Cell-penetrating peptide, conjugate containing same, and composition containing same

Abstract

To provide a cell penetrating carrier peptide, a conjugate of the cell penetrating carrier peptide and an active ingredient, and a composition comprising the same.SOLUTION: In a conjugate of a cell penetrating carrier peptide and an active ingredient, the carrier peptide is a peptide comprising at least one of SEQ ID NO: 1 to SEQ ID NO: 156, a peptide having at least 80% homology with the peptide sequence, or a fragment thereof, and the peptide having at least 80% homology with the sequence or the fragment thereof maintains cell penetrating capacity of at least one amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156. A composition comprises the conjugate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell-penetrating peptide derived from telomerase, a conjugate of the cell-penetrating peptide with an active ingredient, and a composition containing the conjugate. [Background technology]

[0002] Small molecules, nucleic acids, proteins, and nanoparticles have great potential as molecular-level therapeutic agents, but their use is limited due to their resistance to tissue and cell membrane penetration. The development of a system for intracellular delivery of these substances has been an issue in molecular-based therapeutics. While intracellular delivery of small molecules, nucleic acids, and nanoparticles has been achieved using various reagents, electroporation, or heat shock, intracellular delivery of proteins while maintaining their activity has been a challenging problem, and no solution has been found. Meanwhile, in the 1980s, research into HIV cell membrane penetration revealed that the HIV-TAT protein, consisting of a specific 11 amino acid sequence, plays a key role in the intracellular delivery process. Since the 1990s, full-scale research into intracellular protein delivery has been underway.

[0003] Telomeres are genetic material found repeatedly at the ends of chromosomes and are known to prevent damage to the chromosome or its connection to other chromosomes. Each time a cell divides, the length of the telomere shortens slightly. After a certain number of cell divisions, the telomere becomes so short that the cell stops dividing and dies. Meanwhile, lengthening telomeres is known to extend the lifespan of cells. For example, cancer cells secrete an enzyme called telomerase, which prevents telomeres from shortening, allowing the cancer cells to continue growing without dying. Summary of the Invention [Problem to be solved by the invention]

[0004] One aspect of the present invention provides novel peptides.

[0005] One aspect of the present invention provides polynucleotides encoding novel peptides.

[0006] One aspect of the present invention provides a cell-penetrating peptide.

[0007] One aspect of the present invention provides peptides useful as intracellular active ingredient delivery vehicles.

[0008] One aspect of the present invention provides a conjugate in which a cell-penetrating peptide and an active ingredient are conjugated.

[0009] One aspect of the present invention provides a composition comprising a conjugate of a cell-penetrating peptide and an active ingredient.

[0010] One aspect of the present invention provides a pharmaceutical composition comprising a conjugate of a cell-penetrating peptide and an active ingredient.

[0011] One aspect of the present invention provides a functional cosmetic composition comprising a conjugate of a cell-penetrating peptide and an active ingredient.

[0012] One aspect of the present invention provides a health food composition comprising a conjugate of a cell-penetrating peptide and an active ingredient.

[0013] One aspect of the present invention provides an imaging agent comprising a conjugate of a cell-penetrating peptide and an active ingredient. [Means for solving the problem]

[0014] A conjugate according to one aspect of the present invention is a conjugate of a cell-permeable carrier peptide and an active ingredient, wherein the carrier peptide is a peptide comprising any one of SEQ ID NOs: 1 to 156, a peptide having more than 80% sequence identity to the peptide sequence, or a fragment thereof, and the peptide and fragment having more than 80% sequence identity are conjugates that retain the cell permeability of the corresponding peptide of any one of SEQ ID NOs: 1 to 156.

[0015] In the conjugate according to another aspect of the present invention, the fragment may be a fragment consisting of three or more amino acids.

[0016] In the conjugate according to another aspect of the present invention, the delivery peptide is also a peptide consisting of 30 amino acids or less.

[0017] In the conjugate according to another aspect of the present invention, the delivery peptide may have any one or more of the sequences of SEQ ID NO: 1 to SEQ ID NO: 156.

[0018] In a conjugate according to another aspect of the present invention, the delivery peptide may be a peptide consisting of any one of the sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 90, SEQ ID NO: 92, and SEQ ID NO: 121.

[0019] An imaging agent according to one aspect of the present invention is also an imaging agent comprising any one of the conjugates described above.

[0020] In the contrast agent according to another aspect of the present invention, the contrast material is also used to image cells.

[0021] In the contrast agent according to another aspect of the present invention, the cells are also stem cells.

[0022] A composition according to one aspect of the invention may comprise any one of the conjugates described above.

[0023] In the composition according to another aspect of the present invention, the active ingredient is an active ingredient for treating or preventing a disease, and the composition is also a pharmaceutical composition.

[0024] In the composition according to another aspect of the present invention, the active ingredient is an active ingredient of a functional cosmetic, and the composition is also a cosmetic composition.

[0025] In the composition according to another aspect of the present invention, the active ingredient is an active ingredient of a functional health food, and the composition is also a health food composition.

[0026] A method according to one aspect of the present invention is a method for delivering an active ingredient into a cell, comprising administering any one of the conjugates described above to a subject in need thereof, wherein the transport peptide is a cell-penetrating peptide that delivers the active ingredient into the cell, and the peptides and fragments having more than 80% sequence homology also retain the cell permeability of the corresponding peptide of any one of SEQ ID NO: 1 to SEQ ID NO: 156.

[0027] A method for intracellular delivery of an active ingredient according to another aspect of the present invention also involves localized delivery of the active ingredient to intracellular mitochondria.

[0028] A cell-penetrating peptide according to one aspect of the present invention may have one or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 156.

[0029] Another aspect of the present invention is a cell-penetrating peptide comprising any one of the sequences selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 90, SEQ ID NO: 92, and SEQ ID NO: 121.

[0030] A polynucleotide according to one aspect of the present invention is a polynucleotide encoding the cell-penetrating peptide described above.

[0031] A vector according to one aspect of the present invention is a vector comprising the polynucleotide.

