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WO2023008683A1 - Peptide amphipathique de pénétration cellulaire et son utilisation - Google Patents

Peptide amphipathique de pénétration cellulaire et son utilisation Download PDF

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Publication number
WO2023008683A1
WO2023008683A1 PCT/KR2022/004938 KR2022004938W WO2023008683A1 WO 2023008683 A1 WO2023008683 A1 WO 2023008683A1 KR 2022004938 W KR2022004938 W KR 2022004938W WO 2023008683 A1 WO2023008683 A1 WO 2023008683A1
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Prior art keywords
cell
peptide
cells
biologically active
proteins
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English (en)
Korean (ko)
Inventor
오창규
이재호
박순익
박정호
박원진
조윤주
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Celltroy Co Ltd
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Celltroy Co Ltd
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Priority claimed from KR1020220037451A external-priority patent/KR102584294B1/ko
Application filed by Celltroy Co Ltd filed Critical Celltroy Co Ltd
Publication of WO2023008683A1 publication Critical patent/WO2023008683A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to an intracellular delivery technology for delivering biologically active molecules into cells, and specifically, to novel amphiphilic cell-penetrating peptides with improved cell permeability compared to existing cell-penetrating peptides, and uses thereof. .
  • the cell membrane is a barrier to the permeation of proteins, nucleic acids, peptides, and poorly soluble compounds into cells due to its unique impermeability. Intracellular delivery of drugs is a problem that must be solved in the fields of disease treatment and prevention, diagnosis, and the like.
  • methods for permeating biologically active macromolecules into cell membranes include electroporation, membrane fusion using liposomes, transfection using cationic polymers such as polyethyleneimine (PEI), DEAE-dextran, and viral nucleic acid infection. , there are single cell microinjection methods.
  • CPP cell penetrating peptide
  • Cell-penetrating peptides are peptides generally composed of 5 to 30 amino acids and have a function of transporting biologically active molecules such as liposomes, nucleic acids, proteins, and compounds into cells without a specific receptor.
  • Cell-penetrating peptides were first known in 1988 when TAT, a transcription factor of HIV (human immunodeficiency virus), was confirmed to be transmitted into cells. Since then, various cell-penetrating peptides such as penetratin, VP22, and MTS have been developed.
  • the present invention was made to solve the above problems in the prior art, and provides a novel cell-permeable peptide capable of effectively transporting biologically active molecules into cells through cell membranes even when bound to biologically active molecules, and uses thereof, etc. for that purpose
  • the present invention is a cell-permeable peptide consisting of an amino acid sequence represented by the following general formula,
  • A' does not exist or 1 to 8 amino acids are linked
  • B' is absent or 1 to 15 amino acids are linked
  • the peptide provides a cell-permeable peptide consisting of 7 to 25 amino acids.
  • the cell-penetrating peptide may have an alpha-helix structure, but is not limited thereto.
  • the cell-permeable peptide may be an amphipathic peptide, but is not limited thereto.
  • the 1 to 8 amino acids constituting A' may each independently be lysine, alanine, isoleucine, or tyrosine, but is not limited thereto.
  • the 1 to 15 amino acids constituting B' may each independently be lysine, leucine, or isoleucine, but is not limited thereto.
  • the cell-penetrating peptide may consist of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, but is not limited thereto.
  • the cell-penetrating peptide may mediate transport of biologically active molecules bound thereto into cells, but is not limited thereto.
  • the cells are blood-brain barrier endothelial cells, cancer cells, blood cells, epidermal cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells It may be at least one selected from the group consisting of cells, macrophages, microglia, neurons, glial cells, astrocytes, and muscle cells, but is not limited thereto.
  • the present invention provides a polynucleotide encoding the cell-penetrating peptide according to the present invention.
  • the present invention provides a complex comprising the cell-penetrating peptide and the biologically active molecule according to the present invention.
  • the cell-penetrating peptide may be bound to one end or both ends of the biologically active molecule, but is not limited thereto.
  • the complex may be a cell-penetrating peptide of 1 to 5 linked to one end or both ends of the biologically active molecule, but is not limited thereto.
  • the bond may be a chemical bond, a bond using a linker, or a peptide bond, but is not limited thereto.
  • the chemical bond is a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond, a diselenide bond, a maleimide bond, a thioester bond, and a thioether bond. It may be one or more selected from the group consisting of, but is not limited thereto.
