one of the following: γ-hydroxyglutamic acid, γ-methyleneglutamic acid, γ-carboxyglutamic acid, α-aminoadipic acid, 2-aminoheptanedioic acid, α-aminosuberic acid, 4-carboxyphenylalanine, cysteic acid, 4-phosphonophenylalanine, or 4-sulfomethylphenylalanine.

[0064] Histidine-rich domain In some embodiments, the peptide shuttle described herein may further comprise one or more histidine-rich domains. In some embodiments, the histidine-rich domain may be a stretch of at least two, at least three, at least four, at least five, or at least six amino acids, comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% histidine residues. In some embodiments, the histidine-rich domain may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive histidine residues. While not constrained by theory, the histidine-rich domain in the shuttle agent may act as a proton sponge in endosomes by protonating its imidazole group under acidic endosome conditions, providing another mechanism for endosomal membrane destabilization and thus further enhancing the ability of endosome-trapped cargo to reach the cytosol. In some embodiments, the histidine-rich domain may be located at the N and / or C terminus of the peptide shuttle agent or may be oriented toward the N and / or C terminus.

[0065] Linker In some embodiments, the peptide shuttle agents described herein may comprise one or more suitable linkers (e.g., flexible polypeptide linkers). In some embodiments, such linkers may detach two or more amphiphilic alpha-helix motifs (see, for example, shuttle agent FSD18 in Figure 49D of International Publication No. 2018 / 068135). In some embodiments, the linkers may be used to separate two additional domains (CPD, ELD, or histidine-rich domains) from each other. In some embodiments, the linkers may be formed by adding a sequence of small hydrophobic amino acids (e.g., glycine) that do not have rotational ability and polar serine residues that confer stability and mobility. The linkers may be soft and allow the domains of the shuttle agent to move. In some embodiments, proline may be avoided as it may add significant steric rigidity. In some embodiments, the linkers may be serine / glycine-rich linkers (e.g., GS, GGS, GGSGGGS, GGSGGGSGGGS, etc.). In some embodiments, the use of a shuttle agent containing a suitable linker may be advantageous for delivering cargo to suspension cells rather than adherent cells. In some embodiments, the linker includes or consists of -Gn-;-Sn-;-(GnSn)n-;-(GnSn)nGn-;-(GnSn)nSn-;-(GnSn)nGn(GnSn)n-; or (GnSn)nSn(GnSn)n-, where G is the amino acid Gly; S is the amino acid Ser; and n is an integer from 1 to 5. In some embodiments, a short stretch or "linker" of a flexible and / or hydrophilic amino acid (e.g., glycine / serine-rich stretch) may be added to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the shuttle agent described herein, or to the C-terminus cleavage shuttle agent described herein. In some embodiments, such stretching can facilitate the dissolution of shuttle agents that are otherwise insoluble or poorly soluble in aqueous solutions, particularly short shuttle agents (e.g., those having an amphiphilic alpha-helix structure containing a strongly hydrophobic portion).In some embodiments, increasing the solubility of the shuttle agent peptide can avoid the use of organic solvents (e.g., DMSO) which may obscure cargo delivery results and / or make the shuttle agent unsuitable for therapeutic application.

[0066] Domain-based peptide shuttle agents In some embodiments, the shuttle agent described herein may be the shuttle agent described in International Publication No. 2016 / 161516, and comprises an endosomal leakage domain (ELD) operably linked to a cell membrane permeability domain (CPD).

[0067] Endosome leakage domain (ELD) In some embodiments, the peptide shuttle agents described herein may include an endosomal leakage domain (ELD) to facilitate endosomal escape and access to the cytoplasmic compartment. As used herein, the expression “endosomal leakage domain” refers to a sequence of amino acids that confers the ability of cargo captured by endosomes to reach the cytoplasmic compartment. While not theoretically bound, endosomal leakage domains are short sequences (often derived from viral or bacterial peptides) that are thought to induce destabilization of the endosomal membrane and release of endosomal contents into the cytoplasm. As used herein, the expression “endosomal soluble peptide” refers to this general class of peptides having endosomal membrane destabilizing properties. Accordingly, in some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may include an ELD that is an endosomal soluble peptide. The activity of such peptides can be evaluated, for example, using the calcein endosomal escape assay described in Example 2 of International Publication No. 2016 / 161516.

[0068] In some embodiments, ELDs can be peptides that disrupt membranes at acidic pH, such as pH-dependent membrane-active peptides (PMAPs) or pH-dependent soluble peptides. For example, peptides GALA and INF-7 are amphiphilic peptides that form alpha helices when a decrease in pH modifies the charge of the amino acids they contain. While not bound by theory, more specifically, it is suggested that ELDs such as GALA induce endosomal leakage by forming membrane lipid pores and flip-flops after conformational changes due to pH reduction (Kakudo, Chaki et al., 2004, Li, Nicol et al., 2004). In contrast, it has been suggested that ELDs such as INF-7 induce endosomal leakage by accumulating in the endosomal membrane and destabilizing it (El-Sayed, Futaki et al., 2009). Therefore, a simultaneous decrease in pH during endosomal maturation causes changes in peptide structure, which destabilizes the endosomal membrane and leads to the release of endosomal contents. The same principle is thought to apply to Pseudomonas toxin A (Varkouhi, Scholte et al., 2011). After a decrease in pH, the structure of the toxin's translocation domain changes, allowing its insertion into the pore-forming endosomal membrane (London 1992, O'Keefe 1992). This ultimately leads to endosomal destabilization and the movement of the complex to the outside of the endosome. The above ELD is encompassed within the ELD described herein and other mechanisms of endosomal leakage whose mechanisms of action are not yet fully understood.

[0069] In some embodiments, ELDs may be antimicrobial peptides (AMPs), such as linear cationic alpha-helix antimicrobial peptides (AMPs). These peptides play a crucial role in the innate immune response due to their ability to strongly interact with bacterial membranes. While not theoretically bound, these peptides are thought to exist in a disordered state in aqueous solutions but adopt an alpha-helix secondary structure in hydrophobic environments. The latter structure is thought to contribute to their typical concentration-dependent membrane disruption properties. When accumulated in endosomes at specific concentrations, some antimicrobial peptides can induce endosomal leakage.

[0070] In some embodiments, the ELD may be an antimicrobial peptide (AMP), such as the cecropine-A / melittin hybrid (CM) peptide. Such peptides are considered to be among the smallest and most effective AMP-derived peptides with membrane-disrupting ability. Cecropine is a family of antimicrobial peptides that have membrane-disrupting ability against both Gram-positive and Gram-negative bacteria. Cecropine A (CA), the first identified antimicrobial peptide, consists of 37 amino acids with a linear structure. Melittin (M), a 26-amino acid peptide, is a cell membrane-lytic factor found in bee venom. Cecropine-melittin hybrid peptides have been shown to produce short, efficient antibiotic peptides that are non-cytotoxic to eukaryotic cells (i.e., non-hemolytic), a desirable property for any antimicrobial agent. These chimeric peptides have been constructed from various combinations of the hydrophilic N-terminal domain of cecropine A and the hydrophobic N-terminal domain of melittin and have been tested in bacterial model systems. Two 26-mers, CA(1-13)M(1-13) and CA(1-8)M(1-18) (Boman et al., 1989), have been shown to demonstrate a broader, improved potency of natural secropin A without the cytotoxic effects of melittin.

[0071] In an attempt to produce shorter CM series peptides, Andreu et al., authors of 1992, constructed hybrid peptides such as 26-mer(CA,1-8)M,1-18 and compared them with 20-mer(CA,1-8)M,1-12, 18-mer(CA,1-8)M,1-10, and six 15-mer(CA(1-7)M(1-8), CA(1-7)M(2-9), CA(1-7)M(3-10), CA(1-7)M(4-11), CA(1-7)M(5-12), and CA,1-7)M,6-13. The 20-mer and 18-mer maintained similar activity compared to CA(1-8)M(1-18). Among the 15 meridians of the 6 types, CA(1-7)M(1-8) showed low antibacterial activity, while the other 5 types showed similar antibiotic efficacy compared to the 26 meridians that did not exhibit hemolytic effects. Therefore, in some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may include CM series peptide variants such as those mentioned above, or ELDs derived therefrom.

[0072] In some embodiments, ELD may be the CM series peptide CM18, which consists of residues 1-7 of secropine-A (KWKLFKKIGAVLKVLTTG) fused with residues 2-12 of melittin (YGRKKRRQRRR) [C(1-7)M(2-12)]. When fused with the cell membrane permeable peptide TAT, CM18 independently crosses the cell membrane, destabilizes the endosomal membrane, and allows cargo captured by some endosomes to be released into the cytosol (Salomone et al., 2012). However, the use of the CM18-TAT11 peptide fused with the fluorophore (atto-633) in some of the authors' experiments increased uncertainty about the peptide's contribution to the fluorophore, as the use of the fluorophore itself was found to contribute, for example, to endosomal lysis by photochemical disruption of the endosomal membrane (Erazo-Oliveras et al., 2014).

[0073] In some embodiments, ELD may be a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identity with respect to SEQ ID NO: 1 of International Publication No. 2016 / 16516, and possessing endosomal lytic activity.