[0032] A transformed cell according to one aspect of the present invention is also a transformed cell containing the vector. [Effects of the Invention]

[0033] By using the peptide according to the present invention, or a conjugate of the peptide and an active ingredient, active ingredients that are difficult to penetrate into cells can be easily delivered into cells. This enhances the efficacy of the active ingredient, thereby reducing the dosage, thereby minimizing side effects caused by drug administration and improving therapeutic efficiency. In particular, localized delivery to mitochondria can improve the efficacy of improving, preventing, or treating mitochondrial-related diseases or disorders. Even in the case of cosmetics, excellent effects can be achieved with only a small amount of active ingredient. By using a conjugate with a contrast material, the peptide can be used as a contrast agent for monitoring the cell transplantation process or transplanted cells in cell therapy. In particular, it can be effectively used as a contrast agent for stem cells injected into the body. [Brief explanation of the drawings]

[0034] [Figure 1] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 2] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 3]FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 4] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 5] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 6] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 7] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 8] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 9] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 10]FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 11] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 12] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 13] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 14] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 15] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 16] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 17]FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 18] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 19] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 20] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 21] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 22] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 23] FITC was conjugated to peptides of sequence numbers 2 to 156, and then each was applied to a HeLa cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). The control group was treated with FITC only. [Figure 24]FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 25] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 26] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 27] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 28] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 29] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 30] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 31] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 32]FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 33] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 34] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 35] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 36] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 37] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 38] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 39] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 40]FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 41] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 42] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 43] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to the Hur7 cell line. The graph shows the number of cells uptaken as a result of analysis using a flow cytometer (FACS). [Figure 44] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 45] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 46] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 47] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 48]FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 49] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 50] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 51] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 52] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 53] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 54] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 55]FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 56] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 57] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 58] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was applied to a human T lymphocyte cell line (Jurkat), and the results of analysis using a flow cytometer (FACS) showed the number of cells uptaken. [Figure 59] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 60] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 61] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 62] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 63] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 64] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 65] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 66] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 67] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 68] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 69] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 70] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 71] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. [Figure 72] FITC was conjugated to peptides of SEQ ID NO: 1 to SEQ ID NO: 156, and then each was treated with a HeLa cell line and cultured, after which cell viability and toxicity were measured. DETAILED DESCRIPTION OF THE INVENTION

[0035] Proteins, nucleic acids, peptides, and viruses have great potential as therapeutic agents. However, their molecular size limits their ability to penetrate tissues and cell membranes, significantly limiting their use. Furthermore, even small substances often cannot penetrate the lipid bilayer of cell membranes due to their properties or structure. Therefore, attempts have been made to deliver proteins, nucleic acids, peptides, and viruses into cells using electroporation or heat shock, but it has been difficult to deliver these substances into cells without damaging the cell membrane and while maintaining their activity. Meanwhile, it has been discovered that the trans-activating transcriptional activator (TAT) protein derived from the human immunodeficiency virus (HIV) can act as a cell-penetrating peptide capable of delivering large active substances into cells, spurring vigorous research into this topic. Specifically, unlike TAT protein, which is known to cause intracellular toxicity, research has been conducted on substances that do not cause in vivo toxicity and can more effectively transport macromolecules such as proteins, nucleic acids, peptides, or viruses into target cells. Through continuous research, the present inventors have discovered that a peptide derived from telomerase has little cytotoxicity and excellent effects as a cell-penetrating peptide.

[0036] The peptides set forth in SEQ ID NOs: 1 to 156 are as shown in Table 1 below. SEQ ID NO: 157 is the sequence of the full-length human telomerase protein. The "names" in Table 1 below are given to distinguish the peptides. In another aspect of the present invention, one or more of the peptides set forth in SEQ ID NOs: 1 to 156 include "synthetic peptides" synthesized by selecting peptides at the corresponding positions from peptides contained in telomerase. As used herein, "pep" is a general term referring to a peptide having any one of the sequences set forth in SEQ ID NOs: 1 to 156, or a peptide having more than 80% sequence identity to that sequence, or a fragment of the sequence.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3]

[0040] [Table 4]

[0041] One aspect of the present invention provides a polynucleotide encoding a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide having greater than 80% sequence identity to the peptide sequence, or a fragment thereof. The polynucleotide can be used to mass-produce the peptide. For example, the peptide can be mass-produced by introducing a vector containing a polynucleotide encoding the peptide into a host cell and culturing the cell.

[0042] The peptides disclosed herein may include peptides with greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% sequence identity. The peptides disclosed herein may also include peptides comprising any one of SEQ ID NOs: 1 through 156, or fragments thereof, and peptides with one or more amino acid changes, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, or seven or more amino acids.

[0043] In one aspect of the present invention, amino acid changes are made to alter the physicochemical properties of the peptide, for example, to improve the thermal stability of the peptide, alter its substrate specificity, or change its pH optimum.

[0044] As used herein, the term "amino acid" refers not only to the 22 standard amino acids naturally incorporated into peptides, but also to D-isomers and modified amino acids. Accordingly, in one aspect of the present invention, a peptide may also contain a D-amino acid. Meanwhile, in another aspect of the present invention, a peptide may contain a non-standard amino acid that has undergone post-translational modification. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-elimination deimidation, deamidation), and structural changes (e.g., formation of disulfide bridges). It also includes amino acid changes, such as changes at the amino group, carboxylic acid group, or side chain, that occur due to chemical reactions that occur during the process of coupling with a crosslinker to form a peptide conjugate.

[0045] The peptides disclosed herein may be wild-type peptides identified and isolated from natural sources. Alternatively, the peptides disclosed herein may be artificial variants, which contain an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted compared to a peptide that is a fragment of any one of SEQ ID NOs: 1 to 156. Amino acid changes in wild-type polypeptides, as well as artificial variants, include conservative amino acid substitutions that do not significantly affect protein folding and / or activity. Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine, and methionine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, and threonine). Generally, amino acid substitutions that do not alter specific activity are known in the art. The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and their opposites. Other examples of conservative substitutions are set forth in Table 2 below.

[0046] [Table 5]

[0047] Substantial variations in the biological properties of peptides are achieved by selecting substitutions that differ significantly in (a) their effect on maintaining the structure of the polypeptide backbone in the region of substitution, e.g., sheet or helical conformation, (b) their effect on maintaining the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining the bulk of the side chain. Natural residues are divided into the following groups based on common side chain properties:

[0048] (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain direction: gly, pro; and (6) Aromatics: trp, tyr, phe.

[0049] Non-conservative substitutions are made by exchanging a member of one of these classes for another. Any cysteine ​​residue not involved in maintaining the proper conformation of the peptide is generally substituted with serine to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bonds can be added to the peptide to improve its stability.