  • the biologically active molecule is a peptide, protein, glycoprotein, nucleic acid, carbohydrate, lipid, glycolipid, compound, natural product, semi-synthetic drug, microparticle, nanoparticle, liposome, It may be at least one selected from the group consisting of viruses, quantum dots, fluorochromes, and toxins, but is not limited thereto.
  • the protein is a growth factor, enzyme, nuclease, transcription factor, antigenic peptide, antibody, antibody fragment, hormone, carrier protein, immunoglobulin, structural protein, motor function protein, receptor, Signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secreted proteins, viral proteins, protein complexes, chemically modified proteins, and prions ), but may be one or more selected from the group consisting of, but is not limited thereto.
  • the nucleic acid is DNA, RNA, ASO (Antisense oligonucleotide), microRNA (miRNA), small interfering RNA (siRNA), aptamer (aptamer), LNA (locked nucleic acid), peptide nucleic acid (PNA), and morpholino, but may be one or more selected from the group consisting of, but is not limited thereto.
  • ASO Antisense oligonucleotide
  • miRNA microRNA
  • siRNA small interfering RNA
  • aptamer aptamer
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • morpholino but may be one or more selected from the group consisting of, but is not limited thereto.
  • the compound may be at least one selected from the group consisting of therapeutic drugs, toxic compounds, and chemical compounds, but is not limited thereto.
  • the present invention provides a composition for delivery of a biologically active molecule comprising the cell-penetrating peptide or the complex as an active ingredient.
  • the present invention also provides a method of delivering a biologically active molecule into a subject or cell comprising administering a complex according to the present invention to a subject or cell in need thereof,
  • the present invention provides the use of the cell-penetrating peptide for delivering biologically active molecules into cells.
  • the present invention provides the use of the cell-penetrating peptide for preparing a drug for delivery of a biologically active molecule.
  • the present invention provides a use of the cell-permeable peptide for preparing a drug having improved cell-permeability.
  • the cell-permeable peptide according to the present invention is a novel cell-permeable peptide capable of effectively transporting biologically active molecules into cells through cell membranes even when biologically active molecules are bound thereto.
  • the cell permeability that can be transported into the cell is remarkably excellent, and the transported biologically active molecules can effectively maintain their activity in the cell. Therefore, the cell-permeable peptide of the present invention can be very usefully used in various fields such as beauty, diagnosis, drug delivery system, recombinant protein vaccine, DNA/RNA therapy, and gene and protein therapy.
  • FIG. 1a and 1b show the result of comparing the cell permeability of a novel amphiphilic cell penetrating peptide according to an embodiment of the present invention and a control group.
  • Figure 1a shows the result of performing flow cytometry
  • Figure 1b shows the result of quantification.
  • Figures 2a and 2b show the results of confirming the cell penetration efficiency of the Amp.96 peptide according to an embodiment of the present invention using human epidermal cells.
  • Figure 2a shows the result of performing flow cytometry
  • Figure 2b shows the result of quantification thereof.
  • Figures 3a and 3b show the results of confirming the cell penetration efficiency of the Amp.96 peptide according to an embodiment of the present invention using a human neuroblastoma cell line.
  • Figure 3a shows the result of performing flow cytometry
  • Figure 3b shows the result of quantification thereof.
  • Figure 4 shows the results of confirming using a fluorescence microscope whether the Amp.96 peptide according to an embodiment of the present invention substantially delivers biologically active molecules attached to the peptide into cells.
  • FIG. 5a and 5b show the results of comparison of cell penetration efficiency in human epidermal cells by preparing various candidate peptides to confirm the minimum active sequence for the cell penetration ability of the Amp.96 peptide.
  • Figure 5a shows the result of performing flow cytometry
  • Figure 5b shows the result of quantifying it.
  • FIG. 6a and 6b show the result of confirming the cell penetration efficiency of the candidate peptides using a human neuroblastoma cell line.
  • Figure 6a shows the result of performing flow cytometry
  • Figure 6b shows the result of quantification.
  • FIG. 7 shows the result of comparing the delivery effect of biologically active molecules of the candidate peptides into cells using a fluorescence microscope.
  • An object of the present invention is to provide a novel cell-penetrating peptide capable of effectively transporting a biologically active molecule into a cell through a cell membrane even when bound to the biologically active molecule, and uses thereof.