[0074] In some embodiments, ELD may be a peptide derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA), which can cause endosomal membrane destabilization when accumulated in endosomes.

[0075] In some embodiments, the synthetic peptide or polypeptide-based shuttle agents described herein may include ELDs shown in Table I, or ELDs derived therefrom, or variants thereof having endosomal escape activity and / or pH-dependent membrane disruption activity.

[0076] [Table 2]

[0077] In some embodiments, the shuttle agent described herein may comprise one or more ELDs or types of ELDs. More specifically, they may comprise at least two, at least three, at least four, at least five, or more ELDs. In some embodiments, the shuttle agent may comprise 1 to 10 types of ELDs, 1 to 9 types of ELDs, 1 to 8 types of ELDs, 1 to 7 types of ELDs, 1 to 6 types of ELDs, 1 to 5 types of ELDs, 1 to 4 types of ELDs, 1 to 3 types of ELDs, etc.

[0078] In some embodiments, the order or arrangement of ELDs relative to other domains (CPDs, histidine-rich domains) in the shuttle agent described herein may be changed as long as the shuttle agent's reversible transport capability is maintained.

[0079] In some embodiments, the ELD is any one variant or fragment listed in Table I and may have endosomal lytic activity. In some embodiments, the ELD may include, or consist of, any one amino acid sequence of any one of Sequence IDs 1-15, 63, or 64 of International Publication No. 2016 / 16516 or any one of Sequence IDs 1-15, 63, or 64 of International Publication No. 2016 / 16516, and may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to any one of Sequence IDs 1-15, 63, or 64 of International Publication No. 2016 / 16516 and having endosomal lytic activity.

[0080] In some embodiments, the shuttle agents described herein do not contain one or more amino acid sequences from any one of the sequence numbers 1-15, 63, or 64 of International Publication No. 2016 / 16516.

[0081] Cell permeation domain (CPD) In some embodiments, the shuttle agents described herein may include a cell penetration domain (CPD). As used herein, the term “cell penetration domain” means a sequence of amino acids that confers the ability of a CPD-containing polymer (e.g., a peptide or protein) to be introduced into cells.

[0082] In some embodiments, the CPD may be (or may be derived from) a cell membrane-permeable peptide or a protein transduction domain of a cell membrane-permeable peptide. Cell membrane-permeable peptides can function as carriers for the successful delivery of various cargoes (e.g., polynucleotides, polypeptides, small molecule compounds, or other macromolecules / compounds that are otherwise membrane-impermeable) into cells. Cell membrane-permeable peptides are often rich in basic amino acids and, when fused with (or rather operably linked to) macromolecules, contain short peptides that mediate internal translocation into cells (Shaw, Catchpole et al., 2008). The first cell membrane-permeable peptide was identified by analyzing the cell penetration ability of the transcriptional HIV-1 transactivator (Tat) protein (Green and Loewenstein 1988, Vives, Brodin et al., 1997). This protein contains a short hydrophilic amino acid sequence named "TAT" that facilitates its insertion into the cell membrane and pore formation. Since this discovery, numerous other cell membrane-permeable peptides have been reported. In this regard, in some embodiments, the CPD may be a cell membrane-permeable peptide or a variant thereof having cell membrane-permeable activity, as described in Table II.

[0083] [Table 3]

[0084] While not bound by theory, cell membrane-permeable peptides are thought to interact with the cell membrane and then pass through the membrane via pinocytosis or endocytosis. In the case of TAT peptides, their hydrophilicity and charge are thought to promote insertion into the cell membrane and pore formation (Herce and Garcia 2007). Alpha-helix motifs (e.g., SP) within hydrophobic peptides are also thought to form pores within the cell membrane (Veach, Liu et al., 2004).

[0085] In some embodiments, the shuttle agents described herein may comprise one or more CPDs or types of CPDs. More specifically, they may comprise at least two, at least three, at least four, or at least five or more CPDs. In some embodiments, the shuttle agents may comprise CPDs between 1 and 10, CPDs between 1 and 6, CPDs between 1 and 5, CPDs between 1 and 4, CPDs between 1 and 3, and so on.

[0086] In some embodiments, CPD is a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity with respect to SEQ ID NO: 17 of International Publication No. 2016 / 16516, and having cell membrane permeable activity, or an international publication Penetratin having the amino acid sequence of Sequence ID No. 18 of International Publication No. 2016 / 16516, or a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity with Sequence ID No. 18 of International Publication No. 2016 / 16516 and possessing cell membrane permeable activity.

[0087] In some embodiments, CPD may be PTD4 having the amino acid sequence of SEQ ID NO: 65 of International Publication No. 2016 / 16516, or a variant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity with SEQ ID NO: 65 of International Publication No. 2016 / 16516.

[0088] In some embodiments, the order or arrangement of CPDs relative to other domains (ELDs, histidine-rich domains) within the shuttle agent described herein may be changed as long as the ability of the shuttle agent to reverse-introduce is maintained.

[0089] In some embodiments, the CPD is any one variant or fragment listed in Table II and may have cell membrane permeable activity. In some embodiments, the CPD may include or consist of any amino acid sequence of any one of Sequence IDs 16-27 or 65 of International Publication No. 2016 / 16516 or any one of Sequence IDs 16-27 or 65 of International Publication No. 2016 / 16516 that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to any one of Sequence IDs 16-27 or 65 of International Publication No. 2016 / 16516 and has cell membrane permeable activity.

[0090] In some embodiments, the shuttle agents described herein do not contain one or more amino acid sequences from SEQ ID NOs. 16-27 or 65 of International Publication No. 2016 / 16516.

[0091] Methods, kits, uses, compositions, and cells In some embodiments, this description relates to a method for delivering a non-anionic polynucleotide analog cargo from the extracellular space of a target eukaryotic cell to the cytoplasm and / or nucleus. The method includes contacting the target eukaryotic cell with the cargo in the presence of a shuttle agent at a concentration sufficient to increase the delivery efficiency of the cargo compared to the absence of the shuttle agent. In some embodiments, contact between the target eukaryotic cell and the cargo in the presence of the shuttle agent results in an increase of at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold in the delivery efficiency of the non-anionic polynucleotide analog cargo compared to the absence of the shuttle agent.

[0092] In some embodiments, this description relates to a method for increasing the delivery efficiency of a non-anionic polynucleotide analog cargo into the cytoplasm and / or nucleus of target eukaryotic cells. As used herein, the expression “increased delivery efficiency” refers to the ability of the shuttle agent described herein to improve the percentage or proportion of the target cell population to which the cargo of interest (e.g., a non-anionic polynucleotide analog cargo) is delivered into the cell. Cargo delivery efficiency can be evaluated using immunofluorescence microscopy, flow cytometry, and other suitable methods. In some embodiments, the shuttle agent described herein can enable delivery efficiencies of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, as measured, for example, by immunofluorescence microscopy, flow cytometry, FACS, and other suitable methods. In some embodiments, the shuttle agents described herein may enable one of the aforementioned transduction efficiencies, along with cell viability of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, as measured by the assay described in Example 3.3a of International Publication No. 2018 / 068135, or by another suitable assay known in the art.

[0093] In addition to increasing the efficiency of target cell delivery, the shuttle agents described herein may facilitate the delivery of the cargo of interest (e.g., non-anionic polynucleotide analog cargo) to the cytoplasm and / or nucleus of target cells. In this regard, efficiently delivering extracellular cargo to the cytoplasm and / or nucleus of target cells using peptides can be difficult. This is because cargo often becomes trapped in intracellular endosomes after crossing the cell membrane, which can limit its intracellular availability and lead to its eventual metabolic degradation. For example, the use of a protein transduction domain derived from the HIV-1 Tat protein has been reported to result in the large-scale sequestration of cargo into intracellular vesicles. In some embodiments, the shuttle agents described herein may facilitate the ability of endosome-trapped cargo to escape from the endosome and gain access to the cytoplasmic compartment. In this regard, for example, the expression "to the cytoplasm" in the phrase "increases the efficiency of introducing non-anionic polynucleotide analog cargoes into the cytoplasm" is intended to refer to the ability of the shuttle agent described herein to enable the target cargo, once delivered into the cell, to escape from endosomal confinement and gain access to the cytoplasmic and / or nuclear compartments. After the target cargo has gained access to the cytoplasm, it can freely bind to intracellular targets (e.g., in the cytoplasm, nucleus, nucleolus, mitochondria, and peroxisomes). In some embodiments, therefore, the expression "to the cytoplasm" is intended to include not only delivery to the cytoplasm but also delivery to other intracellular compartments that require the cargo to first gain access to the cytoplasmic compartment.

[0094] In some embodiments, the methods described herein are in vitro methods (e.g., for therapeutic and / or diagnostic purposes). In other embodiments, the methods described herein are in vivo methods (e.g., for therapeutic and / or diagnostic purposes). In some embodiments, the methods described herein involve topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of a non-anionic polynucleotide analog cargo and a synthetic peptide shuttle. In some embodiments, the Specified Description describes compositions formulated for topical, enteral / gastrointestinal (e.g., oral), or parenteral administration of a non-anionic polynucleotide analog cargo and a synthetic peptide shuttle.