[0050] Another type of amino acid variant of a peptide is one that alters the glycosylation pattern of the antibody, by which is meant the deletion of one or more carbohydrate residues found in the peptide and / or the addition of one or more glycosylation sites that are not present in the peptide.

[0051] Glycosylation of peptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate residue to the asparagine side chain. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid. Attachment to serine or threonine is most commonly referred to, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0052] Glycosylation sites may be added to the peptide by altering the amino acid sequence such that it contains one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Such alterations may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0053] One aspect of the present invention provides a cell-penetrating peptide, the cell-penetrating peptide comprising any one of SEQ ID NOs: 1 to 156, or a fragment thereof, or a peptide having greater than 80% sequence identity to the peptide sequence. One aspect of the present invention provides a pharmaceutical composition comprising a peptide comprising any one of SEQ ID NOs: 1 to 156, or a fragment thereof, or a peptide having greater than 80% sequence identity to the peptide sequence, as a drug delivery vehicle for delivering one or more active ingredients. A peptide comprising any one of SEQ ID NOs: 1 to 156, or a fragment thereof, or a peptide having greater than 80% sequence identity to the peptide sequence is safe yet acts as an excellent cell-penetrating peptide, and can bind to a drug and effectively deliver the drug into cells.

[0054] One aspect of the present invention provides a conjugate in which a peptide comprising one of SEQ ID NOs: 1 to 156, a peptide that is a fragment thereof, or a peptide that has more than 80% sequence identity with the peptide sequence is conjugated to an active ingredient to be delivered. In one aspect of the present invention, the active ingredient is one or more selected from proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast agents, drugs, and chemical compounds. In one aspect of the present invention, the active ingredient is also a peptide. In one aspect of the present invention, the active ingredient peptide is also a cytokine, antibody, antibody fragment, therapeutic enzyme, soluble receptor, or ligand.

[0055] As used herein, the term "cell penetrating peptide (CPP)" refers to a peptide capable of transporting a cargo into cells in vitro and / or in vivo. The term "cargo" as used herein includes any substance that can be conjugated to a cell penetrating peptide and transported into cells, including, but not limited to, any substance for which increased cell penetration efficiency is desired, specifically, active ingredients in drugs, cosmetics, or health foods, and more specifically, substances that do not easily transport into cells via conventional pathways, including, but not limited to, proteins, nucleic acids, peptides, minerals, glucose, sugars, nanoparticles, biological agents, viruses, contrast agents, or other chemical substances. As used herein, the term "drug" is a broad term that encompasses substances used to alleviate, prevent, treat, or diagnose diseases, injuries, or specific symptoms.

[0056] As used herein, the term "carrier peptide" refers to a peptide that binds to an active ingredient and transports the active ingredient to a desired site.

[0057] In one aspect of the present invention, the protein or peptide to be delivered may include, but is not limited to, one or more of hormones, hormone analogs, enzymes, enzyme inhibitors, signal transduction proteins (or peptides), antibodies, and vaccines. In one aspect of the present invention, the nucleic acid may be a naturally occurring or artificial DNA molecule or RNA molecule, single-stranded or double-stranded. The nucleic acid molecule may be one or more nucleic acid molecules of the same type (e.g., having the same nucleoside sequence), or may be another type of nucleic acid molecule. The nucleic acid molecule may include, but is not limited to, one or more of DNA, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomers, plasmids, and other modified nucleic acids. In one aspect of the present invention, the virus may include the entire virus or a viral core containing viral nucleic acid. In one aspect of the present invention, the chemical substance is a broad concept that includes chemical substances that can function as drugs, and includes natural or synthetic chemical substances.

[0058] In one aspect of the present invention, the drug delivered into a cell by a cell-penetrating peptide may further comprise a drug delivery vehicle such as a liposome, a micelle, a nanoparticle, a magnetic particle, or a quantum dot.

[0059] As used herein, the term "contrast material" is a broad term that encompasses all materials used in medical imaging to visualize biological structures or fluids. Suitable contrast materials include, but are not limited to, radiopaque contrast agents, paramagnetic contrast agents, superparamagnetic contrast agents, computed tomography (CT) contrast agents, and other contrast materials. For example, radiopaque contrast materials (for X-ray imaging) include inorganic and organic iodine compounds (e.g., diatrizoic acid), radiopaque metals and their salts (e.g., silver, gold, platinum, etc.), and other radiopaque compounds (e.g., calcium salts, barium salts such as barium sulfate, tantalum, and tantalum oxide). Suitable paramagnetic contrast materials (for MR imaging) include gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). acid) and its derivatives, and other complexes of gadolinium, manganese, iron, dysprosium, copper, europium, erbium, chromium, nickel, and cobalt, such as 1,4,7,10-tetraazacyclododecane-N,N',N",N'''-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,-N',N"-triacetic acid (DO3A), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,8,10-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA), hydroxybenzylethylenediaminediacetic acid (HBED), and the like. Suitable superparamagnetic contrast materials (for MR imaging) include magnetite, superparamagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO), and monocystailine iron oxide.Other suitable contrast agents are iodinated and non-iodinated, ionic and non-ionic CT contrast agents, as well as contrast agents such as spin-labels or other diagnostically effective agents.

[0060] Other examples of imaging agents include marker genes that encode proteins that are easily detectable when expressed in cells, including, but not limited to, β-galactosidase, green fluorescent protein, blue fluorescent protein, luciferase, etc. A variety of labels are available, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (especially haptens).

[0061] In one embodiment of the present invention, the imaging material may be ferrocenecarboxylic acid, represented by the following Formula 2: The structure of ferrocene is shown in Formula 1.

[0062] [ka]

[0063] [ka]

[0064] In one embodiment of the present invention, the conjugate of the cell-penetrating peptide and the contrast agent is ferrocenecarboxylic acid-pep represented by the following formula 3:

[0065] [ka]

[0066] In one aspect of the present invention, the peptide or composition is fused to one or more detectable labels. The label can be a compound that generates a detectable compound or signal, directly or indirectly, in a chemical, physical, or enzymatic reaction. Labeling and subsequent detection can be accomplished by methods known in the art (e.g., Sambrook, J., and Russell, DW (2001); Lottspeich, F., and Zorbas H. (1998), Bioanalytik, Spektrum Akademischer Verlag, Heidelberg / Berlin, Germany). Labels include, but are not limited to, fluorescent labels, enzyme labels, chromogenic labels, luminescent labels, radiolabels, haptens, biotin, metal complexes, metals, and colloidal gold. All such types of labels are well known in the art and are commercially available from a variety of suppliers.

