Cell-penetrating peptide variants and uses thereof

Abstract

A cell-penetrating peptide is provided. [Solution] A cell-penetrating peptide comprising a cell-penetrating domain and a zinc finger domain is provided, wherein the cell-penetrating domain has a specific amino acid sequence, and the zinc finger domain has an amino acid sequence represented by the following general formula 1: JPEG2026041880000006.jpg13150 (where X1 is C or A, X2 is Y or R, X3 is N, R or D, X4 is C or A, X5 is G or E, X6 is H or A, and X7 is C or A.) The cell-penetrating peptide of the present invention has the effect of improving cell penetration, cell proliferation, cell migration, and angiogenesis, and can be used for cell transmission, wound healing, and angiogenesis promotion. In particular, since it has excellent therapeutic effects on intractable diabetic wounds (ulcers), it can be usefully used for wound healing.

Description

[Technical Field]

[0001] The present invention relates to the use of cell-permeable peptide variants in wound healing. [Background technology]

[0002] The cells of higher animals have a plasma membrane that separates the inside and outside of the cell, thoroughly separating the intercellular transport of substances. The plasma membrane is composed of a phospholipid bilayer, and the hydrophobic nature of this lipid bilayer makes it almost impossible for most peptides, proteins, nucleotides, liposomes, etc. to move into the cell. Therefore, the plasma membrane acts as an obstacle to the intracellular transport of peptides, proteins, compound drugs, and gene therapy agents. To overcome this obstacle, methods such as the use of cationic lipids or polyethyleneimine (PEI), viral vectors, or electroporation are often used. However, these methods have limitations, such as low efficiency, limited cell types, and intracellular toxicity (Lindsay MA (2002) Peptide-mediated cell delivery: application in protein target validation. Curr Opin Pharmacol 2:587-594; Green I, Christison R, Voyce CJ, Bundell KR, Lindsay MA (2003) Protein transduction domains: are they delivering Trends Pharmacol Sci 24:213-215). Furthermore, as the functions of key proteins that play important roles in the development and progression of disease have become clearer, small molecule compounds have been used as the primary drugs to regulate disease-related proteins within cells. However, the large surface area of ​​protein-protein interaction sites has limited the ability of small molecule drugs to bind and regulate target protein activity. To overcome this, efforts are being made to develop peptide- and protein-based drugs that are suitable for modulating protein-protein interactions.For the successful development of protein drugs aimed at regulating the activity of target proteins within cells, intracellular delivery and transport of proteins is essential, but it has generally been known that intracellular transport of proteins is difficult.

[0003] To overcome these shortcomings and limitations, attempts have been made to use protein transduction domains (PTDs), i.e., cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs). CPPs are parts of translocatory proteins, and representative examples include the membrane translocating sequence (MTS), a hydrophobic region in the signal sequence of human fibroblast growth factor 4, and the basic amino acid domain (Tat-PTD) of the Tat protein, one of the HIV viral proteins (Mann DA, Frankel AD (1991) Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J 10:1733-1739; Rojas M, Donahue JP, Tan Z, Lin YZ (1998) Genetic engineering of proteins with cell membrane permeability. Nat Biotechnol 16:370-375).

[0004] However, while MTS is recognized as a good candidate due to its excellent intracellular entry efficiency, it has the disadvantage that inclusion bodies may be generated during the expression process for production because most of its amino acid sequence is a hydrophobic domain, and more complicated steps are required for expression and purification. Even purified proteins can lose their activity due to aggregation during storage due to hydrophobic interactions (Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, Ruley HE (2001) Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase. Nat Biotechnol 19:929-933; DiGiandomenico A, Wylezinski LS, Hawiger J (2009) Intracellular delivery of a cell-penetrating SOCS1 that targets IFN-gamma signaling. Sci Signal 2:ra37). In contrast, basic domains such as Tat-PTD, which are primarily composed of basic amino acids, are hydrophilic and therefore less likely to form insoluble bodies during expression. They can be purified relatively easily from the cytoplasm, and their activity can be maintained for a relatively long period of time even during storage. Numerous other PTDs have also been reported (Lindgren M, Langel U (2011) Classes and prediction of cell-penetrating peptides. Methods Mol Biol 683:3-19).

[0005] Research to identify and sequence multiple cell-penetrating peptides is actively underway worldwide, and both the Tat-PTD series and the MTS series are being used as cell-penetrating drugs for direct therapeutic purposes. However, their effectiveness is still minimal compared to other therapeutic methods such as gene therapy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] (Lindsay MA(2002)Peptide-mediated cell delivery:application in protein target validation.Curr Opin Pharmacol 2:587-594, Green I, Christison R, Voyce CJ, Bundell KR, Lindsay MA(2003)Protein transduction domains:are they delivering Trends Pharmacol Sci 24:213-215) [Non-patent document 2] (Mann DA, Frankel AD (1991) Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J 10:1733-1739, Rojas M, Donahue JP, Tan Z, Lin YZ (1998) Genetic engineering of proteins with cell membrane permeability. Nat Biotechnol 16:370-375) [Non-patent document 3] (Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, Ruley HE(2001)Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase.Nat Biotechnol 19:929-933, DiGiandomenico A, Wylezinski LS, Hawiger J(2009)Intracellular delivery of a cell-penetrating SOCS1 that targets IFN-gamma signaling.Sci Signal 2:ra37) [Non-patent document 4] (Lindgren M, Langel U(2011)Classes and prediction of cell-penetrating peptides.Methods Mol Biol 683:3-19) Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a cell-penetrating peptide.

[0008] It is also an object of the present invention to provide a composition for promoting cell proliferation and migration.