[0095] In some embodiments, the method described herein may include the step of contacting target eukaryotic cells with a shuttle agent or composition as defined herein, and a non-anionic polynucleotide analog cargo. In some embodiments, the shuttle agent or composition may be pre-incubated with the cargo to form a mixture before the target eukaryotic cells are exposed to the mixture. In some embodiments, the type of shuttle agent may be selected based on the identity and / or physicochemical properties of the cargo delivered into the cells. In other embodiments, the type of shuttle agent may be selected to take into account the identity and / or physicochemical properties of the cargo delivered into the cells, the type of cell, the type of tissue, etc.

[0096] In some embodiments, the method may involve multiple treatments of target cells with the shuttle agent or composition (e.g., one, two, three, four or more times per day and / or according to a predetermined schedule). In such cases, lower concentrations of the shuttle agent or composition may be desirable (e.g., to reduce toxicity). In some embodiments, the cells may be suspension cells or adherent cells. In some embodiments, those skilled in the art may adapt the teachings described herein to suit specific needs for delivering non-anionic polynucleotide analog cargoes to specific cells with desired viability, using different combinations of shuttles, domains, uses, and methods.

[0097] In some embodiments, the methods described herein can be applied to methods for delivering non-anionic polynucleotide analog cargoes into cells in vivo. Such methods can be achieved by parenteral administration or direct injection into tissues, organs, or systems.

[0098] In some embodiments, the compositions or synthetic peptide shuttles described herein may be for in vitro or in vivo use to increase the efficiency of introducing non-anionic polynucleotide analog cargoes (e.g., targeting therapeutically or biologically relevant RNA molecules) into target eukaryotic cells, and the synthetic peptide shuttle or synthetic peptide shuttle variant may be used or formulated for use at concentrations sufficient to increase the efficiency of introducing cargoes into target eukaryotic cells and cytoplasmic and / or nuclear delivery compared to the absence of the synthetic peptide shuttle or synthetic peptide shuttle variant.

[0099] In some embodiments, the compositions or synthetic peptide shuttles described herein can be used therapeutically, wherein the synthetic peptide shuttle or synthetic peptide shuttle variant introduces therapeutically relevant non-anionic polynucleotide analog cargoes into the cytoplasm and / or nucleus of target eukaryotic cells, and the synthetic peptide shuttle or synthetic peptide shuttle variant is used (or formulated for use) at a concentration sufficient to increase the efficiency of cargo introduction into target eukaryotic cells compared to the absence of the synthetic peptide shuttle.

[0100] In some embodiments, this specification describes compositions for use in introducing non-anionic polynucleotide analog cargoes into target eukaryotic cells, the compositions comprising a synthetic peptide shuttle formulation with pharmaceutically appropriate excipients, the concentration of the synthetic peptide shuttle in the composition being sufficient to increase the efficiency of cargo introduction into the target eukaryotic cells and cytoplasmic and / or nuclear delivery at administration compared to the absence of the synthetic peptide shuttle. In some embodiments, the composition further comprises cargoes. In some embodiments, the composition may be mixed with the cargoes before administration or therapeutic use.

[0101] In some embodiments, this specification describes compositions for therapeutic use, comprising a synthetic peptide shuttle agent formulated with a non-anionic polynucleotide analog cargo to be introduced into target eukaryotic cells by the synthetic peptide shuttle agent, wherein the concentration of the synthetic peptide shuttle agent in the composition is sufficient to increase the efficiency of cargo introduction into the target eukaryotic cells and cytoplasmic and / or nuclear delivery at administration compared to the absence of the synthetic peptide shuttle agent.

[0102] In some embodiments, this specification describes compositions comprising a non-anionic polynucleotide analog cargo for intracellular delivery and a synthetic peptide shuttle agent that is independent of or not covalently bonded to the non-anionic polynucleotide analog cargo, wherein the synthetic peptide shuttle agent is a peptide comprising an amphiphilic alpha-helix motif having both a positively charged hydrophilic outer surface and a hydrophobic outer surface, and the synthetic peptide shuttle agent increases the cytoplasmic / nuclear delivery of the non-anionic polynucleotide analog cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttle agent. In some embodiments, the compositions and / or shuttle agents described herein are free of organic solvents (e.g., DMSO) or do not contain organic solvents in concentrations unsuitable for therapeutic or human use. In some embodiments, the shuttle agents described herein are advantageously designed with water solubility in mind, thereby eliminating the need for the use of organic solvents.

[0103] In some embodiments, the shuttle agent or composition and the non-anionic polynucleotide analog cargo may be exposed to target cells in or out of the presence of serum. In some embodiments, the method may be suitable for clinical or therapeutic use.

[0104] In some embodiments, this description relates to a kit for delivering a non-anionic polynucleotide analog cargo from the extracellular space of a target eukaryotic cell to the cytoplasm and / or nucleus. In some embodiments, this description relates to a kit for increasing the efficiency of introducing a non-anionic polynucleotide analog cargo into the cytoplasm of a target eukaryotic cell. The kit may comprise a shuttle agent, or a composition as defined herein, and a suitable container.

[0105] In some embodiments, the target eukaryotic cells may be animal cells, mammalian cells, or human cells. In some embodiments, the target eukaryotic cells may be stem cells (e.g., embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, peripheral blood stem cells), primary cells (e.g., myoblasts, fibroblasts), immune cells (e.g., NK cells, T cells, dendritic cells, antigen-presenting cells), epithelial cells, skin cells, gastrointestinal cells, mucosal cells, or lung (lung) cells. In some embodiments, the target cells may include those having a cellular apparatus for endocytosis (i.e., producing endosomes).

[0106] In some embodiments, this description relates to isolated cells containing a synthetic peptide shuttle agent as defined herein. In some embodiments, the cells may be pluripotent stem cells. It is understood that cells that are often resistant to or unsuitable for DNA transfection may be interesting candidates for the synthetic peptide shuttle agents described herein.

[0107] In some embodiments, this description relates to compositions or methods described herein, where the non-anionic polynucleotide analog cargo is a non-anionic antisense oligonucleotide that targets a gene in the Hedgehog pathway. In some embodiments, the non-anionic antisense oligonucleotide targets Gli1 for knockdown. In some embodiments, the non-anionic antisense oligonucleotide hybridizes to one of the polynucleotide sequences of SEQ ID NOs. 365-368 (e.g., in the cytoplasm or under cytoplasmic conditions). In some embodiments, the non-anionic antisense oligonucleotide described herein contains a sequence that hybridizes to one of SEQ ID NOs. 365-368. In some embodiments, this description relates to compositions or methods described herein, compositions or methods for the treatment of Gorin syndrome and / or basal cell carcinoma. [Examples]

[0108] (Example 1) Materials and methods All materials and methods not described or specified herein were generally those as demonstrated in International Publication No. 2018 / 068135, CA 3,040,645, or International Publication No. 2020 / 210916.

[0109] Materials and Reagents

[0110] [Table 4]

[0111] Cell lines and culture conditions The cells were cultured according to the manufacturer's instructions.

[0112] [Table 5]

[0113] Phosphorodiamidate morpholino oligomer introduction protocol Phosphodiamidate morpholino oligomer (PMO-FITC) labeled with the fluorophore FITC was prepared in 1 mM sterile water. HeLa cells were seeded in 96-well dishes (20,000 cells / well) the day before the experiment. Each delivery mixture containing synthetic peptide shuttle (7.5, 10, or 20 μM) and PMO-FITC (6 μM) was prepared and mixed to 50 μL in RPMI-1640 medium. The cells were washed once with PBS and added to the cells with either the shuttle / PMO-FITC or PMO-FITC alone for 5 minutes. Next, 100 μL of DMEM containing 10% FBS was added to the mixture and removed. The cells were washed once with PBS and incubated in DMEM containing 10% FBS. The cells were analyzed by flow cytometry after 2 hours of incubation.

[0114] Antisense GFP-PMO delivery protocol in GFPd reporter HeLa cells Cargo stock solutions were prepared as PMO stock (1 mM in water) and siRNA stock (100 μM in 60 mM KCl, 6 mM HEPES-pH 7.5, and 0.2 mM MgCl2).

[0115] Delivery HeLa-plex-TetO-GFPd cells were seeded in 96-well dishes (20,000 cells / well) the day before the experiment. Each delivery mixture containing synthetic peptide shuttle (7.5 μM) and PMO (0.1 or 10 μM) was prepared and mixed to 50 μL in RPMI-1640 medium. The cells were washed once with PBS, and the shuttle / PMO or PMO alone was added to the cells for 5 minutes. Next, 100 μL of DMEM containing 10% FBS was added to the mixture and removed. The cells were washed once with PBS and incubated in DMEM containing 10% FBS. After 5 hours of incubation by flow cytometry, the cells were analyzed.