[0067] In one aspect of the present invention, a transfer target (cargo) can be directly bound to a peptide. In another aspect of the present invention, a transfer target can be bound to a peptide via a variety of binding methods, including covalent and non-covalent binding. The transfer target can be bound, for example, to the N-terminus or C-terminus of a peptide according to one aspect of the present invention. For example, a transfer target can be bound to a peptide via a disulfide bond or a covalent bond that binds the transfer target to the alpha-amine of the peptide N-glutamate (E) or the amine of the C-terminal lysine (K) residue. Alternatively, a peptide and a transfer target can be bound to each other via a non-covalent bond in which one of the peptide and the transfer target encapsulates the other in a form, such as a capsule.

[0068] In another aspect of the present invention, a peptide can be linked to a transfer target via a linker. For example, a linker such as 6-hydrazinopyridine-3-carboxylic acid (hynic) linker can be introduced into the α-amine of peptide N-glutamate or the amine of a C-terminal lysine residue, and then the transfer target can be linked to the linker.

[0069] In yet another aspect of the present invention, when the target of transfer is DNA or RNA, an SH group (thiol group) is introduced into the peptide, and a maleimide group is introduced into the DNA or RNA, and then the SH group of the peptide is bonded to the maleimide group of the DNA or RNA, thereby binding the target of transfer to the peptide.

[0070] In another aspect of the present invention, when the transfer target is a protein or peptide, the transfer target can be linked to DNA expressing a delivery peptide by ligating the DNA expressing the transfer target and then expressing the ligated DNA in the form of a fusion protein of the transfer target and the peptide. A specific example of ligation using a fusion protein is as follows: when preparing a primer for producing the fusion protein, nucleotides encoding the delivery peptide are added before the nucleotides expressing the transfer target. The resulting nucleotides are inserted into a vector, such as a pET vector, using a restriction enzyme, and then transformed into cells, such as BL-21(DE3), for expression. The fusion protein can then be effectively expressed by treating with an expression inducer, such as IPTG (isopropyl-1-thio-β-D-galactopyranoside). The expressed fusion protein can then be purified using a method such as His tag purification, dialyzed with PBS, placed in a kit, and concentrated by centrifugation, for example, at 2,000 to 4,000 rpm for 5 to 20 minutes.

[0071] In one aspect of the present invention, the delivery peptide can be conjugated to a dye or a fluorescent substance, specifically, fluorescein isothiocyanate (FITC) or green fluorescent protein (GFP). In one aspect of the present invention, FITC binds to the amino group (NH3) of the N-terminal Lys or C-terminal Lys of the delivery peptide. + When the peptide does not have a Lys residue at its terminus, the peptide can be linked to FITC via a linker containing Lys.

[0072] Peptides disclosed herein that function as delivery peptides, including any one of SEQ ID NOs: 1 to 156, or peptides that are fragments thereof, or peptides that share more than 80% sequence identity with the peptide sequences, can bind to a transport target (cargo) at a molar ratio of 1:1, but other molar ratios are also possible. For example, the CPP:transfer target molar ratio can be 2:1 or greater. Specifically, the molar ratio can be 2:1 or greater, 3:1 or greater, 4:1 or greater, 5:1 or greater, 6:1 or greater, 7:1 or greater, 8:1 or greater, 9:1 or greater, or 10:1 or greater. This means that multiple delivery peptide molecules can bind to a single delivery target molecule. Multiple delivery peptide molecules can be linked to each other in series or parallel. "Linked in series" means that the delivery peptides are linked to each other at the terminal amino acid positions, while "linked in parallel" means that the delivery peptides are linked to each other at positions other than the terminal amino acid positions. Conversely, the molar ratio of delivery peptide:transfer target can be 1:2 or greater. This means that multiple target molecules can be bound to one delivery peptide molecule. For example, the molar ratio of delivery peptide to target molecules can be 1:2. More specifically, the molar ratio can be 1:2 or greater, 1:3 or greater, 1:4 or greater, 1:5 or greater, 1:6 or greater, 1:7 or greater, 1:8 or greater, 1:9 or greater, or 1:10 or greater.

[0073] Since the transport pathway of a peptide conjugated with fluorescein isothiocyanate can be easily identified, the delivery peptide according to one aspect of the present invention is utilized for cell imaging or tracking intracellular drug delivery pathways.

[0074] One aspect of the present invention provides the use of a peptide comprising any one of SEQ ID NO: 1 to SEQ ID NO: 156, or a peptide that is a fragment thereof, or a peptide that has more than 80% sequence identity with said peptide sequence, as a drug delivery vehicle for delivering one or more active ingredients.

[0075] One aspect of the present invention provides a method for delivering a drug into cells of a subject, comprising applying to the subject a composition comprising a drug and a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a peptide having more than 80% sequence identity to the peptide sequence.

[0076] One aspect of the present invention provides a method for tracking the delivery pathway of a drug applied to a subject, comprising applying to the subject a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a peptide having more than 80% sequence identity to the peptide sequence, and a contrast agent.

[0077] One aspect of the present invention provides a method for tracking the delivery pathway of a drug administered to a subject, comprising administering to the subject a conjugate of a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a peptide having more than 80% sequence homology to the peptide sequence, and a contrast agent.

[0078] One aspect of the present invention provides a kit for intracellular drug delivery to a subject, comprising a composition comprising a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a peptide having more than 80% sequence identity to the peptide sequence, and a conjugate of a drug to be delivered, and instructions disclosing one or more of the dosage, administration route, administration frequency and indications of the composition.

[0079] One aspect of the present invention provides a cosmetic or food composition comprising an active ingredient and a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a peptide that has more than 80% sequence identity to the peptide sequence. Another aspect of the present invention provides a cosmetic or food composition comprising a conjugate of an active ingredient and a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a peptide that has more than 80% sequence identity to the peptide sequence.

[0080] One aspect of the present invention provides a pharmaceutical, cosmetic, or food composition that comprises a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a conjugate of an active ingredient with a peptide that has more than 80% sequence homology to the peptide sequence, and that is highly effective in delivering the active ingredient into cells.

[0081] Mitochondria are central organelles for energy metabolism in nucleated cells and were the first intracellular organelles to be linked to human disease (Luft R, Ikkos D, Palmieri G, Ernster L, Afzelius B: A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical, and morphological study, J Clin Invest 41: 1776-804, 1962).