  • known human and viral proteins are screened as a whole, and cell permeability of peptides satisfying specific conditions such as length and property is confirmed, resulting in higher cell permeability than previously known cell penetrating peptides
  • a novel peptide (Amp.96) having the same was selected (Example 1).
  • candidate peptides in which a part of the Amp.96 peptide is deleted are prepared, and then their As a result of comparing cell penetrability and biologically active molecule delivery efficiency, it was confirmed that the 7 amino acid sequence inside Amp.96 is the least active sequence of Amp.96 (Example 4).
  • the novel cell-permeable peptide according to the present invention is composed of at least 7 amino acids and can effectively deliver biologically active molecules into cells, so it is expected to be usefully used in various industrial fields such as beauty, diagnosis, vaccine, and treatment.
  • the first letter (three letter) of an amino acid refers to the following amino acids according to standard abbreviation conventions in the field of biochemistry: A (Ala), alanine; C (Cys), cysteine; D (Asp), aspartic acid; E (Glu), glutamic acid; F (Phe), phenylalanine; G (Gly), glycine; H (His), histidine; I (IIe), isoleucine; K (Lys), lysine; L (Leu), leucine; M (Met), methionine; N (Asn), asparagine; O (Ply), pyrrolysine; P (Pro), proline; Q (Gln), glutamine; R (Arg), arginine; S (Ser), serine; T (Thr), threonine; U (Sec), selenocysteine; V (Val), valine; W (Trp), tryptophan; and Y (Ty (Ty
  • the present invention is a cell-permeable peptide consisting of an amino acid sequence represented by the following general formula,
  • A' does not exist or 1 to 8 amino acids are linked
  • B' is absent or 1 to 15 amino acids are linked
  • the peptide provides a cell-permeable peptide consisting of 7 to 25 amino acids.
  • cell permeability means the ability or property of a substance to permeate a cell (membrane) and penetrate into the cell.
  • cell membrane refers to a lipid-containing barrier that separates a cell or cell population from the extracellular space.
  • Cell membranes include, but are not limited to, plasma membranes, cell walls, organelle membranes such as mitochondrial membranes, nuclear membranes, and the like.
  • a “peptide” is a polymer of amino acids, and a form in which a small number of amino acids are linked is usually called a peptide, and a form in which many amino acids are linked is called a protein.
  • the linkage between amino acids in a peptide or protein structure is composed of an amide bond or a peptide bond.
  • a peptide bond refers to a bond between a carboxyl group (-COOH) and an amino group (-NH 2 ) in which water (H 2 O) escapes and forms a -CO-NH- form.
  • cell penetrating peptide is a peptide having cell penetrability, and means a peptide having the ability to deliver a cargo into cells in vitro and/or in vivo . .
  • “cargo” includes all substances that can be transported into cells by binding to cell-penetrating peptides, and may include all types of biologically active molecules.
  • the transport target is, for example, any substance desired to increase cell permeation efficiency, specifically an effective substance for drugs, cosmetics or health food, more specifically a substance that is not easily moved into cells through a general route, and more specifically Examples include proteins, nucleic acids, peptides, minerals, sugars such as glucose, nanoparticles, biological agents, viruses, contrast materials, or other chemical substances, but are not limited thereto.
  • the cell-penetrating peptide according to the present invention is characterized by comprising the amino acid sequence of SEQ ID NO: 1 (KIITILI).
  • the cell-penetrating peptide according to the present invention is characterized in that it consists of the amino acid sequence of KIITILI.
  • amino acid sequences throughout this specification are written in the direction from the N- to the C-terminus.
  • the amino acid sequence of SEQ ID NO: 1 is the least active sequence of the cell-permeable peptide according to the present invention, and in a specific embodiment, the present inventors found that the peptide containing the amino acid sequence of SEQ ID NO: 1 has superior cell-penetrating ability compared to conventional cell-permeable peptides. And it was confirmed that it has the intracellular delivery ability of the substance.
  • the cell-permeable peptide according to the present invention may further include various amino acids at the N-terminus and/or C-terminus of the peptide having the amino acid sequence of SEQ ID NO: 1.
  • the cell-permeable peptide may further include various amino acids (preferably, A' peptide) at the N-terminus of the peptide consisting of the amino acid sequence of SEQ ID NO: 2.
  • the cell-permeable peptide may further include various amino acids (preferably, B' peptide) at the C-terminus of the peptide consisting of the amino acid sequence of SEQ ID NO: 3.