[0116] Transfection HeLa-plex-TetO-GFPd cells were seeded in 96-well dishes (20,000 cells / well) the day before the experiment. siRNA (2.5 pmol) was transfected using Lipofectamine® RNAiMax reagent according to the manufacturer's instructions. Lipofectamine® RNAiMax was diluted in Opti-MEM (0.3 μL in 25 μL). The siRNA stock was first diluted in 10 μM RNAse-free water, then 2.5 pmol (0.25 μL) was added to 25 μL Opti-MEM. The diluted Lipofectamine® RNAiMax was mixed with the diluted siRNA (final concentration 50 nM) and incubated at room temperature for 5 minutes. The cells were washed once with PBS and 100 μL of DMEM containing 10% FBS was added to the cells. The diluted siRNA in Lipofectamine® RNAiMax was added to the cells. After 24 hours, the culture medium was replaced with 100 μL of fresh DMEM containing 10% FBS. Cells were analyzed by flow cytometry 48 hours after transfection.

[0117] Antisense PMO delivery protocol for knockdown of Gli1 expression in DU145 cells DU145 cells were trypsin-treated and seeded in 24-well dishes (500,000 cells / well) the day before the experiment. Each delivery mixture was prepared containing synthetic peptide shuttle agent (5 μM) and PMO-FITC (6 μM) alone or with antisense PMO (6 μM) designed to knock down the expression of the target protein, and mixed to 1 mL in plain RPMI-1640 medium. Cells were washed once with PBS and the delivery mixture was added to the cells for 5 minutes. Next, 2 mL of RPMI-1640 containing 10% FBS was added to the mixture and removed. Untreated cells were incubated with RPMI-1640 alone. Cells were incubated in fresh RPMI-1640 containing 10% FBS. After 48 hours, the medium was removed and cells were washed once with PBS before trypsin treatment. Cells were harvested, recovered by centrifugation, washed with PBS, and resuspended. Next, PMO-FITC-positive and PMO-FITC-negative cells were sorted and collected by FACS (BD FACS Aria Fusion). FITC-positive and FITC-negative cell samples were collected by centrifugation and resuspended in 50 μL of protein extraction RIPA buffer (150 mM NaCl, 1% Nonidet® P-40, 0.1% SDS, 0.5% sodium deoxycholate, 25 mM Tris). Total protein concentration was measured using a BCA protein assay kit. For all conditions, 10 μg of protein was prepared to a final dose of 40 μL using 4X Laemmeli and RIPA buffer. The protein samples were then heated at 90°C and separated through an 8% SDS-PAGE gel. The proteins were then transferred to a 0.2 μM PVDF membrane overnight (25 volts). The membrane was blocked for 1 hour using 5% bovine serum albumin, Tris-buffered saline, and 0.1% Tween® 20 solution (5% BSA / TBS-T). After blocking, the membrane was incubated for 1 hour with a 5% BSA / TBS-T solution containing anti-alphaactinin (D6F6) primary antibody at a 1:1000 ratio. Alpha-actinin protein is present in the cell lysates and serves as a loading control. Subsequently, the membrane was incubated overnight with a 5% BSA / TBS-T solution containing anti-Gli1 primary antibody diluted to 1:500.Next, the membrane was incubated for 1 hour in a 1:5000 dilution of a 5% BSA / TBS-T solution of HRP-conjugated goat anti-rabbit secondary antibody. Chemiluminescence detection was performed using Clarity® Western ECL Substrate and ChemiDoc® XRS instruments. Gli1 and alpha-actinin densitometry were evaluated using ImageJ® software.

[0118] Propidium iodide or GFP-NLS introduction protocol HeLa cells were seeded in 96-well dishes (20,000 cells / well) the day before the experiment. Each delivery mixture containing synthetic peptide shuttle agent (10 μM) and propidium iodide (PI) (10 μg / mL) or GFP-NLS (10 μM) was prepared and brought to 50 μL in phosphate-buffered saline (PBS) for PI or RPMI-1640 medium for GFP-NLS. The cells were washed once, and the shuttle agent / PI or shuttle agent / GFP-NLS was added to the cells for 1 minute (PI) or 5 minutes (GFP-NLS). Next, 100 μL of DMEM containing 10% FBS was added to the mixture and removed. The cells were washed once with PBS and incubated in DMEM containing 10% FBS. After 2 hours of incubation by flow cytometry, the cells were analyzed. Cells were analyzed 1 hour after PI or GFP-NLS treatment.

[0119] (Example 2) Synthetic peptide shuttle agents: A novel class of intracellular delivery peptides. Synthetic peptides called shuttle agents represent a novel class of intracellular delivery peptides that possess the ability to rapidly introduce polypeptide cargoes into the cytoplasmic / nuclear compartment of eukaryotic cells. In contrast to traditional intracellular delivery strategies based on cell membrane-permeable peptides, synthetic peptide shuttle agents are not covalently bound to their polypeptide cargoes. In fact, covalently binding shuttle agents to their cargoes by non-cleavable means generally has adverse effects on their delivery activity.

[0120] First-generation synthetic peptide shuttle agents, described in International Publication No. 2016 / 161516, consist of peptides based on multiple domains having operably linked endosomal leakage domains (ELDs) to cell membrane permeability domains (CPDs), and optionally further comprising one or more histidine-rich domains. While shuttle agent-mediated cargo delivery was initially thought to occur via a mechanism similar to that of conventional cell membrane permeability peptides, the rate and efficiency of cargo delivery to the cytoplasmic / nuclear compartment suggest a strong contribution from a more direct delivery mechanism that crosses the cell membrane without requiring complete endosome formation (DelGuidice et al., 2018). Therefore, a large-scale, iterative design and screening program using first-generation shuttle agents as a starting point was attempted to optimize the shuttle agents for rapid and efficient polypeptide cargo delivery while reducing cytotoxicity. The program included manual and computer-aided design / modeling of approximately 11,000 synthetic peptides, as well as the synthesis of hundreds of different peptides and testing their ability to rapidly and efficiently deliver various polypeptide cargoes in multiple cells and tissues. Rather than considering shuttle agents as fusions of cell membrane-permeable peptides (CPDs) and endosomal-soluble peptides (ELDs) as is well known from the literature, each peptide was considered holistically based on its predicted three-dimensional structure and physicochemical properties. The design and screening program determined a rational design for shuttle agents with an improved delivery / toxicity profile over first-generation shuttle agents for polypeptide cargoes, resulting in second-generation synthetic peptide shuttle agents defined by a set of 15 parameters described in International Publication 2018 / 068135. These second-generation synthetic peptide shuttle agents were designed and empirically screened for rapid (i.e., typically less than 5 minutes) delivery of polypeptide cargoes, and were primarily designed to lack prototypical CPDs.

[0121] (Example 3) Inefficient delivery of naked DNA / RNA cargo to cytoplasmic / nuclear compartments by synthetic peptide shuttles. Cell membrane-permeable peptides (CPPs) have been used for decades in transfection strategies for delivering DNA / RNA into cells. Polynucleotide delivery using CPPs can be divided into two categories: those where the CPP is covalently or electrostatically bound to the polynucleotide cargo. The increased complexity in the synthesis of the former presents a significant hurdle, while the latter is relatively simple given the cationic nature of CPPs and the negatively charged phosphate backbone of DNA / RNA. Therefore, during the screening of first-generation synthetic peptide shuttles, experiments were conducted to determine whether the shuttle could efficiently introduce plasmid DNA cargo for gene expression into the nucleus. Example 7.2 of International Publication 2016 / 161516 reports the results of these transfection experiments, demonstrating that the first-generation shuttle CM18-TAT-Cys successfully enabled intracellular delivery of fluorescently labeled plasmid DNA encoding GFP. However, GFP expression was detected in only 0.1% of cells (see Table 7.1 in International Publication 2016 / 161516), strongly suggesting that the internally translocated plasmid DNA remained trapped in endosomes without gaining access to the cytoplasmic / nuclear compartment. Example 7.3 in International Publication 2018 / 068135 re-examined the ability of several first and second-generation synthetic peptide shuttles to successfully transfect cells with GFP-encoding plasmids. The results were similar—GFP expression was detected in less than 1% of cells for all shuttles (see Table 7.2 in International Publication 2018 / 068135). These results suggest that synthetic peptide shuttles are not suitable for introducing DNA / RNA into the cytoplasm / nucleus of eukaryotic cells.

[0122] Several strategies have been attempted to deliver DNA / RNA cargo to the cytoplasmic / nuclear compartment, but have been unsuccessful. Interestingly, in experiments attempting to simultaneously introduce both GFP and polynucleotide cargoes, it was observed that the presence of polynucleotides reduced the efficiency of GFP cargo introduction in a concentration-dependent manner. Assuming that the inhibitory effect of polynucleotides is likely due to their negatively charged phosphate backbone, we attempted to neutralize the negative charge by coating the polynucleotides with positively charged small molecules before introduction. The positively charged small molecules considered included 1,3-diaminoguanidine monohydrochloride; 3,5-diamino-1,2,4-triazole; guanidine hydrochloride; and L-arginineamide dihydrochloride at concentrations ranging from 100 nM to 10 mM. However, these strategies continued to suffer from the endosomal capture problem and failed to significantly improve cytoplasmic / nuclear delivery of DNA / RNA cargo, potentially suggesting that something more than simple charge neutralization was needed for shuttle agent-mediated introduction.