[0082] Mitochondria play a key role in regulating cellular energy metabolism and apoptosis, making them a major target for various therapeutic drugs. Furthermore, mitochondria are involved in regulating intracellular calcium levels, and the mitochondrial respiratory chain functions as an electron transport chain important for energy production and causes the production of reactive oxygen species. Consequently, mitochondrial dysfunction is closely related to adult diseases such as diabetes, cardiomyopathy, infertility, blindness, kidney / liver disease, and stroke (Modica-Napolitano KS, Singh KK: April mitochondria as targets for detection and treatment of cancer, Expert Rev Mol Med 11: 1-19, 2002). At the same time, mutations in mitochondrial genes have been suggested to be involved in the pathogenesis of aging, degenerative neurological disorders, and cancer.

[0083] One aspect of the present invention provides a mitochondria-targeting active ingredient delivery system, which includes any one of the conjugates described above, wherein the carrier peptide is a peptide that locally transports to intracellular mitochondria and performs localized mitochondrial delivery of the active ingredient, and the peptide or fragment thereof having more than 80% sequence homology retains the mitochondrial targeting ability of the corresponding peptide of any one of SEQ ID NOs: 1 to 156.

[0084] Furthermore, according to one aspect of the present invention, a composition for regulating mitochondrial activity is provided, which comprises any one of the conjugates described above, and the transport peptide is a peptide that moves locally to mitochondria within cells and performs local mitochondrial delivery of the active ingredient, and the peptide and fragment thereof having more than 80% sequence homology is also a composition for regulating mitochondrial activity that possesses the mitochondrial targeting ability of the corresponding peptide of any one of SEQ ID NO: 1 to SEQ ID NO: 156.

[0085] In a composition for regulating mitochondrial activity according to one aspect of the present invention, the composition is a pharmaceutical composition for treating, preventing, inhibiting the progression of, or alleviating symptoms of a mitochondrial-related disease or disorder, and the active ingredient is also an ingredient that exhibits the function of treating, preventing, inhibiting the progression of, or alleviating symptoms of a mitochondrial-related disease or disorder.

[0086] "Mitochondrial-related diseases" referred to herein include, but are not limited to, Huntington's disease; amyotrophic lateral sclerosis; MELAS (mitochondrial encephalomyopathy with lactic academia and stroke-like episodes); MERRF (myoclonus, epilepsy, and myopathy with ragged red fibers); NARP / MILS (neurogenic muscular weakness, ataxia, retinitis pigmentosa / maternally inherited Leigh syndrome); LHON (Lebers hereditary optic neuropathy, mitochondrial blindness); KSS (Kearns-Sayre syndrome); PMPS (Pearson marrow-pancreas syndrome). syndrome; Pearson myeloid-pancreatic syndrome; CPEO (chronic progressive external opthalnoplegia); Reye's syndrome; Alpers syndrome; multiple mtDNA deficiency syndromes; mtDNA wasting syndromes; complex I defects; complex II (SDH) defects; complex III defects; cytochrome c oxidase (COX, complex IV) defects; complex V defects; atenin nucleotide transporter (ANT) defects; pyruvate dehydrogenase (PDH) defects; ethylmalonic aciduria with lactic acidemia; 3-methylglutaconic aciduria with lactic acidemia; unresponsive epilepsy with a decline during infection; Asperger's syndrome with a decline during infection; autism with a decline during infection; attention deficit hyperactivity disorder (ADHD); cerebral palsy with a decline during infection; dyslexia with a decline during infection; maternally inherited thrombocytopenia; leukemia;MNGIE (mitochondrial myopathy, peripheral and autonomic neuropathy, gastrointestinal dysfunction, and epilepsy); MARIAHS syndrome (mitochondrial ataxia, recrudescent infection, aphasia, hypouricemia / hypomyelination, seizures, and dicarboxylic acid aciduria) Mitochondrial ataxia, recurrent infections, aphasia, hypouricemia / hypomyelinosis, seizures, and dicarboxylic aciduria); ND6 dystonia; cyclic vomiting syndrome with decline during infection; 3-hydroxyisobutyric aciduria with lactic acidemia, diabetes mellitus with lactic acidemia; uridine-responsive neurological syndrome (URNS); familial bilateral striatal necrosis (FBSN); aminoglycoside-associated hearing loss; attenuated cardiomyopathy; splenic lymphoma; Wolfram syndrome; multiple mitochondrial DNA defects syndrome; and renal tubular acidosis / diabetes / ataxia syndrome.

[0087] In another aspect, the present invention provides a nucleic acid molecule encoding the polypeptide, the base sequence of which is, for example, GAA GCG CGC CCG GCG CTG CTG ACC AGC CGC CTG CGC TTT ATT CCG AAA (SEQ ID NO: 181). The nucleic acid molecule can be introduced into host cells by techniques known to those skilled in the art, such as calcium phosphate transfection, liposome transfection, electroporation, transformation by contacting cells with viruses, or direct intracellular microinjection. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell. Examples of prokaryotic hosts suitable for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Mycobacteria.

[0088] The vector containing the nucleic acid molecule is typically a recombinant expression vector and may include an origin of replication and a selectable marker (e.g., dihydrofolate reductase for eukaryotic cell culture, or neomycin resistance, or tetracycline resistance or ampicillin resistance in E. coli, or the S. cerevisiae TRP1 gene) to enable transformation of the host cell, and a promoter controlling transcription of the protein coding sequence. Useful expression vectors include, for example, SV40; derivatives of pcDNA; known bacterial plasmids such as colE1, pCR1, pBR322, pMal-C2, pET, and pGEX (Smith, et al., Gene 67:31-40 (1988)); plasmids such as pMB9 and its derivative RP4; phage DNA, including numerous derivatives of phage I, such as NM989; phage DNA, including M13 and filamentous single-stranded phage DNA; yeast plasmids, e.g., phage DNAb, or vectors derived from combinations of plasmids and phage DNA modified to utilize expression control sequences. Mammalian expression vectors contain an origin of replication, a suitable promoter, and an enhancer. They may also contain necessary ribosome binding sites, polyadenylation sites, splice donor and splice acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences. Mammalian expression vectors include vectors containing inducible promoters, such as the dihydrofolate reductase promoter, any expression vector containing a DHFR expression cassette, or a DHFR / methotrexate coamplification vector such as pED (Randal J, Kaufman, 1991, Randal J. Kaufman, Current Protocols in Molecular Biology, 16, 12 (1991)).Alternatively, glutamine synthetase / methionine sulfoximine co-amplification vectors, such as pEE14 (Celltech), or vectors directing episomal expression under the control of Epstein-Barr virus (EBV) or EBNA, such as pREP4 (Invitrogen), pCEP4 (Invitrogen), pMEP4 (Invitrogen), pREP8 (Invitrogen), pREP9 (Invitrogen), and pEBVHis (Invitrogen), can be used. Selectable mammalian expression vectors include Rc / CMV (Invitrogen) and pRc / RSV (Invitrogen). Vaccinia virus mammalian expression vectors that can be used in the present invention include pSC11, pMJ601, and pTKgptF1S.