  • the cell-penetrating peptide may be characterized in that it contains lysine at one end or both ends.
  • A' of the general formula may not exist or may be additionally linked with 1 to 8 amino acids. That is, the cell-permeable peptide according to the present invention may be one in which 1 to 8 amino acids are additionally bound to the N-terminus of the amino acid sequence represented by SEQ ID NO: 1.
  • A' in the above general formula does not exist or is 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 amino acids may be connected.
  • B' of the general formula may not exist or may be additionally linked with 1 to 15 amino acids. That is, the cell-permeable peptide according to the present invention may be one in which 1 to 15 amino acids are additionally bound to the C-terminus of the amino acid sequence represented by SEQ ID NO: 1.
  • B' of the above general formula does not exist or is 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, or 2 to 7 amino acids may be linked.
  • the cell-penetrating peptide according to the present invention may consist of 7 to 25 amino acids as a whole. That is, 7 to 25, 7 to 24, 7 to 23, 7 to 22, 7 to 21, 7 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 9 to 24, 9 to 22, 9 to 20, 9 to 19, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 11 to 24, 11 to 22, 11 to 20, 11 to 19, 11 to 18, 11 to 17, 11 to 16, 11 to 15, 11 to 14, Or it may consist of 11 to 13 amino acids.
  • cell-penetrating peptide according to the present invention may have an alpha-helix structure.
  • cell penetrating peptide according to the present invention may be an amphipathic peptide.
  • an amphiphilic peptide refers to a peptide having both polar and non-polar characteristics including a hydrophilic moiety and a hydrophobic moiety.
  • Derivatives and analogues (mimetics, or peptidomimetics) of the cell-penetrating peptide are included within the scope of the present invention.
  • “derivative” refers to a generic term for similar peptides obtained by changing a part of the cell-penetrating peptide composed of the amino acid sequence represented by SEQ ID NO: 1 of the present invention, and preferably one or more amino acids are different from each other. It may be substituted with an amino acid, added with one or more amino acids, deleted with one or more amino acids, or fused with a compound that increases the half-life of the peptide (eg, polyethylene glycol, etc.). In the present invention, the derivative may maintain, increase, or decrease the function or characteristic (eg, cell permeability) of the cell-permeable peptide according to the present invention.
  • the derivative may have one or more amino acid substitutions while maintaining properties as a cell penetrating peptide according to the present invention.
  • amino acid substitutions may generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
  • glycine, alanine, valine, leucine, and isoleucine having aliphatic side chains may be substituted for each other; Glycine and alanine, which have relatively short side chains, may be substituted for each other; Valine, leucine, and isoleucine, which have larger aliphatic side chains that are hydrophobic, may be substituted for each other; Phenylalanine, tyrosine, and tryptophan having aromatic side chains may be substituted for each other; Lysine, arginine, and histidine having basic side chains may be substituted for each other; Aspartate and glutamate having acidic side chains may be substituted for each other; Cysteine and methionine having sulfur-containing side chains may be substituted for each other.
  • 1 to 8 amino acids constituting A' in the general formula may each independently be lysine, arginine, histidine, alanine, glycine, valine, leucine, isoleucine, tyrosine, phenylalanine, or tryptophan, and most preferably Each may independently be lysine, alanine, isoleucine, or tyrosine.
  • A' does not exist or 1 to 4 amino acids are linked, and the 1 to 4 amino acids are each independently lysine, alanine, isoleucine, or tyrosine, but A' is It may be characterized in that it does not contain two or more identical amino acids.
  • A' is composed of 4 amino acids and includes all of lysine, alanine, isoleucine, and tyrosine; Consisting of three amino acids, including all of alanine, isoleucine, and tyrosine; consisting of two amino acids, including airoleucine and tyrosine; Or it may consist of only any one amino acid of lysine, alanine, isoleucine, and tyrosine. Most preferably, A' may be selected from the group consisting of KAIY, AIY, IY, and Y.
  • 1 to 15 amino acids constituting B' in the general formula are each independently isoleucine, valine, leucine, lysine, arginine, histidine, leucine, glycine, valine, alanine, threonine, tyrosine, phenylalanine, or tryptophanyl It may be, and most preferably each independently may be lysine, leucine, or isoleucine.