[0009] It is also an object of the present invention to provide a cell-transmitting vehicle.

[0010] Another object of the present invention is to provide a composition for tissue regeneration.

[0011] Another object of the present invention is to provide a composition for promoting angiogenesis.

[0012] It is also an object of the present invention to provide a pharmaceutical composition for wound healing.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating angiogenesis-dependent diseases.

[0014] It is also an object of the present invention to provide a method for wound healing.

[0015] It is also an object of the present invention to provide a method for treating angiogenesis-dependent diseases.

[0016] Another object of the present invention is to provide a use for producing a composition for wound healing.

[0017] Another object of the present invention is to provide a use for producing a composition for preventing or treating angiogenesis-dependent diseases. [Means for solving the problem]

[0018] To solve the above problems, the present invention provides a cell-penetrating peptide.

[0019] The present invention also provides a composition for promoting cell proliferation and migration, which comprises a cell-penetrating peptide.

[0020] The present invention also provides a cell-penetrating vehicle comprising a cell-penetrating peptide.

[0021] The present invention also provides a composition for tissue regeneration comprising a cell-penetrating peptide.

[0022] The present invention also provides a composition for promoting angiogenesis, which comprises a cell-penetrating peptide.

[0023] The present invention also provides a pharmaceutical composition for wound healing comprising a cell-penetrating peptide.

[0024] The present invention also provides a pharmaceutical composition for preventing or treating angiogenesis-dependent diseases, comprising a cell-penetrating peptide.

[0025] The present invention also provides a method for wound healing, comprising the step of administering a pharmaceutically effective amount of a cell-penetrating peptide to a wound.

[0026] The present invention also provides a method for treating an angiogenesis-dependent disease, comprising the step of administering a pharmaceutically effective amount of a cell-penetrating peptide to an individual having an angiogenesis-dependent disease.

[0027] The present invention also provides a use of the cell-penetrating peptide for the preparation of a composition for wound healing.

[0028] In addition, the present invention provides a use of the cell-penetrating peptide for producing a composition for preventing or treating angiogenesis-dependent diseases. [Effects of the Invention]

[0029] The cell-penetrating peptide of the present invention has the effect of improving cell penetration, cell proliferation, cell migration, and angiogenesis, and can be used for cell transmission, wound healing, and angiogenesis promotion. In particular, since it has excellent therapeutic effects on intractable diabetic wounds (ulcers), it can be usefully used for wound healing. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 shows the structure of the cell-penetrating peptide LPX. [Figure 2] FIG. 1 shows the cell permeability of the LPX101 peptide. [Figure 3] FIG. 1 shows the cell permeability properties of the LPX102 and LPX103 peptides. [Figure 4] FIG. 1 shows the cell growth and migration promoting effect of the LPX101 peptide. [Figure 5] FIG. 1 shows the cell migration promoting effects of LPX101, LPX102 and LPX103 peptides. [Figure 6] FIG. 1 shows the angiogenic effect of the LPX101 peptide. [Figure 7]FIG. 1 shows graphs confirming the improvement of glucose absorption capacity by the LPX101, LPX102, and LPX103 peptides. [Figure 8] FIG. 1 shows the improved glucose metabolism by LPX101, LPX102 and LPX103 peptides. [Figure 9] FIG. 1 is a graph confirming the wound-healing effect of the LPX101 peptide on diabetic model mice. [Figure 10] FIG. 1 shows histological analysis of the diabetic wound healing effect of LPX101 peptide. [Figure 11] This figure confirms the acceleration of remodeling of the regenerated cortex by LPX101 peptide in diabetic wounds. [Figure 12] FIG. 1 shows the wound-healing-enhancing effects of LPX101, LPX102, and LPX103 peptides confirmed in an STZ-induced diabetic mouse model. [Figure 13] FIG. 1 is a graph comparing the wound regenerating activity of LPX101, LPX102, and LPX103 peptides in a diabetic mouse model (db / db). [Figure 14] FIG. 1 shows histological evaluation of wound healing and angiogenesis effects in diabetic mouse models treated with LPX103 or EGF. [Figure 15] FIG. 1 shows histological evaluation of re-epithelialization by LPX101, LPX102 and LPX103 peptides. [Figure 16] FIG. 1 shows the effects of combined treatment with LPX101, LPX102 and LPX103 peptides, and EGF. [Figure 17] FIG. 11 summarizes the mode of action of LPX peptide to improve regenerative effects in diabetic wounds. [Figure 18] FIG. 1 shows the amino acid sequence for the LPX peptide variant. [Figure 19] FIG. 1 shows the amino acid sequence for the LPX peptide variant. [Figure 20] FIG. 1 shows the sequences of LPX peptide mutants of the present invention. [Figure 21]FIG. 1 shows the effects of LPX peptide mutants (CPP domain mutants) on peptide stability, cell migration, and cell growth in in vitro cultured cells. [Figure 22] This figure confirms the effects of LPX peptide mutants (HIS-tagged mutants) for endosomal escape on intracellular peptide stability, cell migration, and cell growth. [Figure 23] This figure confirms the intracellular stability of LPX peptide mutants (mutations in the RNA binding site and zinc finger sequence). [Figure 24] FIG. 1 shows confirmation of stabilization by mutation of residues in the screened LPX103 peptide. [Figure 25] FIG. 10 confirms the effects on cell proliferation and migration of mutants of the LPX103 peptide screened for protein stability. [Figure 26] FIG. 1 is a schematic diagram showing changes in activity due to mutations in the LPX peptide. [Figure 27] FIG. 1 shows LPX105 peptides selected for improved biological activity. [Figure 28] FIG. 1 shows the cell migration promoting effect of mutations in the LPX105 peptide. [Figure 29] This figure confirms the diabetic wound healing effect of mutations in the LPX105 peptide (A: Confirmation of reduction in diabetic wound area, B: Confirmation of reduction in wound area on day 8). DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention will be described in detail below with reference to the following examples, but the following examples are presented as examples of the present invention and are not intended to limit the present invention, and various modifications and applications of the present invention are possible within the scope of the claims below and the scope of equivalents that can be analyzed therefrom.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0033] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. Also, amino acids referred to in the present invention by abbreviations are described according to the IUPAC-IUB nomenclature as follows: Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y and Valine: V.