[0089] Yeast expression systems that can be used in the present invention include the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), and the pRS vector.

[0090] The vector can be introduced into a variety of mammalian cells, particularly human cells, as well as bacteria, yeast, fungi, insects, nematodes, and plant cells. Examples of suitable cells include VERO cells, HELA cells (e.g., ATCC No. CCL2), CHO cell lines (e.g., ATCC No. CCL61), COS cells (e.g., COS-7 cells and ATCC No. CRL1650 cells), W138, BHK, HepG2, 3T3 (e.g., ATCC No. CRL6361), A549, PC12, K562 cells, 293 cells, Sf9 cells (e.g., ATCC No. CRL1711), and Cv1 cells (e.g., ATCC No. CCL70).

[0091] Other suitable cells for use in the present invention include prokaryotic host cell strains such as Escherichia coli (e.g., strain DH5-α), Bacillus subtilis, Salmonella typhimurium, or strains belonging to the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0092] A composition according to one aspect of the present invention may contain a peptide comprising any one of SEQ ID NOs: 1 to 156, or a peptide that is a fragment thereof, or a peptide that has greater than 80% sequence identity to the peptide sequence, in an amount of 0.1 μg / mg to 1 mg / mg, specifically 1 μg / mg to 0.5 mg / mg, and more specifically 10 μg / mg to 0.1 mg / mg. This range is not only suitable for achieving the intended effects of the present invention, but also for ensuring both the stability and safety of the composition, and is also suitable from the perspective of cost-effectiveness.

[0093] The compositions according to one aspect of the present invention may be applied to all animals including humans, dogs, chickens, pigs, cows, sheep, guinea pigs or monkeys.

[0094] The pharmaceutical composition according to one aspect of the present invention may be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intraosseously, intrathecally, or subcutaneously, and the like.

[0095] Dosage forms for oral administration include, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, liquids, or emulsions.Dosage forms for parenteral administration include, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays.

[0096] The pharmaceutical composition according to one aspect of the present invention may contain, if necessary, additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavors, or sweeteners. The pharmaceutical composition according to one aspect of the present invention may be prepared by a method commonly used in the art.

[0097] The active ingredient of the pharmaceutical composition according to one aspect of the present invention varies depending on the age, sex, and weight of the recipient, the pathological condition and its severity, the route of administration, and the discretion of the prescriber. Determining the dosage based on such factors is within the skill of one of ordinary skill in the art. The daily dosage may be, for example, but is not limited to, 0.1 μg / kg / day to 1 g / kg / day, specifically 1 μg / kg / day to 10 mg / kg / day, more specifically 10 μg / kg / day to 1 mg / kg / day, and even more specifically 50 μg / kg / day to 100 μg / kg / day. The pharmaceutical composition according to one aspect of the present invention is administered once to three times daily, but is not limited thereto.

[0098] The cosmetic composition according to one aspect of the present invention may be provided in any dosage form suitable for topical application, such as a solution, an emulsion obtained by dispersing an oily phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oily phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol. Such dosage forms may be prepared by methods commonly known in the art.

[0099] The cosmetic composition according to one aspect of the present invention may contain other ingredients that can provide a synergistic effect to the main effect, as long as the ingredients do not impair the main effect. The cosmetic composition according to one aspect of the present invention may further contain a moisturizer, an emollient, a surfactant, an ultraviolet absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic or inorganic pigment, a fragrance, a cooling agent, or an antiperspirant. The amount of the ingredients can be easily determined by those skilled in the art within a range that does not impair the purpose and effects of the present invention, and the amount may be 0.01 to 5 wt %, specifically 0.01 to 3 wt %, based on the total weight of the cosmetic composition.

[0100] The dosage form of the food composition according to one aspect of the present invention is not particularly limited, and may be, for example, a tablet, granule, powder, liquid, solid preparation, etc. In addition to the active ingredient, each dosage form may be formulated by a person skilled in the art by appropriately selecting and incorporating ingredients commonly used in the art according to the dosage form or intended use, and when used simultaneously with other ingredients, a synergistic effect occurs.

[0101] Determining the dosage of the active ingredient is within the level of a person skilled in the art, and the daily dosage may be, for example, but not limited to, 1 μg / kg / day to 10 mg / kg / day, more specifically 10 μg / kg / day to 1 mg / kg / day, and even more specifically 50 μg / kg / day to 100 μg / kg / day, and may vary depending on various factors such as the age, health condition, and complications of the subject to be administered.

[0102] The terminology used herein is intended only for the purpose of describing particular embodiments and is not intended to limit the invention. The use of terms preceded by a noun without a number does not denote a limitation of quantity, but rather indicates that there are one or more of the referenced item. The terms "comprise," "have," and "contain" are to be construed as open terms (i.e., meaning "including, but not limited to").

[0103] Reciting a range of values ​​is merely an easy way of referring to each separate value falling within the range individually, and unless expressly stated otherwise, each separate value is incorporated herein as if it were individually set forth in the specification. All range endpoints are included within the range and are independently combinable.

[0104] All methods referred to herein may be performed in any suitable manner unless otherwise specified or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as"), unless otherwise included in the claims, is merely to better describe the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs.