  • B' in the general formula does not exist or 1 to 7 amino acids are linked, and the 1 to 7 amino acids are each independently lysine, leucine, or isoleucine, but all amino acids constituting B'
  • the ratio of lysine to lysine may be 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55%. More preferably, the ratio of leucine to all amino acids constituting B' may be 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, or 20% to 35%. .
  • B' may be composed of 7 amino acids and include all of lysine, leucine, and isoleucine
  • B' may be composed of 7 amino acids and include all of lysine, leucine, and isoleucine. However, it may contain 4 lysines, and more preferably may contain 2 leucines.
  • B' may be composed of 5 amino acids, including lysine and leucine, but including 3 lysines.
  • B' may be composed of two amino acids and include lysine and leucine.
  • B' may be selected from the group consisting of KLKKLIK, KLKKLI, KLKKL, KLKK, KLK, KL, and K.
  • the cell-permeable peptide according to the present invention comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or more preferably any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12 It may consist of, but is not limited thereto, and variants of the amino acid sequence are included within the scope of the present invention. That is, the cell-penetrating peptide according to the present invention is a functional equivalent of the polypeptide constituting it, for example, although some amino acid sequences of the polypeptide have been modified by deletion, substitution or insertion, It is a concept including variants capable of functionally the same action as the polypeptide.
  • the cell-permeable peptide according to the present invention is 70% or more, more preferably 80% or more, even more preferably 90% or more, most of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12
  • it may include an amino acid sequence having 95% or more sequence homology.
  • the “percentage of sequence homology” for a polypeptide is determined by comparing two optimally aligned sequences with a region of comparison, wherein a portion of the sequence of the polypeptide in the region of comparison is a reference sequence (additional or may include additions or deletions (i.e., gaps) compared to not including deletions).
  • the cell-penetrating peptide according to the present invention itself has cell-penetrating ability, so it can be used as a delivery system that can effectively introduce any substance bound to the peptide into cells.
  • the cell is not limited to a specific type, but for example, blood-brain barrier endothelial cells, cancer cells, blood cells, epidermal cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells, macrophages, microglia, neurons, glial cells, astrocytes, and muscle cells.
  • blood-brain barrier endothelial cells cancer cells, blood cells, epidermal cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells, macrophages, microglia, neurons, glial cells, astrocytes, and muscle cells.
  • the "cancer” means or describes a physiological state characterized by unregulated cell growth in mammals, which rapidly grows while infiltrating surrounding tissues and spreads to various parts of the body. It may include, without limitation, a malignant tumor that has become or metastasized and is life-threatening. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy.
  • cancer More specific examples include breast cancer, chronic myelogenous leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, lung cancer, stomach cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, cervical cancer, ovarian cancer Cancer, colorectal cancer, small intestine cancer, rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, liver cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer , prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal pelvic cancer, hematological cancer, brain cancer, central nervous system tumor, spinal cord tumor, brainstem glioma, neuroblasto
  • the present invention provides a polynucleotide encoding the cell-penetrating peptide according to the present invention and a recombinant vector containing the polynucleotide.
  • the polynucleotide may include or consist of any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14 to 37, but is not limited thereto. That is, the above sequence is only one preferred embodiment of the present invention, and the polynucleotide according to the present invention can be included without limitation as long as it can produce the cell-permeable peptide according to the present invention as a result through the process of transcription and translation. .
  • the polynucleotide may be in the form of RNA or DNA, and the DNA includes cDNA and synthetic DNA.
  • DNA can be single-stranded or double-stranded. If single-stranded, it may be the coding strand or the non-coding (antisense) strand, wherein the coding sequence encodes the same polypeptide as a result of degeneracy or redundancy of the genetic code.
  • the polynucleotides of the present invention may also include variants of the polynucleotides described above, which variants of the polynucleotides are naturally occurring allelic variants of the polynucleotide or non-naturally occurring variants of the polynucleotide.
  • can be An allelic variant is an alternating form of a polynucleotide sequence that may have a substitution, deletion, or addition of one or more nucleotides that does not substantially alter the function of the polynucleotide being encoded (encoding). It is well known in the art that a single amino acid can be encoded by more than one nucleotide codon and that the polynucleotide can be readily modified to produce alternating polynucleotides encoding the same peptide.
  • the polynucleotide according to the present invention is 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably, any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14 to 37 Preferably, it may include nucleic acid sequences having 95% or more sequence homology.