[0123] (Example 4) Synthetic peptide shuttle agents enable intracellular delivery of FITC-labeled charged-neutral polynucleotide analogs. Phosphorodiamidate morpholino oligomers (PMOs) are short, single-stranded polynucleotide analogs useful as antisense oligonucleotides for modifying gene expression via steric hindrance. Their molecular structure contains DNA / RNA nucleic acid bases linked to a methylenemorpholin ring backbone via phosphorodiamidate groups. Due to their charge-neutral structure, many intracellular delivery systems developed for DNA / RNA (e.g., electrostatic coupling using cationic lipids; cationic membrane-permeable peptides) are unsuitable for intracellular delivery of PMOs. Thus, modified forms of PMOs have been developed for intracellular delivery, including covalent bonding of PMOs to eight guanidium heads (Vivo-morpholinos) or membrane-permeable peptides (PPMOs). However, apart from direct injection strategies, there are few reports of successful cytoplasmic delivery of unmodified PMOs capable of modifying gene expression.

[0124] To investigate whether synthetic peptide shuttles can deliver PMO into cells, HeLa cells were exposed for 5 minutes in plain RPMI medium containing 6 μM 25-mer PMO molecules covalently labeled with fluorophores ("PMO-FITC"; SEQ ID NO: 348) in the presence of representative members of first and second-generation synthetic peptide shuttles at 7.5, 10, or 20 μM concentrations. The results are shown in Figure 1, and mean cell viability ("Mean viability") and "Mean % of PMO+ cells" (number of viable cells positive for PMO-FITC cargo) were evaluated by flow cytometry as described in International Publication 2018 / 068135. The "Mean delivery score" provides a further indicator of the total amount of cargo (PMO-FITC) delivered per cell among all cargo-positive cells and was calculated by multiplying the mean fluorescence intensity measured for viable PMO-FITC+ cells (at least for duplicate samples) by the mean percentage of viable PMO-FITC+ cells and dividing by 100,000. Finally, a "delivery-survival score" was calculated for each peptide by multiplying the average survival rate by the average delivery score and then by 10, allowing for the ranking of shuttle agents in terms of both their induction activity and toxicity. The rows in Figure 1 are sorted from lowest to highest in terms of their delivery-survival scores.

[0125] Negative controls included "Cargo Alone" (cells incubated with PMO-FITC in the absence of the peptide) and "FSD10 Scramble" (a control peptide with the same amino acid composition as the shuttle agent FSD10, except that its primary amino acid sequence was "shuffled" to disrupt the cationic amphipathic structure common to all shuttle agents). The results in Figure 1 show that second-generation shuttle agents designed for protein cargo delivery are generally superior to first-generation shuttle agents in terms of both delivery efficiency (average %) of PMO+ cells and the average amount of cargo delivered to each cell (delivery score). Assuming that the effects of PMO and other antisense oligonucleotide analogs are dependent on intracellular concentration, the latter are particularly advantageous for the purpose of modifying gene expression. The first-generation shuttle agents CM18-Penetratin-cys, CM18-TAT, and His-CM18-PTD4 included in the experiment showed higher toxicity (i.e., average survival rate of less than 50%) at the lowest concentration tested (7.5 μM) and were therefore excluded from Figure 1. This result was unexpected, given that such first-generation shuttle agents have been used at high concentrations to deliver GFP-NLS to the same HeLa cells without significant adverse effects on viability. Since numerous other shuttle agents tested showed much higher average % and average PMO delivery scores in PMO+ cells without the same level of toxicity, the increased toxicity is unlikely to be solely due to the PMO-FITC cargo. These results suggest that the toxicity may be due to the interaction between each shuttle agent and the PMO-FITC cargo, and that shuttle agent / cargo "matching" may be considered for toxicity purposes as well as induction activity.

[0126] In summary, the results in Figure 1 show that the second-generation shuttle generally performs better than the first-generation shuttle in terms of delivery efficiency, cargo volume delivered per cell, and toxicity, demonstrating that synthetic peptide shuttle agents can deliver PMO-FITC cargo into cells.

[0127] (Example 5) The synthetic peptide shuttle FSD10 introduces antisense PMO into the cytoplasm, enabling knockdown of GFP gene expression in HeLa cells. Endosome capture was found to be problematic for shuttle agent-mediated delivery of naked DNA / RNA cargo (Example 3). Flow cytometry results shown in Example 4 and Figure 1 examine cells positive for PMO-FITC cargo, regardless of whether the cargo was captured in the cytoplasm / nucleus or endosomes. Furthermore, recent reports from other groups have shown that hydrophobic fluorophores (e.g., tetramethylrhodamine) may interact with the lipid bilayer and promote membrane destabilization (Brock et al., 2018). Therefore, experiments were conducted to confirm that the shuttle agent could deliver unlabeled PMO cargo to the cytoplasmic / nuclear compartment in a biologically active form that knocks down the expression of the target gene.

[0128] A "HeLa plex TetO GFPd" cell line was created, consisting of HeLa cells stably expressing a GFP variant ("GFPd") engineered to have a short half-life of approximately 2 hours. HeLa plex TetO GFPd cells were exposed for 5 minutes in plain RPMI medium containing, with or without a synthetic peptide shuttle, either an antisense PMO molecule designed to knock down GFPd expression ("PMO-GFP"; SEQ ID NO: 345) or an off-target antisense PMO molecule targeting GLI1 expression ("PMO-GLI1"; SEQ ID NO: 346). As a further control, siRNA ("siRNA-GFP"; SEQ ID NO: 349) and non-specific ("siRNA-Gli1"; SEQ ID NO: 350) PMO molecules with the same sequence as the antisense were delivered via a commercially available cationic lipid-based delivery system (Lipofectamine® RNAiMax). Following introduction / transfection, cells were washed, cultured in growth medium, and then analyzed by flow cytometry to assess the effect on GFPd expression at appropriate times (5 hours for PMO treatment or 48 hours for siRNA treatment) to observe the knockdown effect.

[0129] As shown in Figure 2, untreated HeLa plex TetO GFPd cells had a baseline mean of 65% GFP+ cells, and exposure of cells to PMO cargo alone (without the shuttle agent) did not alter this. Interestingly, exposure of cells to 10 μM PMO-GFP cargo in the presence of 7.5 μM shuttle agent FSD10 resulted in a 34% reduction in the percentage of GFP+ cells. The effect was specific to the PMO-GFP cargo, as no effect on GFPd expression was observed using a negative control PMO nonspecific cargo. Ultimately, the effect was dose-dependent, as knockdown in GFPd expression was lost by reducing the PMO-GFP cargo concentration to 0.1 μM.

[0130] (Example 6) The synthetic peptide shuttle FSD250 introduces antisense PMO into the cytoplasm, enabling knockdown of Gli1 expression in DU145 cells. Human DU145 cells were exposed for 5 minutes in plain RPMI medium containing 6 μM of either an antisense PMO molecule designed to knock down Gli1 protein expression ("PMO-Gli1"; SEQ ID NO: 346) or an antisense PMO molecule designed to knock down Wnt1 protein expression ("PMO-Wnt1"; SEQ ID NO: 347), in the presence of 5 μM synthetic peptide shuttle FSD250. Tracer PMO-FITC molecule (6 μM) was also included in both conditions to allow fluorescence-excited cell sorting (FACS) of introduced cells from cells not introduced within the same cell population. 48 hours after introduction, cells were analyzed by flow cytometry and then separated into FITC-positive and FITC-negative populations by FACS.

[0131] For cells treated with PMO-Gli / PMO-FITC, the average percentage of %PMO-FITC+ cells was 37%, and the survival rate was 95.8%. For cells treated with PMO-Wnt1 / PMO-FITC, the average percentage of %PMO-FITC+ cells was 36.8%, and the survival rate was 75.7%. For untreated cells, the average percentage of %PMO-FITC+ cells was 0.6%, and the survival rate was 91.3%.

[0132] Next, FITC+ and FITC- cell populations were lysed, separated by SDS-PAGE, and subjected to Western blotting analysis using an anti-Gli1 polyclonal antibody, an anti-actinin polyclonal antibody as a loading control, and an appropriate enzyme-conjugated second antibody. The results, including densitometry scan values ​​for each band, are shown in Figure 3. Figure 3 clearly shows that Gli1 protein expression was knocked down in transduced cells ("FITC+ cells") compared to non-transduced cells ("FITC- cells") by shuttle agent-mediated intracellular delivery of antisense PMO-Wnt1 and antisense PMO-Gli1. As predicted, the Gli1 knockdown effect was stronger with direct knockdown of Gli1 mRNA via PMO-Gli1 than with indirect knockdown of Gli1 via knockdown of Wnt1 mRNA, which is part of the same signaling pathway.

[0133] (Example 7) The synthetic peptide shuttle FSD250 introduces antisense PMOs into the cytoplasm, enabling knockdown of Gli1 expression in DU145 cells. Human DU145 cells were exposed for 5 minutes in plain RPMI medium containing 6 μM of either PMO-Gli1 (SEQ ID NO: 346) or PMO-GFP (SEQ ID NO: 345) in the presence of 3.75 μM synthetic peptide shuttle FSD250. Tracer PMO-FITC molecules were also included in both conditions (and alone as an additional negative control) to allow FACS separation of transfected cells from untransfected cells. 48 hours after transfection, cells were separated into FITC-positive and FITC-negative populations by FACS.