[0105] The preferred embodiment of the present invention includes the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiment will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will take advantage of such variations and will implement the invention in ways other than as described herein. Accordingly, this invention includes equivalents and all modifications of the subject matter recited in the appended claims as permitted by the patent laws. Furthermore, any combination of the above-described elements, within all possible variations, is encompassed by this invention unless expressly stated to the contrary herein or clearly contradicted by context. While the invention has been particularly shown and described with reference to illustrative embodiments, it will be readily apparent to those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention as defined by the appended claims. [Example]

[0106] Example 1: Synthesis of peptides Any one of the peptides represented by SEQ ID NO: 1 to 156 was prepared by a conventional solid phase peptide synthesis method. Specifically, the peptide was synthesized by coupling amino acids one by one from the C-terminus via Fmoc solid phase peptide synthesis (SPPS) using an ASP48S (Peptron, Inc., Daejeon, Republic of Korea). The peptides were used with the first amino acid at the C-terminus attached to the resin as follows. For example:

[0107] NH2-Lys(Boc)-2-chloro-Trityl Resin NH2-Ala-2-chloro-Trityl Resin NH2-Arg(Pbf)-2-chloro-Trityl Resin

[0108] All amino acid starting materials used in peptide synthesis were protected at the N-terminus with Fmoc, and the residues were protected with Trt, Boc, t-butylester (t-Bu), 2,2,4,6,7-pentamethyl dihydrobenzofuran-5-sulfonyl (Pbf), etc., all of which can be removed with acid. For example:

[0109] Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, F moc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH, Trt-Mercaptoaceticacid

[0110] The coupling reagents used were HBTU [2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate], HOBt [N-Hydroxxybenzotriazole], and NMM [4-Methylmorpholine]. Fmoc removal was performed using 20% ​​piperidine in DMF. The synthesized peptide was separated from the resin and the protecting groups of the residues were removed using a cleavage cocktail [TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / HO = 92.5 / 2.5 / 2.5 / 2.5].

[0111] Each peptide was synthesized by repeatedly reacting the starting amino acids with protecting groups bound to the solid support, washing with solvent, and deprotecting the amino acids. The synthesized peptides were cleaved from the resin, purified by HPLC, and freeze-dried after confirming their integrity by MS.

[0112] Taking PEP 1 (EARPALLTSRLRFIPK) of SEQ ID NO: 164 as an example, the specific synthesis process will be described as follows.

[0113] 1) Coupling An amino acid (8 equivalents) protected by NH2-Lys(Boc)-2-chloro-Trityl Resin and coupling reagents HBTU (8 equivalents), HOBt (8 equivalents), and NMM (16 equivalents) were dissolved in DMF and added, and the mixture was reacted at room temperature for 2 hours. The mixture was then washed with DMF, MeOH, and DMF, in that order.

[0114] 2) Fmoc deprotection 20% piperidine in DMF was added, and the mixture was reacted twice for 5 minutes at room temperature, and then washed with DMF, MeOH, and DMF in that order.

[0115] 3) Reactions 1 and 2 were repeated to produce the peptide backbone NH2-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Boc)-2-chloro-Trityl Resin.

[0116] 4) Cleavage: After synthesis, a cleavage cocktail was added to the peptide resin, and the peptide was separated from the resin.

[0117] 5) Cooling diethyl ether is added to the resulting mixture, which is then centrifuged to precipitate the resulting peptide.

[0118] 6) After purification by Prep-HPLC, the molecular weight was confirmed by LC / MS, and the product was frozen and made into powder.

[0119] Example 2: Preparation of CPP-FITC conjugate (1) Preparation of FITC-CPP conjugate A conjugate of any one of peptides represented by SEQ ID NO: 1 to 156 conjugated with FITC was prepared as follows: For example, a conjugate of pep1 represented by SEQ ID NO: 157 with FITC, i.e., FITC-linker-pep1, was prepared as follows.

[0120] FITC was reacted with the peptide backbone (NH2-linker-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Boc)-2-chloro-trityl resin) obtained in Example 1. Specifically, fluorescein-5-isothiocyanate (FITC) (8 equivalents) and N,N-diisopropylethylamine (DIPEA) (16 equivalents) were dissolved in DMF and added, followed by reaction at room temperature for 2 hours. The mixture was then washed sequentially with DMF, MeOH, and DMF. The resulting product was FITC-linker-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Boc)-2-chloro-trityl resin. Here, the linker was 6-aminohexanoic acid (Ahx). TFA / TIS / HO (95 / 2.5 / 2.5) was added to the synthesized peptide resin to separate the conjugate from the resin. Cooling diethyl ether was added to the resulting mixture, followed by centrifugation to precipitate the resulting conjugate. The product was then purified by prep-HPLC, followed by analytical HPLC to confirm its purity and LC / MS to confirm its molecular weight. The resulting material was confirmed to be FITC-pep1 through molecular weight confirmation. The product was then lyophilized. Conjugates of peptides of SEQ ID NO: 1 to SEQ ID NO: 156 conjugated with FITC were also prepared, along with pep1 of SEQ ID NO: 157.

[0121] (2) Preparation of CPP-FITC conjugate As in Example 1, the peptide backbone (NH-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Dde)-2-chloro-trityl resin) was prepared. To selectively introduce FITC into the C-terminus of the prepared peptide backbone, the N-terminus was protected with Boc. Then, a solution of di-tert-butyl dicarbonate (30 equivalents) and DIPEA (30 equivalents) in DMF was added, followed by reaction at room temperature for 2 hours. The resulting mixture was washed sequentially with DMF, MeOH, and DMF to yield Boc-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Dde)-2-chloro-trityl resin. To attach FITC to the C-terminus K residue, the protecting group Dde at the C-terminus Lys residue was removed by treatment with hydrazine in 2% DMF. This resulted in Boc-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(NH2)-2-chloro-trityl resin. FITC (8 equiv.) and DIPEA (16 equiv.) were then dissolved in DMF and added. The reaction mixture was incubated at room temperature for 2 hours and washed sequentially with DMF, MeOH, and DMF. This resulted in Boc-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(FITC)-2-chloro-trityl resin. After the synthesis was completed, TFA / TIS / HO (95 / 2.5 / 2.5) was added to the peptide resin to separate the peptide from the resin. Cooling diethyl ether was added to the resulting mixture, which was then centrifuged to precipitate the resulting peptide. The peptide was then purified by prep-HPLC, and its purity was confirmed by analytical HPLC, and its molecular weight was confirmed by LC / MS. The resulting substance was confirmed to be pep1-FITC. It was then dried. Peptide-FITC conjugates of SEQ ID NOs: 1 to 156 were also prepared in the same manner as for pep1-FITC.

[0122] Example 3: Cell penetration experiment of pep-FITC conjugate (1) Cell penetration test using HeLa cell line Cell line culture HeLa cells obtained from ATCC were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (Invitrogen, USA), Earle's salts, non-essential amino acids, sodium pyruvate, 100 μg / ml penicillin, and 100 units / ml streptomycin in a 5% CO2 incubator at 37°C.