  • the present invention provides a complex comprising the cell-penetrating peptide and the biologically active molecule according to the present invention.
  • the present invention provides a method for producing a biologically active molecule having cell permeability, comprising the step of attaching (binding) the cell permeable peptide according to the present invention to the biologically active molecule.
  • biologically active molecules preferably collectively refer to substances having biological or pharmacological activity, which penetrate into cells (cytoplasm or nucleus) to be involved in physiological activity regulation or to express pharmacological effects. It means a substance that has biological activity in various parts of the body such as cells, tissues, interstitial cells, blood, etc. that are present or transported to act. Such biologically active molecules may be used interchangeably with the term "macromolecule”.
  • the biologically active molecules are peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, fluorescence It may be selected from the group consisting of a fluorochrome, a toxin, and a complex thereof.
  • Non-limiting examples of the protein include growth factors, enzymes, nucleases, transcription factors, antigenic peptides, antibodies (eg, monoclonal, chimeric, humanized antibodies, etc.), antibody fragments, hormones, Transport proteins, immunoglobulins, structural proteins, motor function proteins, receptors, signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secreted proteins, viruses Included are proteins, protein complexes, chemically modified proteins, and prions, and the like.
  • the nucleases include CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1 (CxxCfinger protein-1), ZEN (zinc-finger nucleases), and TALEN (Transcription activator-like effector nuclease). included
  • Non-limiting examples of the nucleic acid include DNA, RNA, ASO (Antisense oligonucleotide), microRNA (miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid) , PNA (peptide nucleic acid), and morpholino, and the like.
  • ASO Antisense oligonucleotide
  • miRNA microRNA
  • siRNA small interfering RNA
  • aptamer aptamer
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • morpholino and the like.
  • Non-limiting examples of such compounds include therapeutic drugs, toxic compounds, chemical compounds, and the like.
  • the "drug” is a broad concept that includes substances for alleviating, preventing, treating, or diagnosing a disease, injury, or specific symptom. That is, the cell-penetrating peptide according to the present invention can be used as a drug delivery system for preventing or treating diseases.
  • the biologically active molecules of the present invention include cholesterol, chemotherapeutics, vitamins, co-factors, 2,5-A chimeras, allozymes, aptamers, , molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of polymers such as polyamines, polyamides, polyethylene glycols, and polyethers.
  • Complexes according to the present invention include those formed by simply mixing a peptide and a substance, those formed by mixing a peptide and a substance, or those formed by linking or conjugating these by chemical bonds or the like.
  • the complex may be connected by physical bond, chemical bond, covalent bond, non-covalent bond, peptide bond, or self-assembly, or may be connected in an integrated or fused form using a mediator (eg, linker).
  • the chemical bond is selected from the group consisting of a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond, a diselenide bond, a maleimide bond, a thioester bond, and a thioether bond.
  • a disulfide bond a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond, a diselenide bond, a maleimide bond, a thioester bond, and a thioether bond.
  • the cell-penetrating peptide according to the present invention may be bound to one end or both ends of the biologically active molecule.
  • the complex may be a single or a plurality of cell-penetrating peptides bound to one end or both ends of the biologically active molecule. That is, the complex is 1 to 5, 1 to 4, 2 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, or 3 to 4 Cell penetrating peptides may be linked to one end or both ends of the biologically active molecule.
  • the complex may be a complex produced by expressing the peptide and the biologically active material in a fused state.
  • a gene encoding the peptide and a gene expressing a biologically active substance are inserted into one vector, and then an organism is transformed with the vector to express the gene inserted into the vector, the peptide and the biological activity Substances can be expressed as fusion proteins.
  • an optional linker may be incorporated between the peptide and the biologically active substance.
  • the present invention provides a composition for delivery of biologically active molecules comprising the complex according to the present invention as an active ingredient.
  • the composition includes a pharmaceutical composition, food composition, health functional food composition, cosmetic composition, and / or feed composition.
  • composition according to the present invention may contain, as an active ingredient, a complex comprising a pharmaceutically, food-wise, or veterinarily-acceptable salt of the cell-penetrating peptide and a biologically active molecule. That is, the scope of the cell-penetrating peptide of the present invention may include all isomers, hydrates and solvates that can be prepared by conventional methods as well as pharmaceutically/food/veterinarily acceptable salts.
  • acids examples include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and the like.