[0034] In one aspect, the present invention relates to a cell-penetrating peptide comprising a cell-penetrating domain and a zinc finger domain.

[0035] The zinc finger domain can comprise an amino acid sequence of general formula 1:

[0036] [ka]

[0037] In the above formula, X1 is C or A; X2 is Y or R; X3 is N, R or D; X4 is C or A; X5 is G or E; X6 is H or A; and X7 is C or A.

[0038] In one embodiment, the peptide may be a first zinc finger, and more preferably, X1 is C, X3 is N, X4 is C, X6 is H, and X7 is C.

[0039] In one embodiment, when X2 is R and X5 is E, stability may be improved.

[0040] In one embodiment, the peptide may be CRNCGELDHHAKEC, which is a modified version of CYNCGGLDHHAKEC to improve stability.

[0041] In one embodiment, the peptide of the present invention may comprise an amino acid sequence of the following general formula 2:

[0042] [ka]

[0043] In the above formula, X8 is D or R; X2 is Y or R; X5 is G or E; and X9 is L or W.

[0044] In one embodiment, when X8 is R, X5 is E, and X9 is W in Formula 2, intracellular stability may be improved.

[0045] In one embodiment, the peptide of general formula 2 may be a GDRCYNCGGLDHHAKECKL peptide of SEQ ID NO: 3, wherein mutations D2R, C4A, Y5R, N6R, N6D, C7A, G9E, H12A, C17A and L19W may be present, and mutations D2R, Y5R, G9E and L19W may improve stability, while mutations C4A, N6R, N6D, C7A, H12A and C17A may decrease stability.

[0046] In one embodiment, in the peptide of general formula 2 and SEQ ID NO: 2, X8, X5, and X9 may be stability residues; the 4th C, 7th C, 12th H, and 17th C may be zinc finger residues; the 3rd R, X2, and 15th K may be affinity residues; and the 6th N may be an RNA binding residue.

[0047] In one embodiment, the N-terminus (amino terminus) may further comprise the amino acid sequence of SEQ ID NO: 4, more preferably added to the N-terminus of the peptide of general formula 2 and SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 4 may be a CPP domain, which may be changed to SEQ ID NO: 5 or 6.

[0048] In one embodiment, the amino acid sequence of SEQ ID NO: 5 may further be included at the N-terminus, and more preferably added to the N-terminus of the peptide of general formula 2 and SEQ ID NO: 2.

[0049] In one embodiment, the amino acid sequence of SEQ ID NO: 6 may further be included at the N-terminus, and more preferably added to the N-terminus of the peptide of general formula 2 and SEQ ID NO: 2.

[0050] In one embodiment, the N-terminus may further comprise amino acid E or GSE, more preferably added to the N-terminus of the peptide of general formula 2 and SEQ ID NO:2.

[0051] In one embodiment, the peptide may further include a His tag at the N-terminus, and the His tag may be 6xHis repeated six times, and is more preferably added to the N-terminus of the peptide of general formula 2 and SEQ ID NO:2.

[0052] In one embodiment, the C-terminus (carboxy terminus) may further comprise the amino acid sequence of SEQ ID NO: 7, more preferably added to the C-terminus of the peptide of general formula 2 and SEQ ID NO: 2.

[0053] In one embodiment, the C-terminus may further comprise the amino acid sequence of SEQ ID NO: 8, more preferably added to the C-terminus of the peptide of general formula 2 and SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 8 may be a zinc finger (second zinc finger).

[0054] In one embodiment, the C-terminus may further comprise the amino acid sequence of SEQ ID NO: 9, more preferably added to the C-terminus of the peptide of general formula 2 and SEQ ID NO: 2.

[0055] In one embodiment, the amino acid sequence of SEQ ID NO: 10 may further be included at the C-terminus, and more preferably, it is added to the C-terminus of the peptide of general formula 2 and SEQ ID NO: 2.

[0056] In one embodiment, the amino and carboxy termini of the peptide may be modified or protected with multiple organic terminals to protect them from in vivo protease cleavage and increase their stability, for example, the C-terminus may be amidated.

[0057] In one embodiment, the peptide may consist of any one of the amino acid sequences of SEQ ID NOs: 37 to 40 (LPX105 and its variants).

[0058] In one embodiment, the peptide may be a peptide variant selected from the group consisting of LPX101 consisting of the amino acid sequence of SEQ ID NO: 11, LPX102 consisting of the amino acid sequence of SEQ ID NO: 12, LPX103 consisting of the amino acid sequence of SEQ ID NO: 13, and LPX104 consisting of the amino acid sequence of SEQ ID NO: 14.

[0059] In one embodiment, the LPX103 peptide variant may be a peptide variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

[0060] In one embodiment, the LPX104 peptide variant may be a peptide variant consisting of one or more amino acid sequences selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0061] In one embodiment, the cell-penetrating peptide may consist of the amino acid sequence of SEQ ID NO: 37, and may further include the amino acid sequence of SEQ ID NO: 38, the amino acid sequence of SEQ ID NO: 39, and the amino acid sequence of SEQ ID NO: 40.