[0123] Flow cytometry The cell line was treated with the peptides of the present invention, pep (CPP), represented by SEQ ID NO: 1 to SEQ ID NO: 156, and flow cytometry analysis was performed to compare the uptake levels with those of the control.

[0124] Cell lines were plated into 6-well plates and cultured in a medium supplemented with 10% fetal bovine serum (Invitrogen, USA), 100 μg / ml penicillin, and 100 units / ml streptomycin at 37°C in a 5% CO2 incubator for 12 hours. After washing with PBS, the cells were starved in MEM (minimum essential medium) for 1 hour. 20 μM of each delivery peptide was treated and cultured at 37°C for 1 hour. The cells were washed three times with PBS and treated with trypsin-EDTA at 37°C for 10 minutes to remove delivery peptides attached to the extracellular surface. After harvesting the cells with chilled PBS, the cells were washed three times via centrifugation. The cells were then suspended in 0.5 ml of 4% paraformaldehyde in PBS and analyzed for fluorescence using a FACS Calibur (Becton Dickinson). The cellular uptake patterns of cells not treated with the delivery peptide (control group) and various FITC-conjugated peptides were compared and analyzed using MFI (mean fluorescence intensity).

[0125] The results are shown in Figures 1 to 23. The analysis results shown in Figures 1 to 23 are detailed in Table 3 below.

[0126] [Table 6]

[0127] [Table 7]

[0128] (2) Cell penetration test in Huh7 cell line Cell line culture We used a suspension cell line of human hepatocellular carcinoma (Huh7) (purchased from ATCC (American Type Cell Culture)). The Huh7 cell line uses RPMI 1640 medium. The medium was supplemented with 10% fetal bovine serum (Invitrogen, USA), 100 μg / ml penicillin, and 100 units / ml streptomycin, and the cells were cultured at 37°C in a 5% CO2 incubator.

[0129] Cell permeability screening via flow cytometry To examine the cell penetration ability of the peptides, flow cytometry was performed on Huh7 cell lines treated with peptides of SEQ ID NO: 1 to SEQ ID NO: 156. The analytical method was as described in the Hela cell line analysis in (1) above. The analytical results are shown in Figures 24 to 43.

[0130] (3) Cell penetration test using human T lymphocyte cell lines Cell line culture Suspension cells of the human T-cell leukemia cell line Jurkat (purchased from ATCC (American Type Cell Culture)) were used. Jurkat cells were grown in RPMI 1640 medium. The medium was supplemented with 10% fetal bovine serum (Invitrogen, USA), 100 μg / ml penicillin, and 100 units / ml streptomycin, and cultured at 37°C in a 5% CO2 incubator.

[0131] Human lymphocytes were isolated by collecting 50 ml of blood from a healthy individual, and then using Biocoll Separating Solution (Biochrom AG, Berlin, Germany), the PBMC (peripheral blood mononuclear cells) layer and lymphocytes were collected.

[0132] Cell penetration analysis of human T lymphocyte cell lines To examine the cell-penetrating ability of the 185 peptide, flow cytometry was performed on a human T lymphocyte cell line treated with peptides of SEQ ID NO: 1 to SEQ ID NO: 156. The analytical method was as described in the HeLa cell line analysis in (1) above. The analytical results are shown in Figures 44 to 58.

[0133] (4) Cell viability and toxicity analysis Meanwhile, the HeLa cell line cultured in Example 3(1) was dispensed into a 96-well plate and cultured in a medium supplemented with 10% fetal bovine serum (Invitrogen, USA), 100 μg / ml penicillin, and 100 units / ml streptomycin in a 5% CO2 incubator at 37°C for 12 hours. After washing with PBS, the cells were starved in MEM (minimum essential medium) for 1 hour. After treatment with 20 μM of each delivery peptide and culturing at 37°C for 24 hours, cell viability and toxicity were analyzed using an MTT assay. The results are shown in Figures 59 to 72.

Claims

1. A composition for intracellular delivery of an active ingredient, comprising: The present invention comprises a conjugate of a cell-permeable delivery peptide and an active ingredient, The delivery peptide is (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 66, or (2) A peptide having a sequence homology of more than 90% with the peptide of (1) above and maintaining the cell permeability of the peptide of (1) above. That is, The composition.

2. The composition according to claim 1 , wherein the active ingredient is one or more selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, nanoparticles, biological agents, contrast agents, drugs, and chemical substances.

3. The composition of claim 1 , wherein the delivery peptide and the active ingredient are linked by a covalent bond, optionally mediated by a linker.

4. The composition of claim 1 , wherein the delivery peptide and the active ingredient are linked by a non-covalent bond.

5. The composition of claim 2 , wherein the active ingredient is a protein or peptide.

6. The composition of claim 2 , wherein the active ingredient is a cytokine, an antibody, an antibody fragment, a therapeutic enzyme, a soluble receptor, or a ligand.

7. 3. The composition of claim 2, wherein the contrast agent is selected from the group consisting of a radiopaque contrast agent, a paramagnetic contrast agent, a superparamagnetic contrast agent, and a CT contrast agent.

8. The composition of claim 2 , wherein the contrast agent is iron-based.

9. The composition of claim 8 , wherein the imaging agent is ferrocenecarboxylic acid.

10. A contrast agent comprising the composition of any one of claims 1 to 9.

11. The contrast agent of claim 10 for imaging cells.

12. The imaging agent of claim 11 , wherein the cells are stem cells.

13. The composition according to any one of claims 1 to 9, wherein the active ingredient is an active ingredient for treating or preventing a disease, and the composition is a pharmaceutical composition.

14. The composition according to any one of claims 1 to 9, wherein the active ingredient is an active ingredient of a functional cosmetic, and the composition is a cosmetic composition.

15. The composition according to any one of claims 1 to 9, wherein the active ingredient is an active ingredient of a functional health food, and the composition is a health food composition.

16. A cell-penetrating delivery peptide, the delivery peptide comprising: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 66, or (2) A peptide having a sequence homology of more than 90% with the peptide of (1) above and maintaining the cell permeability of the peptide of (1) above. That is, The cell-permeable delivery peptide.

17. A polynucleotide encoding the peptide of claim 16.

18. A vector comprising the polynucleotide of claim 17.

19. A transformed cell comprising the vector of claim 18.

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