[0134] For cells introduced with PMO-FITC alone, the average percentage of PMO-FITC+ cells was 44.1%, and the survival rate was 77.7%. For cells introduced with PMO-Gli1 / PMO-FITC, the average percentage of PMO-FITC+ cells was 36.0%, and the survival rate was 61.1%. For cells introduced with PMO-GFP / PMO-FITC, the average percentage of PMO-FITC+ cells was 48.9%, and the survival rate was 77.0%. For untreated cells, the average percentage of PMO-FITC+ cells was 0.1%, and the survival rate was 84.5%.

[0135] Next, FITC+ and FITC- cell populations were lysed, separated by SDS-PAGE, and subjected to Western blot analysis using an anti-Gli1 polyclonal antibody, an anti-actinin polyclonal antibody as a loading control, and an appropriate enzyme-conjugated second antibody. The results, including relative densitometry scan values ​​for each band standardized to the respective actinin loading control band, are shown in Figure 4. Figure 4 clearly shows that Gli1 protein expression was knocked down by shuttle agent-mediated intracellular delivery of antisense PMO-Gli1, but not in cells introduced with tracer PMO-FITC alone or with PMO-GFP.

[0136] (Example 8) Large-scale screening of candidate peptide shuttle agents for propidium iodide (PI) and GFP-NLS delivery activity. A proprietary library of over 300 candidate peptide shuttles was screened in parallel for both propidium iodide (PI; small molecule cargo) and GFP-NLS transduction activity in HeLa cells using flow cytometry, as generally described in Example 1. PI was used as a cargo because it exhibits 20-30-fold enhanced fluorescence and a detectable shift in the maximum excitation / emission spectrum only after binding to genomic DNA—a property particularly suitable for distinguishing endosome-trapped cargo from endosome-released cargo that accesses the cytoplasmic / nuclear compartment. Therefore, since no PI remaining trapped in endosomes reaches the nucleus and exhibits neither enhanced fluorescence nor spectral shift, both intracellular delivery and endosomal escape are measurable by flow cytometry.

[0137] For the numerous peptides screened, negative controls were performed in parallel for each experimental batch, including “untreated” (NT) controls in which cells were not exposed to the shuttle peptide or cargo, and “cargo-only” controls in which cells were exposed to the cargo in the absence of the shuttle agent. The results are shown in Figure 5, where “transmission efficiency” refers to the percentage of overall viable cells that were positive for the cargo (PI or GFP-NLS). “Mean delivery score” provides a further indicator of the total amount of cargo delivered per cell among all cargo-positive cells. The mean PI or GFP-NLS delivery score was calculated by multiplying the mean fluorescence intensity measured for viable PI+ or GFP+ cells (at least for duplicate samples) by the mean percentage of viable PI+ or GFP+ cells, and dividing by 100,000 for GFP delivery, or by 10,000 for PI delivery. Next, the mean delivery scores for PI and GFP-NLS for each candidate shuttle agent were standardized by dividing by the mean delivery score for the “cargo-only” negative controls performed in parallel for each experimental batch. Therefore, the "standardized mean delivery score" in Figure 5 shows that the mean delivery score doubled compared to the negative control "cargo alone".

[0138] The batch-to-batch variability observed in the negative control was relatively small with GFP-NLS, but considerably higher with PI as the cargo. For example, the variability in the introduction efficiency of the "cargo alone" negative control ranged from 0.4% to 1.3% with GFP-NLS and from 0.9% to 6.3% with PI. Furthermore, the introduction efficiency of several negative control peptides (i.e., peptides known to have low or no GFP introduction activity) tested in parallel (e.g., FSD174 scramble; data not shown) sometimes showed that PI (but not GFP-NLS) had introduction efficiency that was sometimes as low as 5% lower than the "cargo alone" negative control, likely due to nonspecific interactions between PI and the peptide. This phenomenon was not observed in the GFP-NLS introduction experiments. The above suggests that the introduction efficiency of the shuttle agent, at least for PI, can be more appropriately compared to that of the negative control peptide rather than under the "cargo alone" condition.

[0139] Among the candidate peptide shuttle agents in Figure 5 that had an average PI delivery efficiency of at least 20%, were peptides with a length of less than 20 residues: FSD390 (17aa), FSD367 (19aa), and FSD366 (18aa). Furthermore, candidate peptide shuttles with an average PI delivery efficiency of at least 20% included peptides containing either non-physiological amino acid analogs (e.g., FSD435, corresponding to FSD395 except for the lysine residue (K) replaced by an L-2,4-diaminobutyric acid residue) or chemical modifications (e.g., FSD438, corresponding to FSD10 except for the N-terminal octanoic acid modification; FSD436, corresponding to FSD222 except for the phenylalanine residue (F) replaced by a (2-naphthyl)-L-alanine residue; and FSD171, corresponding to FSD168 except for having an N-terminal acetyl group and a C-terminal cysteamide group). These results confirm the reliability of the peptide shuttle platform technology to withstand the use of non-physiological amino acids or their analogs instead of physiological amino acids and / or chemical modifications.

[0140] Additional screening assays were performed using further shuttle agents as shown in Figure 6. Among the results were several active shuttle agents that are cleavage forms of long shuttle agents previously shown to have induction activity: FSD10-15 (15aa) and FSD418-12-2 (12aa), FSD418 (15aa), CM18 (18aa), and penetratin (16aa).

[0141] (Example 9) The synthetic peptide shuttle agent efficiently introduces PMO and PNA into HeLa cells. HeLa cells were introduced using 10 μM PMO-FITC or fluorescently labeled peptide nucleic acid (PNA) (PNA TelC-Alexa 488; catalog number F1004; PNA BIO Inc.) as generally described in the introduction protocol described in Example 1, with some modifications. The shuttle peptide used was FSD250 (5 μM), and the cells were contacted with the cargo and shuttle for 2 minutes. After 1 hour of incubation, the cells were analyzed by flow cytometry. Furthermore, since the presence of DMF in the culture medium resulted in a cell viability of less than 50%, PNA was resolubilized in water instead of dimethylformamide (DMF), as recommended by the manufacturer. The cargo delivery results in Figure 7A demonstrate that FSD250 significantly increased intracellular delivery of both PMO ("FSD250+PMO" delivery) and PNA ("FSD250+PNA" delivery) cargoes compared to the absence of a shuttle agent ("PMO alone" and "PNA alone"; "NT" = untreated cells). Cell viability remained high under all tested conditions, as shown in Figure 7B. Parallel experiments using FSD250 in attempts to introduce fluorescently labeled siRNA as cargo resulted in only 5% intracellular delivery (data not shown). These results suggest that, in addition to PMO, synthetic peptide shuttle agents can also rapidly and efficiently introduce other non-anionic polynucleotide analogs such as PNA.

[0142] (Example 10) The effect of naked DNA / RNA on shuttle agent-mediated delivery of PMO cargo. Naked DNA / RNA cargoes have been shown to be inadequate cargoes for synthetic peptide shuttle agents (Examples 3 and 9), but this example evaluates their potential dominant-negative effect on the trans-transfer of PMO cargoes to shuttle agent-mediated delivery. Briefly, RH-30 cells (150,000 cells / well in 24-well dishes) were brought into contact with a delivery mixture of 6 μM PMO-FITC and 5 μM synthetic peptide shuttle agent FSD250 for 2 minutes in RPMI in the presence of increased amounts of DNA oligonucleotides or sgRNA added to the medium. The cells were then washed, incubated in growth medium, and then harvested for analysis by flow cytometry after 1 hour. The results in Figure 8 show that a reduction in PMO-FITC delivery efficiency was observed in the presence of 1.5 μg of DNA oligo (3 μg / mL) (Figure 8A) and 2 μg of sgRNA (4 μg / mL) (Figure 8B). Cell viability remained above 75% under all conditions tested.

[0143] (Example 11) Comparison of PMO cargo introduction using first and second-generation synthetic peptide shuttle agents. Generally, second-generation synthetic peptide shuttles exhibit higher cargo delivery efficiency than first-generation shuttles. This example compares the PMO delivery activity of a prototype CPD containing a first-generation shuttle with that of two reasonably designed second-generation synthetic peptide shuttles. Briefly, RH-30 cells (20,000 cells / well in 96-well dishes) were contacted for 2 minutes in RPMI with a delivery mixture containing 6 μM PMO-FITC and increasing concentrations of two second-generation synthetic peptide shuttles (FSD250 and FSD10) lacking the first-generation shuttle His-CM18-PTD4 or CPD. The cells were washed, incubated in complete medium, and then harvested after 1 hour for analysis by flow cytometry. PMO-FITC delivery efficiency is shown in Figure 9A, and cell viability is shown in Figure 9B. The results in Figure 9A show that the FSD250 and FSD10 provided higher deployment efficiency for PMO-FITC cargo than the His-CM18-PTD4.