[0062] The above-mentioned protecting groups protect the peptides of the present invention from attack by in vivo protease enzymes. Furthermore, the modified peptides exhibit excellent thermal stability and stability against physicochemical factors such as acid and alkali due to the protecting groups. Therefore, the peptides of the present invention can be modified to have excellent long-term storage stability, and thus can be advantageously applied to products that require long-term storage, such as pharmaceuticals, quasi-pharmaceuticals, cosmetics, and oral care products.

[0063] The above-mentioned amino acid modifications (or variations or mutations) greatly improve the stability of the peptides of the present invention, and the term "stability" referred to in the present invention refers not only to in vivo stability but also to storage stability (e.g., storage stability at room temperature).

[0064] In the present invention, even if a peptide is described as "a peptide consisting of a specific sequence number," it does not exclude meaningless sequence additions or naturally occurring mutations or silent mutations before or after the amino acid sequence of the sequence number, as long as it has the same or corresponding activity as a peptide consisting of the amino acid sequence of the sequence number, and it is self-evident that even if it has such sequence additions or mutations, it falls within the scope of the present application.

[0065] In the present invention, the alteration of a peptide sequence may involve partial amino acid substitution, addition, deletion, and modification, or a combination of these methods. Such alterations include the use of L- or D-amino acids and / or non-natural amino acids; and / or modifications of the natural sequence, such as modifications of side chain functional groups, intramolecular covalent bonds (e.g., side chain intercyclization), methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. The substituted or added amino acids can include not only the 20 amino acids commonly found in human proteins, but also atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing such amino acids and typical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0066] The term "peptide" as used herein refers to a linear molecule formed by the binding of amino acid residues to each other via peptide bonds. The peptides of the present invention can be produced by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)), or by genetic engineering techniques.

[0067] Specifically, the peptides of the present invention can be produced by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, the peptides of the present invention can be synthesized in a number of ways, including, for example, the following: (a) a method for synthesizing peptides stepwise or by fragment assembly by means of solid-state or liquid-state methods, followed by isolation and purification of the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) performing cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; or A method of obtaining peptide fragments by any combination of (a), (b), and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0068] As a more specific example, a fusion gene encoding a fusion protein containing a fusion partner and a peptide of the present invention can be prepared through genetic engineering, transformed into a host cell, and expressed in the form of a fusion protein. The peptide of the present invention can then be cleaved and separated from the fusion protein using a protease or a compound to produce the desired peptide of the present invention. For example, a DNA sequence encoding amino acid residues that can be cleaved by a protease such as Factor Xa or enterokinase, or a compound such as CNBr or hydroxylamine, can be inserted between the fusion partner and the polynucleotide encoding the peptide of the present invention.

[0069] In one aspect, the present invention relates to a polynucleotide encoding a peptide of the present invention, a vector comprising said polynucleotide sequence, or a host cell transformed with said vector. The term "nucleotide" as used herein refers to a dioxyribonucleotide or ribonucleotide that may exist in single- or double-stranded form, and includes analogs of natural nucleotides unless otherwise specified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0070] The term "vector" as used herein refers to any nucleic acid containing a competent nucleotide sequence that can be inserted into a host cell to recombine with the host cell genome and replicate autonomously either as an insert or as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, and viral vectors.

[0071] The term "host cell," as used herein, refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors have been introduced, and should be understood to refer not only to the particular subject cell but also to its progeny or potential progeny. Because some variations can occur in successive generations due to mutation or environmental influences, the progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0072] In one aspect, the present invention relates to a composition for promoting cell proliferation and migration, comprising the peptide of the present invention.

[0073] In one aspect, the present invention relates to a cell-penetrating vehicle comprising the peptide of the present invention.

[0074] In one embodiment, the cell-penetrating vehicle of the present invention can be used as a drug delivery vehicle. Typically, a cell-penetrating peptide can be linked to a target cargo via a non-covalent or covalent bond. It is particularly preferred that the cell-penetrating peptide and the target cargo form a covalent conjugate through chemical cross-linking (Zatsepin, TS, et al., Curr. Pharm. Des., 11:3639-3654 (2005)). Thus, the peptides of the present invention can be linked to a target cargo via a covalent or non-covalent bond to deliver the target cargo into cells or tissues.

[0075] The term "cargo of interest" as used herein refers to a substance to be delivered into cells by conjugating with the peptide of the present invention through a covalent or non-covalent bond, including, but not limited to, chemical substances, nanoparticles, peptides, polypeptides, antisense oligonucleotides, siRNA, shRNA, miRNA, and PNA (peptide-nucleic acid). Furthermore, when the cargo of interest is a polypeptide, the drug delivery system of the present invention may further comprise a linker between the cell-penetrating delivery system comprising the peptide of the present invention and the cargo of interest. Various linkers known in the art may be used as the linker in the present invention. The linker may have a length and / or sequence specifically selected to maximize the activity of the peptide of the present invention, i.e., cell-penetrating activity. Specifically, the linker is a linker consisting of multiple amino acid residues. Linkers consisting of amino acid sequences are disclosed in Huston, et al., Methods in Enzymology, 203:46-88 (1991) and Whitlow, et al., Protein Eng., 6:989 (1993), which are incorporated herein by reference.