  • Acid addition salts can be prepared by conventional methods, for example, by dissolving a compound in an aqueous solution of excess acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can also be prepared by heating equimolar amounts of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or suction filtering the precipitated salt.
  • a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile.
  • Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • An alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate.
  • the metal salt it is particularly suitable for pharmaceutical purposes to prepare a sodium, potassium or calcium salt, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).
  • a suitable silver salt eg, silver nitrate
  • the scope of the peptide of the present invention may include not only pharmaceutically acceptable salts, but also all isomers, hydrates and solvates that can be prepared by conventional methods.
  • the content of the peptide or the complex in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition. It may be % by weight, but is not limited thereto.
  • the content ratio is a value based on the dry amount after removing the solvent.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • the excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.
  • compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods.
  • tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate It can be formulated and used in the form of an external agent such as a warning agent, lotion, pasta agent, spray, inhalant, patch, sterile injection solution, or aerosol, and the external agent is a cream, gel, patch, spray, ointment, warning agent , lotion, liniment, pasta, or cataplasma may have formulations such as the like.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.
  • the pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.
  • “individual” means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. of mammals.
  • administration means providing a given composition of the present invention to a subject by any suitable method.
  • prevention refers to any action that suppresses or delays the onset of a desired disease
  • treatment means that the desired disease and its resulting metabolic abnormality are improved or improved by administration of the pharmaceutical composition according to the present invention. All actions that are advantageously altered are meant, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administration of the composition according to the present invention.
  • the complex of the present invention When the complex of the present invention is used as a food additive, the complex may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).
  • the composite of the present invention when preparing food or beverage, the composite of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material.
  • the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
  • Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in a conventional sense.
  • the formulation of the cosmetic composition according to the present invention is skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, mist, moisture cream, hand cream, hand lotion, foundation, It may be in the form of essence, nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, cleansing oil, cleansing balm, body lotion, or body cleanser.
  • the cosmetic composition of the present invention may further include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, and sphingolipids.
  • CPPs novel amphipathic cell penetrating peptides
  • all human (homo sapiens) and virus proteins registered in UniProt were searched, and a total of 494,889 protein sequences were obtained.
  • the obtained protein sequences were extracted so that the length (window size) was 18mer, and among the obtained 18mer peptides, peptides having an alpha-helix structure were primarily selected.
  • the cell permeation activity of the firstly selected peptide sequences was measured using the C3pred program (iGEM Tuebingen), and the top 1,000 peptides were secondarily selected.
  • the secondly selected 1,000 peptides were displayed by Helical Wheel Projection using Helixvis (HELIQUEST server), and 15 peptide sequences having amphiphilicity were selected.
  • Peptides were synthesized based on the selected 15 peptide sequences.
  • the synthesis of peptides was prepared by coupling one by one from the C-terminus using the generally known Fmoc SPPS (Solid Phase Peptide Synthesis) method.
  • Fmoc SPPS Solid Phase Peptide Synthesis
  • FITC Fluorescein isothiocyanate
  • iSpyBio one of the biologically active molecules, to the peptide
  • lysine K, Lysine
  • FITC was bound to the free-amine residue of lysine.
  • the FITC-conjugated peptides were purified using HPLC, and the molecular weight of the peptides was confirmed using a mass spectrometer, and then lyophilized and stored frozen until use.
  • the peptides were prepared at concentrations of 0.125, 0.25, 0.5, and 1 ⁇ M, respectively, and the HaCAT cell line, a human epidermal cell line, was treated with each peptide. More specifically, HaCAT cells were dispensed to 1 ⁇ 10 7 cells/well in a 24-well plate, and DMEM medium supplemented with 10% fetal bovine serum (FBS) was maintained at 37° C. and 5% CO 2 conditions. After incubation for 24 hours, each peptide was treated and incubated for another hour. As a control group, FITC-conjugated TAT was used.
  • FBS fetal bovine serum
  • the cells were transferred to a sterile tube using 0.25% trypsin, and the medium was removed by centrifugation. Then, the medium was completely removed by washing twice with 500 ⁇ L of D-PBS (Dulbecco's phosphate-buffered saline), and finally resuspended using 400 ⁇ L of D-PBS, followed by flow cytometry (FACS machine, BD science). Intracellular fluorescence was measured using FACSCanto II). The results are shown in Figures 1a and 1b.