[0144] (Example 12) Comparison of synthetic peptide shuttle-mediated PMO delivery with self-transferring VivoPMO. Gli1 knockdown experiments were performed as generally described in Example 6 to compare shuttle agent-mediated delivery of commercially available self-transporting Vivo-Morpholino (VivoPMO), which is a PMO chemically modified with terminal octaganidine dendrimers to facilitate cell entry, with unmodified PMO. Briefly, RH-30 cells were contacted for 2 minutes with a delivery mixture of 6 μM cargo (either PMO-Gli1 or VivoPMO-Gli1) in the presence or absence of 5 μM synthetic peptide shuttle agent FSD250 in RPMI. The cells were then washed and incubated in complete medium and subsequently harvested after 24 hours for Gli1 protein expression analysis by Western blotting using anti-Gli1 polyclonal Ab (Abcam ab273018, 150 kDa), anti-GAPDH Ab (Abcam ab181602, 37 kDa), and anti-rabbit HRP secondary Ab. Western blot results are shown in Figure 10A, and the corresponding densitometry scanning analysis is shown in Figure 10B. Gli1 protein knockdown was observed only in cells treated with both FSD250 and PMO-Gli1 cargo. The absence of Gli1 knockdown observed in cells exposed to VivoPMO-Gli1 alone suggests that the 2-minute incubation time in Figure 10 was insufficient for the autotransport of VivoPMO. Indeed, this is consistent with the 2-4 hour incubation time recommended by the manufacturer for VivoPMO to achieve sufficient autotransport via endocytosis. Nevertheless, the results in Figure 10 highlight a significant difference in internalization kinetics between the slow endocytosis-dependent intracellular delivery of VivoPMO versus the rapid and efficient PMO delivery observed using synthetic peptide shuttles.

[0145] (Example 13) Comparison of PMO delivery via synthetic peptide shuttle and intracellular PMO delivery via Endoporter. PMO cargo delivery experiments in HeLa cells were conducted to directly compare synthetic peptide shuttle-mediated PMO delivery with Endoporter-mediated intracellular PMO delivery. For shuttle-mediated delivery, HeLa cells were exposed to 10 μM PMO-FITC in RPMI in the presence of 2.5, 5, 7.5, or 10 μM second-generation shuttle FSD396 for 5 minutes. For Endoporter-mediated delivery, HeLa cells were exposed to 10 μM PMO-FITC in growth medium in the presence of 2.5, 5, 7.5, or 10 μM commercially available Endoporter® peptide (GeneTools, LLC) for at least 24 hours, the shortest incubation time recommended by the manufacturer. After washing, delivery results were compared by observing intracellular PMO-FITC fluorescence via immunofluorescence microscopy, and the results are shown in Figure 11. HeLa cells exposed to PMO-FITC cargo alone (Figure 11B) showed only minimal intracellular delivery after 48 hours compared to untreated cells (Figure 11A). FSD396 induced robust intracellular delivery of PMO-FITC after a 5-minute incubation (Figure 11C), whereas comparable levels of intracellular PMO-FITC were only achieved with the Endoporter peptide after a 48-hour incubation (Figure 11D). Furthermore, Endoporter-treated cells exhibited undesirable morphological changes not observed in untreated cells (Figure 11A), cells treated with cargo alone (Figure 11B), or cells treated with the shuttle agent (Figure 11C). Large PMO-FITC aggregates were also observed in Endoporter-treated cells and could not be removed from the cells by washing.

[0146] (Example 14) Gli1 knockdown induces increased apoptosis in BCC cell lines but not in normal human skin cell lines. Gorin syndrome, also known as basal cell carcinoma nevus or basal cell carcinoma nevus syndrome (BCCNS), is a genetic disorder associated with abnormal Hedgehog (Hh) pathway signaling, resulting in a high frequency of basal cell carcinoma (BCC) growths on the face, hands, back, and neck. Patients with Gorin syndrome may develop up to 30 lesions per year originating from the basal cell layer of the skin, located between the epidermis and dermis. Gorin patients have a gene mutation that causes constitutive activation of the Hh pathway. Gli1 is a transcription factor involved in the expression of Hh pathway determinants and can therefore be considered a major regulator of Hh signaling.

[0147] The effects of Gli1 knockdown on two human skin cell lines were evaluated: a normal human skin-derived cutaneous epithelial-like cell line (NCTC-2544) and a human basal cell carcinoma cell line (UW-BCC1). Normal-derived NCTC-2544 cells and BCC-derived UW-BCC1 cells were exposed to autotransported VivoPMO-Gli1 (15 μM) in complete cell culture medium for 24 or 48 hours. Approximately 60% knockdown of Gli1 protein expression was observed by Western blotting after 48 hours. In parallel, the percentage of cell apoptosis was measured by flow cytometry using fluorescently labeled annexin-V. Interestingly, treatment with VivoPMO-Gli1 resulted in 68–72% apoptotic UW-BCC1 cells after 48 hours, compared to only 11% apoptotic cells treated with the negative control VivoPMO. In contrast, treatment with VivoPMO-Gli1 resulted in apoptosis in only 3–6% of NCTC-2544 cells and only 2% of cells treated with negative control VivoPMO after 48 hours. These results support Gli1 knockdown via intracellular delivery of Gli1-specific PMO for the treatment of basal cell carcinoma.

[0148] (Example 15) Design, synthesis, and shuttle-mediated introduction of PMO for Gli1 knockdown. Four different PMOs were designed and synthesized, targeting different regions of the human Gli1 gene either in the 5' untranslated region or proximal to the start codon. In ascending order of their distance from the Gli1 start codon, the four synthesized PMOs were: PMO-Gli1_Opt (binding to SEQ ID NO: 365 and straddling the Gli1 start codon); PMO-Gli1_Opt1 (binding to SEQ ID NO: 366); PMO-Gli1_Opt2 (binding to SEQ ID NO: 367); and PMO-Gli1_Opt3 (binding to SEQ ID NO: 368). RH-30 cells were introduced with the shuttle agent FSD250 using each of the four PMO cargoes (6 μM) or with the negative control PMO-FITC (6 μM) as described in Example 12. The overall transduction efficiency in the transduction experiments was approximately 80%, as estimated by flow cytometry of cells transduced with the PMO-FITC control cargo. Cells were harvested 24 hours after delivery, and Gli1 protein expression knockdown was evaluated by Western blotting (Figure 12). All four PMOs knocked down Gli1 protein expression when introduced with a shuttle agent, but minimal knockdown was observed in the absence of the shuttle agent. Densitometry analysis of Gli1 and control actinin bands revealed that all four PMOs knocked down Gli1 expression to 27–45% of that of untreated cells (Figure 12). The introduction experiments were repeated with increasing concentrations of PMO-Gli1_Opt, and it was found that Gli1 knockdown was dose-dependent: 0.325, 0.75, 1.5, 3, and 6 μM PMO-Gli1_Opt knocked down Gli1 levels to 48%, 43%, 34%, 33%, and 22% of those of untreated cells, respectively.

[0149] (Example 16) Shuttle-mediated introduction of PMO for Gli1 knockdown in basal cells of patient-derived tumor explants. Basal cell carcinoma tumors newly obtained after Mohs-type surgery were incubated in complete DMEM culture medium with the surface exposed to air on a wire mesh. The tumors were washed with PBS 1X to ensure cargo delivery to the epidermis and dermis, and the stratum corneum of the explants was transmissively treated with a Pantec PLEASE® laser according to the following parameters: pore density 2.5%, 1 pulse / pore, array size 14 mm, pore depth 20 μm (4.9 J / cm²). 2 (0.8W). Next, the explants were divided in half, and one half was treated with a solution of PBS 1x-2% hydroxyethylcellulose containing 25 μM PMO-Gli1-Cy5 and 40 μM FSD250, while the other half was treated with the same solution containing only PMO-Gli1-Cy5 (without the shuttle agent; control). After treatment, the tumors were incubated at 37°C for 4 hours, fixed with 4% paraformaldehyde (PFA), then treated with 30% sucrose, and frozen at OCT (optimal cutting temperature). 10 μm sections were transferred to coverslips and treated with ProLong® Diamond for fluorescence microscopy analysis. As shown in Figure 13, an increase in fluorescence levels was observed in the half of the tumor treated with the combination of PMO-Gli1-Cy5 and the shuttle agent, particularly at the epidermal level, compared to the other half of the tumor treated with PMO-Gli1-Cy5 alone. Furthermore, after tumor incubation, dermal and epidermal cells were isolated by enzymatic treatment with thermolysin and trypsin and further cultured at 37°C in 5% CO2. Gli1 protein levels were measured by cell fluorescence at various time points in culture after labeling of fixed and isolated skin and epithelial cells with anti-Gli1-Alexa 647 antibody. During the 24 to 48 hours of incubation after tumor treatment, Gli1 protein levels were significantly reduced (50%) in cultured cells treated with a combination of PMO-Gli1 and a shuttle agent, as measured by flow cytometry (data not shown).