[0076] The drug delivery vehicles of the present invention can be used for a variety of purposes. Specifically, the drug delivery vehicles of the present invention can be used to deliver substances such as chemicals, nucleic acids, and nanoparticles. The nucleic acids include, but are not limited to, antisense oligonucleotides, siRNA, shRNA, miRNA, and PNA. Furthermore, depending on the cargo to be delivered, the drug delivery vehicles of the present invention can be used for treating diseases or disorders, detecting specific cells, diagnosing diseases (e.g., cancer), tracking the location of specific cells, and in vivo imaging. The term "nucleic acid molecule" as used herein comprehensively encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic building blocks of nucleic acid molecules, can include not only natural nucleotides but also analogs with modified sugar or base moieties (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0077] In addition, various labeling substances can be used for the peptides and cargoes of interest that constitute the drug delivery vehicles of the present invention, including, for example, fluorescent substances [e.g., fluorescein, FITC (fluoresein isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), and Coumarin], fluorescent proteins (e.g., GFP, RFP, CFP, YFP, BFP, luciferase, or variants thereof), radioisotopes (e.g., C14, I125, P32, and S35), chemical substances (e.g., biotin), luminescent substances, chemiluminescent substances, and FRET (fluorescence resonance energy transfer). When the drug delivery vehicle of the present invention is configured to contain a radioisotope (e.g., the peptide and nanoparticle configuration of the present invention), it can be applied to single photon emission computed tomography (SPECT) or positron emission tomography (PET) and used for tissue imaging.

[0078] In one aspect, the present invention relates to a composition for tissue regeneration comprising the peptide of the present invention.

[0079] In one aspect, the present invention relates to a composition for promoting angiogenesis, which comprises the peptide of the present invention as an active ingredient.

[0080] In one aspect, the present invention relates to a pharmaceutical composition for wound healing comprising the peptide of the present invention as an active ingredient.

[0081] In one embodiment, the wound may be a non-healing traumatic wound, tissue destruction due to radiation, abrasion, osteonecrosis, laceration, avulsion, penetrating wound, gunshot wound, cut, frostbite, contusion or bruise, skin ulcer, dry skin, skin keratosis, cracking, tearing, dermatitis, pain due to dermatophytosis, surgical wound, vascular disease wound, corneal wound, etc., pressure sore, bed sore, conditions related to diabetes and poor circulation such as diabetic skin erosion, chronic ulcer, suture site after plastic surgery, spinal cord injury wound, gynecological wound, chemical wound, or acne. In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating angiogenesis-dependent diseases, which comprises the peptide of the present invention as an active ingredient.

[0082] In one embodiment, the angiogenesis-dependent disease may be one or more selected from the group consisting of ischemic disease, wounds, burns, psoriasis, chronic ulcers, myocardial infarction, angina pectoris, pressure ulcers, hair loss, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, diabetic ulcers, pulmonary hypertension, and cerebrovascular dementia.

[0083] In one embodiment, the ischemic disease may be one or more selected from the group consisting of cerebral ischemia, cardiac ischemia, diabetic vascular and cardiointestinal disease, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis, ischemic acute renal failure, stroke, cerebral trauma, and neonatal hypoxia.

[0084] In the present invention, the term "prevention" means any action of suppressing or delaying the occurrence, spread, and recurrence of angiogenesis-dependent diseases by administering the protein of the present invention or a fragment thereof, or a composition containing the same.

[0085] The therapeutically effective amount of the composition of the present invention may vary depending on several factors, such as the administration method, the target site, and the patient's condition. Therefore, the dosage for use in humans must be determined appropriately, taking into account both safety and efficacy. The dosage for use in humans can also be estimated from the effective dosage determined through animal experiments. These factors to be considered when determining the effective dosage are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin, ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0086] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment and that does not cause side effects. The effective dose level can be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method of administration, the time of administration, the route of administration and excretion rate, the duration of treatment, coadministered or concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect at the minimum dose without side effects, which can be easily determined by one skilled in the art.

[0087] The pharmaceutical compositions of the present invention may contain carriers, diluents, excipients, or combinations of two or more thereof commonly used in biological preparations. The term "pharmaceutically acceptable" as used herein means that the composition is non-toxic to cells exposed to the composition or to humans. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition. For example, the carrier may be a compound listed in the Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, may be added as needed. Furthermore, the compositions may be formulated into injectable dosage forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets, by adding diluents, dispersants, surfactants, binders, and lubricants. Furthermore, formulations can be prepared appropriately depending on the disease or ingredients using appropriate methods in the art or the methods disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA, 18th, 1990).

[0088] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group including oral dosage forms, topical preparations, suppositories, sterile injection solutions, and sprays.

[0089] The compositions of the present invention may also contain carriers, diluents, excipients, or combinations of two or more thereof commonly used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition. For example, compounds listed in the Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components may be used. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, formulations may be prepared according to the disease or ingredients, using appropriate methods in the art or the methods disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA, 18th 1990).

[0090] The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention contains the protein in an amount of 0.0001 to 10 wt %, preferably 0.001 to 1 wt %, based on the total weight of the composition.

[0091] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive, such as starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive of the present invention is preferably included in an amount of 0.1 to 90 parts by weight of the composition, but is not limited thereto.

[0092] The composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. The daily dosage of the composition of the present invention is 0.0001 to 10 mg / m, preferably 0.0001 to 5 mg / m, and more preferably administered once or several times a day in divided doses.