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Claims

1. A composition comprising a non-anionic polynucleotide analog cargo for intracellular delivery, and a synthetic peptide shuttle agent that is independent of or not covalently bonded to the non-anionic polynucleotide analog cargo, wherein the non-anionic polynucleotide analog cargo is a charged-neutral polynucleotide cargo having a phosphorodiamidate skeleton, an amide skeleton, a methylphosphonate skeleton, a neutral phosphotryester skeleton, a sulfone skeleton, or a triazole skeleton, and the synthetic peptide shuttle agent is a peptide comprising an amphiphilic alpha-helix motif having both a positively charged hydrophilic outer surface and a hydrophobic outer surface, wherein the synthetic peptide shuttle agent increases the cytoplasmic / nuclear delivery of the non-anionic polynucleotide analog cargo in eukaryotic cells compared to the absence of the synthetic peptide shuttle agent. (A) The shuttle agent is (1) A peptide having a minimum length of 15 amino acids and a maximum length of 150 amino acids, (2) An amphiphilic alpha helix motif, (3) A positively charged hydrophilic outer surface having an alpha helix with a rotation angle of 100 degrees between consecutive amino acids and / or an alpha helix having 3.6 residues per turn, and having (i) at least two adjacent positively charged K and / or R residues on a helical wheel projection; and (ii) a positively charged hydrophilic outer surface having a segment of six adjacent residues containing three to five K and / or R residues on a helical wheel projection. (4) A hydrophobic outer surface comprising a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12–50% of the amino acids of the peptide, based on an open cylinder representation of an alpha helix having 3.6 residues per turn, and comprising (a) at least two adjacent L residues on a helical wheel projection; and / or (b) a segment of 10 adjacent residues containing at least five hydrophobic residues selected from L, I, F, V, W, and M on a helical wheel projection. It is an amphiphilic alpha helix motif having, The following parameters (5) to (15): (5) The peptide has a hydrophobic moment (μ) of 3.5 to 11; (6) The peptide has a predicted effective charge of at least +3 or +4 at physiological pH; (7) The peptide has an isoelectric point (pI) of 8 to 13; (8) The peptide is composed of 35% to 65% of any combination of amino acids: A, C, G, I, L, M, F, P, W, Y, and V; (9) The peptide is composed of any combination of amino acids: N, Q, S, and T, from 0% to 30%; (10) The peptide is composed of 35% to 85% of any combination of amino acids: A, L, K, or R; (11) The peptide is composed of any combination of amino acids A and L, between 15% and 45%, provided that at least 5% of L is present in the peptide; (12) The peptide is composed of any combination of amino acids K and R, between 20% and 45%; (13) The peptide is composed of any combination of amino acids D and E, from 0% to 10%; (14) The difference between the percentage of A and L residues in the peptide (A+L%) and the percentage of K and R residues in the peptide (K+R%) is 10% or less; and (15) The peptide is composed of 10% to 45% of any combination of amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H. At least four of the following conditions are met, or (B) The shuttle agent comprises an endosomal leakage domain (ELD) operably linked to a cell membrane permeability domain (CPD), composition.

2. The composition according to claim 1, wherein the non-anionic polynucleotide analog cargo is a phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid (PNA), methylphosphonate oligomer, or short-chain interfering ribonucleic acid neutral oligonucleotide (siRNN).

3. The composition according to claim 1 or 2, wherein the non-anionic polynucleotide analog cargo is 5 to 50 m, 5 to 75 m, or 5 to 100 m.

4. The composition according to any one of claims 1 to 3, wherein the non-anionic polynucleotide analog cargo is not covalently bound to a cell membrane-permeable peptide, octaganidine dendrimer, or other intracellular delivery portion.

5. (a) The shuttle agent satisfies at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all of parameters (5) to (15); (b) The shuttle agent is a peptide having a minimum length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, or 140 amino acids; (c) The amphiphilic alpha helix motif is 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, It has a hydrophobic moment (μ) between a lower limit of 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; (d) The amphiphilic alpha-helix motif includes a positively charged hydrophilic outer surface which, on the helical wheel projection, includes at least three or four adjacent positively charged K and / or R residues; (e) The amphiphilic alpha-helix motif includes a hydrophobic outer surface, which, based on an alpha-helix having a rotation angle of 100 degrees between consecutive amino acids and / or an alpha-helix having 3.6 residues per turn, includes (i) at least two adjacent L residues on a helical wheel projection; and (ii) a segment of 10 adjacent residues on a helical wheel projection, which includes at least five hydrophobic residues selected from L, I, F, V, W, and M; (f) The hydrophobic outer surface includes a highly hydrophobic core composed of spatially adjacent L, I, F, V, W, and / or M amino acids corresponding to 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20%, 25%, 30%, 35%, 40%, or 45% of the amino acids of the shuttle agent; (g) The shuttle agent has a hydrophobic moment (μ) between a lower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5; (h) The shuttle agent has a predicted effective charge of +3, +4, +5, +6, +7, +8, +9 to +10, +11, +12, +13, +14, or +15; (i) The shuttle agent has a predictive pI of 10 to 13; or A composition according to any one of claims 1 to 4, which is any combination of (j)(a) to (i).

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

7. The aforementioned shuttle agent contains a histidine-rich domain, and the histidine-rich domain is (i) The shuttle agent is positioned toward the N-terminus and / or C-terminus; (ii) A stretch of at least 3, at least 4, at least 5, or 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 composition according to any one of claims 1 to 6, which is both (i) and (ii).

8. The composition according to any one of claims 1 to 7, wherein the shuttle agent comprises a flexible linker domain rich in serine and / or glycine residues.

9. The shuttle agent, (i) Any one amino acid sequence of SEQ ID NOs: 35, 2, 4-6, 12, 13, 15-17, 23, 29, 32, 127, 136, 156, 212, 246, 277, or 305; or (ii) Amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 35, 2, 4-6, 12, 13, 15-17, 23, 29, 32, 127, 136, 156, 212, 246, 277, or 305. A composition according to any one of claims 1 to 8, comprising or consisting of the following.

10. The shuttle agent, (a) at least one synthetic amino acid; (b) Cyclic peptide; (c) One or more D-amino acids; (d) Chemical modification of one or more amino acids that does not impair the introduction activity of the synthetic peptide shuttle agent; (e) Any combination of (a) to (d) including, The composition according to any one of claims 1 to 8.

11. The composition according to any one of claims 1 to 10, wherein the shuttle agent satisfies at least four of the parameters (1) to (4) and (5) to (15) of (A), and does not contain a cell membrane permeable domain (CPD), a cell membrane permeable peptide (CPP), or a protein delivery domain (PTD).

12. The shuttle agent is as specified in (B), (i) The ELDs include: endosomal soluble peptides; antimicrobial peptides (AMP); linear cationic alpha-helix antimicrobial peptides; secropine-A / melittin hybrid (CM) peptides; pH-dependent membrane-active peptides (PAMP); amphiphilic peptides; peptides derived from the N-terminus of the HA2 subunit of influenza hemagglutinin (HA); CM18; diphtheria toxin T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1; VSVG; pseudomonas toxin; melittin; KALA; JST-1; C(LLKK) 3 C;G(LLKK) 3 G or any combination thereof, or derived from them; (ii) The CPD is a cell membrane permeable peptide or a protein transduction domain derived from a cell membrane permeable peptide; TAT; PTD4; Penetratin; pVEC; M918; Pep-1; Pep-2; Xcentri; Arginine stretch; Transportan; SynB1; SynB3; or any combination thereof or derived therefrom; or (iii) Both (i) and (ii), The composition according to any one of claims 1 to 10.

13. The composition according to any one of claims 1 to 12, wherein the concentration of the non-anionic polynucleotide analog cargo and / or synthetic peptide shuttle agent in the composition is at least 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 μM.

14. (i) For use in regulating gene expression in eukaryotic cells; (ii) For therapeutic use, wherein the non-anionic polynucleotide analog cargo modulates the gene expression of a therapeutically relevant target RNA in eukaryotic cells; (iii) For use as an antiviral agent, in which the non-anionic polynucleotide analog cargo modulates the gene expression of viral genes necessary for virulence and / or pathogenicity; (iv) For use in delivering non-therapeutic non-anionic polynucleotide analog cargoes as diagnostic agents; (v) For use in the manufacture of pharmaceuticals or diagnostic agents; (vi) for use in the treatment of cancer, inflammation or inflammation-related diseases, pain, or diseases affecting the lungs; or (vii) For any combination of (i) to (vi), The composition according to any one of claims 1 to 13.

15. The composition according to any one of claims 1 to 13, or the composition for use according to claim 14, wherein the eukaryotic cells are animal cells, mammalian cells, human cells, stem cells, primary cells, immune cells, T cells, NK cells, dendritic cells, epithelial cells, skin cells, gastrointestinal cells, lung cells, or eye cells.

16. An in vitro method for modifying gene expression in eukaryotic cells, (a) A step of providing a non-anionic polynucleotide analog cargo for intracellular delivery as defined in claim 1, wherein the non-anionic polynucleotide analog cargo is designed to hybridize to the RNA of interest in a eukaryotic cell; (b) A step of providing a synthetic peptide shuttle agent that is independent of or not covalently bonded to the non-anionic polynucleotide analog cargo as defined in claim 1; and (c) A step of contacting eukaryotic cells with non-anionic polynucleotide analog cargoes in the presence of a synthetic peptide shuttle at a concentration sufficient to increase the delivery efficiency and / or cytoplasmic / nuclear delivery of the non-anionic polynucleotide analog cargoes compared to the absence of the synthetic peptide shuttle. A method comprising a non-anionic polynucleotide analog cargo that hybridizes to the target RNA upon cytoplasmic / nuclear delivery, thereby influencing the modification of gene expression.

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