[0093] Liquid formulations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0094] MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0095] Example 1. Characterization of LPX peptide 1-1. Cell penetration effect We investigated the cell penetration effects of three penetrating peptides, LPX101 (SEQ ID NO: 11), LPX102 (SEQ ID NO: 12), and LPX103 (SEQ ID NO: 13), which contain a signal peptide, an RNA-binding affinity residue, and an RNA-binding residue (Figure 1) and two zinc finger motifs. Specifically, to measure cell penetration efficiency, we applied the heparin and trypsin washing method used by the Kaplan group to NIH3T3 cell line and mMSCs (mouse mesenchymal stem cells) to remove any peptides that may be attached to the cell membrane surface [Kaplan IM, Wadia JS, Dowdy SF (2005) Cationic TAT peptide transduction domain enters cells by macropinocytosis. J Controlled release 102:247-253]. Next, LPX101 (20 μg / ml), LPX103, and LPX102 (0, 0.1, 1, 2, 4 μg / ml) peptides were synthesized by LifeTein (USA) in FITC-conjugated forms. Each peptide was then added to the cell culture medium at a concentration of 1 μM and incubated at 37°C for 1 hour. The cells were then washed three times with PBS, detached with trypsin, and subjected to FACS analysis. Furthermore, the presence of each peptide in the nucleus or cytoplasm of the cells was confirmed 18 hours after peptide treatment using a confocal microscope, and the peptides were labeled with PKH for membrane binding.

[0096] As a result, LPX101 showed high cell permeability at both 2 μM and 5 μM concentrations, confirming that most of LPX101 was present in the cytoplasm (Figure 2). Furthermore, both LPX102 and LPX103 showed high cell permeability and similar intracellular retention at concentrations of 2 μM or higher, similar to that of LPX101 (2 μM) (Figure 3).

[0097] 1-2. Cell growth and migration promoting effect The effect of LPX101 peptide on cell growth was examined using the NIH3T3 cell line, and the effects of LPX101, LPX102, and LPX103 peptides on cell migration were examined using the diabetic human epidermal keratinocyte (DHEK) cell line. Specifically, mMSCs or NIH3T3 cells were treated with LPX101 peptide, cultured for 1 hour, and then washed. After 3 days of treatment, the number of cells was counted and the relative proliferation rate compared to the control group was calculated as a percentage to confirm the increase in cell growth due to LPX treatment (n=3). To examine the effect on cell migration, DHEK cells were treated with LPX101, LPX102, and LPX103 peptides, respectively, cultured for 1 hour, and then washed. Cell migration across the scratched border was then measured 72 hours after treatment. The number of cells that migrated across the scratch line was determined using image analysis using ImageJ.

[0098] The results showed that the LPX101 peptide promoted cell growth and migration of DHEK cells at both 2 μM and 5 μM concentrations (Figure 4). Furthermore, the LPX102 and LPX103 peptides promoted cell migration at lower concentrations to a similar extent as LPX101 (Figure 5).

[0099] 1-3. Promoting angiogenesis Human umbilical vein endothelial cells (HUVECs) were seeded onto cytokine-free Matrigel and treated with LPX101 for 1 day (n=3, 1 expt). After 19 days, angiogenesis was assessed microscopically, and angiogenesis parameters, including the number of nodules, intravascular junctions, and master segments, were quantified.

[0100] The results showed that endothelial cells formed more mature capillary rings upon treatment with LPX101 peptide (FIG. 6).

[0101] Example 2. Confirmation of the anti-diabetic effect of LPX peptide 2-1.Improvement of glucose absorption Human dermal fibroblasts (HDF) and diabetic cell lines (DHEK) were cultured in glucose-free medium for 24 hours, then treated with 2 μM (20 / ml) LPX101, 1 μM LPX102, or 1 μM LPX103 for 18 hours, followed by 2-deoxyglucose (2DG). The amount of absorbed glucose was measured using a luminescence kit (Promega) and then measured at 470 nm using a Spectra MaxL instrument. Relative glucose absorption was determined (mean SEM, n=3; *: p<0.05, **: p<0.01).

[0102] As a result, it was shown that the LPX peptide improved glucose absorption in various mammalian cells, including diabetic cell lines (Figure 7).

[0103] 2-2.Improvement of glycolysis NIH3T3 cells were cultured in glucose-free medium for 1 day and then sequentially treated with glucose, oligomycin, and 2-DG. The extracellular acidification rate (ECAR) was measured as a measure of lactate production using a Seahorse extracellular flux (XF) analyzer. The quantification and profile of ECAR in cells treated with the LPX peptide were confirmed (mean SEM n = 3, *; p < 0.05).

[0104] As a result, it was shown that the LPX101, LPX102 and LPX103 peptides promote glucose metabolism (glycolysis) (FIG. 8).

[0105] 2-4. Diabetic wound healing effect 2-4-1. Confirmation of diabetic wound healing effect The skin of wild-type (WT) mice and diabetic model mice (db / db) (n = 14 each) was punched with an 8 mm diameter puncher and treated daily for 14 days with LPX101 (2 mg / ml, 20 μl) or PBS. Wound healing was assessed by analyzing the initial wound area and re-epithelialized area using ImageJ (mean SEM n = 3, *; p < 0.05). Wound healing in LPX101-treated wounds was also assessed histologically. Specifically, diabetic mice (db / db) were stained with H / E or Trichrome staining at 7 and 14 days after wound induction, and microscopic examination was performed. The re-epithelialized and granulated tissue was quantified using ImageJ (n = 3, ***; p < 0.001). Additionally, wounds in db / db mice were treated with LPX101 and the epidermis was analyzed histologically 14 days later.

[0106] The results showed that the LPX101 peptide accelerated wound healing in WT mice and significantly accelerated diabetic wound healing in the db / db mouse model (Figure 9). Furthermore, treatment with the LPX101 peptide induced early granulation tissue formation in diabetic wounds on day 7 (the middle of the healing process) and early contraction (remodeling) of granulation tissue on day 14 (the end of the healing process) (Figure 10). Histological analysis of the epidermis on day 14 also showed that LPX101 treatment rapidly reduced hyperkeratosis, parakeratosis, and acanthosis, accelerating remodeling of the regenerated wound epithelium (Figure 11).

[0107] 2-4-2. Wound healing effect in STZ-induced diabetic mouse model The wound healing effects of LPX101, LPX102, and LPX103 were examined in an STZ-induced diabetic mouse model. Specifically, mice were injected with STZ (streptozotocin) for 5 days to induce diabetes, resulting in hyperglycemia. Subsequently, mice were treated with PBS (negative control group), EGF (positive control group) at 50 μg / ml, LPX101 at 2 mg / ml (20 μl), LPX102, and LPX103 at 2 mg / ml (20 μl) according to the schedule shown in Figure 12A. The wound healing effects in this diabetic mouse model were assessed by imaging, re-epithelialization, and diabetic phenotype (hyperglycemia and impaired glucose tolerance test (IPGTT)) (n = 10 per group, *; p < 0.05). To compare the regenerative activity of LPX peptides in diabetic mouse models, we also examined wound healing kinetics measured by the % non-epithelialized area relative to the initial wound area (n = 8 for each group), and quantified re-epithelialization analyzed as the % re-epithelialized area relative to the initial wound site.

[0108] The results showed that all LPX peptides had improved wound healing effects compared to EGF in an STZ-induced diabetic wound model (a mouse selection with pronounced hyperglycemia), with LPX102 and LPX103 demonstrating even greater efficacy than LPX101 (Figure 12). Furthermore, quantitative analysis of wound healing kinetics and re-epithelialization showed that LPX102 and LPX103 were superior to LPX101 in diabetic wound healing in the db / db mouse model (Figure 13).

[0109] 2-4-3. Diabetic wound healing through promotion of angiogenesis Diabetic ulcers are difficult to treat due to impaired angiogenesis and a lack of vascular supply caused by ischemic necrosis. Therefore, we investigated whether LPX peptide contributes to angiogenesis in diabetic wound healing. Specifically, diabetic model mice (db / db) were wounded and treated with LPX103 or EGF once daily. Angiogenesis recovery was analyzed on day 7 after wound initiation. To visualize the extravasation of intravascular red blood cells, images of the wound and the entrance (red dotted box) in each group were magnified.

[0110] As a result, the LPX103-treated group showed more mature capillary formation and less hemorrhage (extravascular outflow of RBCs) than the EGF-treated group (FIG. 14).

[0111] 2-4-4. Histological evaluation of re-epithelialization induced by LPX peptide To histologically evaluate the re-epithelialization of wounds in diabetic mice treated with LPX peptides 101, 102, and 103, wound tissues from diabetic mice (db / db) treated with each LPX were stained with H / E and examined microscopically (50X and 100X) on day 10 after the initial injury.

[0112] The results showed that both LPX peptides 101, 102 and 103 significantly enhanced the re-epithelialization of diabetic wounds (FIG. 15).

[0113] 2-4-5. Combined administration The diabetic wound healing effect of combined treatment with LPX peptide and EGF was confirmed. Specifically, a diabetic mouse model (db / db) was punched with an 8 mm diameter and treated with a combination of LPX peptide (101, 102, or 103) and / or EGF according to the schedule shown in Figure 15A. Images were observed under a microscope to confirm the effect on re-epithelialization after initial wound formation.

[0114] As a result, it was shown that each of LPX101, LPX102, and LPX103 improved the diabetic wound healing effect when treated in combination with EGF (FIG. 16).

[0115] Through this, it was found that LPX peptide improves the regeneration of diabetic wounds (ulcers), which were previously difficult to treat, in multiple ways (Figure 17).

[0116] Example 3. Confirmation of activity by modifying LPX peptide 3-1. Preparation of peptide mutants Various variants of the LPX peptide were prepared as shown in Figure 18. Specifically, various mutations (substitution of the CPP region, amino acid mutations in the RNA binding domain and zinc finger structure, and addition of a His tag) were added to the LPX101 structure shown in Figure 19 to prepare various constructs (26 types including 101) (Figure 20). Furthermore, the cell migration effects of LPX101, LPX102, LPX103, and LPX104 (SEQ ID NO: 14) on NIH3T3 cells were compared, and the results showed that they were at similar levels, as shown in Table 1 below.

[0117] [Table 1]

[0118] 3-2. Confirmation of activity changes due to CPP domain substitution To confirm the change in biological activity due to the substitution of the CPP domain sequence, the CPP domain of LPX104 (#4) containing sequence number 7, which includes the RNA binding site of the zinc finger 2 domain, was substituted with the HIV TAT or Arg repeat (R9) sequence (TAT-104 and R9-104, respectively) as shown in Figure 21A. Then, the cell permeation effect, peptide stability 24 hours after cell permeation, cell migration effect, and cell growth effect on NIH3T3 cells were compared.

[0119] As a result, substitution of the CPP region of the LPX peptide with TAT or Arg(9) residues showed similar effects on cell permeation, cell proliferation, and cell migration as the original LPX104 (Figure 21B), but the stability of the peptide was significantly higher than that of the original LPX104 (Figure 21C).

[0120] 3-3. Confirmation of activity change by HIS tag insertion To select mutations with improved stability through endosomal escape, a repeated HIS tag sequence (6XHis) was added to the N-terminus of the CPP region of the LPX peptide (LPH103 or LPX104) as shown in Figure 22A, and the stability, cell migration-promoting effect, and cell growth-promoting effect were compared 24 hours after cell infiltration.

[0121] The results showed that adding a 6XHis tag to the LPX peptide significantly increased its intracellular stability by promoting endosomal escape (Figure 22B), and also enhanced cell proliferation and migration (Figures 22C-D).

[0122] 3-4. Confirmation of activity changes due to mutations in the RNA binding domain and zinc finger domain We confirmed the protein stability of mutants with mutations in the RNA-binding and zinc finger regions. Specifically, LPX103 mutants with mutated amino acid residues responsible for RNA binding during cell migration were treated with NIH3T3 cells for one day. The intracellular stabilization effect of each peptide was analyzed after 72 hours using a molecular modeling program. The mutation energy of each amino acid residue was determined, and mutants that could induce a substitution of <-1 kcal / mol were selected as potential mutations that could increase peptide stability and activity (Figures 23 and 24). The effects of the selected mutants on cell proliferation and cell migration were confirmed in NIH3T3 cells (Figure 25). Through this, we were able to identify mutations in the RNA-binding and zinc finger regions that either improved or decreased intracellular peptide stability (Figure 26). Therefore, we selected LPX103, LPH103, and LPS1O3, which were confirmed to have high stability and biological activity.

[0123] Throughout the above examples, potential substitutions of residues that can similarly maintain or improve the biological activity of the LPX peptide are as follows: 1) Addition of N-terminal histidine repeats improves stability; 2) substitution of CPP with TAT or R9 is equally effective; 3) residue mutations that cause protein stabilization (mutation energy <-1 Kcal / mole) are D16R, Y19R, N20R, G23E, L33E, and L33W (see Figure 26); and 4) Any combination of residues from other categories.

[0124] Additionally, the following residues, which may cause a decrease in biological activity, must be conserved in the LPX peptide variant: 1) 18C, 21C, 26H and 31C of the zinc finger motif (based on LPX101 of SEQ ID NO: 11); and 2) 20N in the RNA binding region (based on LPX101 of SEQ ID NO: 11). Through this, we were able to identify amino acid residues that could be substituted or added to maintain or increase the stability and biological activity of the LPX peptide, as well as residues that must be conserved to reduce stability and biological activity.

[0125] Example 4: Confirmation of activity by modifying LPX105 peptide 4-1. Preparation of LPX105 peptide mutants LPX105 mutants were prepared using the method described in Example 3. Specifically, to confirm the effects of mutations in the zinc finger region of LPX105 (SEQ ID NO: 37) and histidine tag insertion, LPS105 (SEQ ID NO: 38), LPH105 (SEQ ID NO: 39), and LPHS105 (SEQ ID NO: 40) were prepared by mutations in the zinc finger region of LPX105 and insertion of a His tag (Figure 27).

[0126] 4-2. Confirmation of cell migration promotion by LPX105 mutants The cell migration promotion effects of LPX105, LPH105, and LPHS105 were confirmed using the method described in Examples 1-2. Specifically, to confirm the cell migration effect, fibroblast 3T3 cells were treated with LPX105, LPH105, and LPHS105 peptides, respectively, and then cultured for 1 hour and washed. Cell migration across the scratched border was then measured 72 hours after treatment. The number of cells that migrated across the scratch line was determined by image analysis using ImageJ.

[0127] As a result, it was confirmed that LPX105 significantly promoted cell migration compared to the control group PBS, and that LPX105 mutants LPH105 and LPHS105 increased cell migration more than LPX105 (Figure 28).

[0128] 4-3. Effect of LPX105 mutants on diabetic wound healing The diabetic wound healing effects of LPX105, LPH105, and LPHS105 were evaluated using the methods described in Examples 2-4. Specifically, the skin of diabetic model mice (db / db) (n=14 each) was punched with an 8 mm diameter puncher, and the wounds were dressed with LPX105, LPH105, or LPHS105 (3 μg) or PBS daily for 10 days. The wound healing effects were confirmed by analyzing the initial wound area and re-epithelialized area using ImageJ (mean SEM n=3, *; p<0.05).

[0129] The results showed that LPX105 accelerated diabetic wound healing in the db / db mouse model compared with the control group (PBS). Furthermore, we confirmed that the LPX105 mutants LPH105 and LPHS105 accelerated diabetic wound healing more significantly than LPX105 (Figure 29).

Claims

1. A cell-penetrating peptide comprising a cell-penetrating domain and a zinc finger domain, the cell-penetrating domain is an amino acid sequence of any one of SEQ ID NOs: 4 to 6; The zinc finger domain is a peptide having an amino acid sequence represented by the following general formula 1: 【Chemistry 1】 (where, X1 is C or A, X2 is Y or R, X3 is N, R or D, X4 is C or A, X5 is G or E, X6 is H or A, X7 is C or A.

2. The cell-penetrating peptide of claim 1, wherein G-X8-R is added to the N-terminus of the zinc finger domain, and X8 is D or R.

3. The cell-penetrating peptide of claim 1, wherein K-X9 is added to the C-terminus of the zinc finger domain, and X9 is L or W.

4. The cell-penetrating peptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:

37.

5. The cell-penetrating peptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:

38.

6. The cell-penetrating peptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:

39.

7. The cell-penetrating peptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:

40.

8. The cell-penetrating peptide of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 11 to 36.

9. The cell-penetrating peptide of claim 1, further comprising an amino acid E, GSE, or His tag at the N-terminus of the cell-penetrating domain.

10. The cell-penetrating peptide according to claim 1, wherein the C-terminus of the zinc finger domain comprises an amino acid sequence of any one of SEQ ID NOs: 7 to 10.

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