  • D-PBS Dynabecco's phosphate-buffered saline
  • the Amp.96 peptide showed increased cell permeability compared to the control, TAT. More specifically, even when the Amp.96 peptide was treated at the lowest concentration of 0.125 ⁇ M, cell permeability was increased by more than 2 times compared to TAT, and at 1 ⁇ M, cell permeability was increased by about 20 times compared to TAT. Confirmed.
  • experiments were conducted using human epidermal cells. More specifically, HaCAT cells, a human epidermal cell line, were dispensed to 1 ⁇ 10 7 cells/well in a 24-well plate, and cultured in DMEM medium supplemented with 10% fetal calf serum at 37°C and 5% CO 2 for 24 hours. cultured for a while. Then, FITC-coupled Amp.96 peptide and FITC-coupled TAT were treated with 4 ⁇ M each, followed by incubation for one hour. After the culture was terminated, the cells were transferred to a sterile tube using 0.25% trypsin, and the medium was removed by centrifugation.
  • the Amp.96 peptide has cell permeability increased 63 times or more in human epidermal cells compared to TAT, a previously known cell penetrating peptide.
  • the Amp.96 peptide showed significantly higher cell permeability compared to conventional cell penetrating peptides in various cell types even in the state in which biologically active molecules were bound thereto.
  • Example 1 In order to confirm whether the Amp.96 peptide selected in Example 1 substantially delivers biologically active molecules into cells, the location of the biologically active molecules was confirmed using a fluorescence microscope. More specifically, a circular cover glass for a microscope (Microscope Cover Glasses, 12mm ⁇ ) was laid on a 24-well plate, and HaCAT cells or SH-SY5Y cells were dispensed to be 5 ⁇ 10 4 cells/well, and 10% fetal bovine serum was added. The cells were adhered to the cover glass by culturing for 18 hours at 37° C. and 5% CO 2 in the added DMEM medium.
  • a fluorescence microscope More specifically, a circular cover glass for a microscope (Microscope Cover Glasses, 12mm ⁇ ) was laid on a 24-well plate, and HaCAT cells or SH-SY5Y cells were dispensed to be 5 ⁇ 10 4 cells/well, and 10% fetal bovine serum was added. The cells were adhered to the cover glass by culturing for
  • the Amp.96 (A96) peptide effectively passed through the cell membrane in human epidermal cells and human neuroblastoma cells and effectively delivered the biological molecules bound to the peptide into the cells, and the biological molecule delivery of the A96 peptide
  • TAT cell penetrating peptide
  • a HaCAT cell line a human epidermal cell
  • the cell membrane penetrating efficiency according to the peptide type of FITC-CPP was analyzed using flow cytometry. Specifically, 1 ⁇ 10 7 HaCAT cells were dispensed per well in a 24-well plate, and then cultured in a cell culture medium for 24 hours. Then, FITC-labeled Amp.96 minimally active sequence candidates (Amp.96-1, 2, 3, 4, 5, 6, 8, and 10) and 1 ⁇ M of TAT peptide were each injected into HaCAT cell lines. treated over time.
  • SH-SY5Y cells which are human neuroblastoma cells, in the same manner. Specifically, 1 ⁇ 10 7 SH-SY5Y cells were dispensed per well in a 24-well plate, and then cultured in a cell culture medium for 24 hours. Then, FITC-labeled Amp.96 minimally active sequence candidates (Amp.96-1, 2, 3, 4, 5, 6, 8, and 10), and 1 ⁇ M of the TAT peptide, respectively, were added to the SH-SY5Y cell line. treated for 1 hour.
  • FITC is attached to each candidate peptide, then treated with human epidermal cells (HaCAT) or human glioblastoma cell line (SH-SY5Y), and fluorescence Intracellular FITC delivery efficiency was analyzed by observing under a microscope.
  • HaCAT human epidermal cells
  • SH-SY5Y human glioblastoma cell line
  • a circular cover glass for a microscope (Microscope Cover Glasses, 12mm ⁇ ) was laid in advance, and cells were dispensed at 5 ⁇ 10 4 and then maintained for 18 hours.
  • 500 ⁇ L of D-PBS (WELGENE) was added thereto, followed by treatment with 300 ⁇ L of DMEM (not containing FBS) to which FITC-CPP was added at a concentration of 1 ⁇ M. And incubated for 1 hour under conditions of 37°C and 5% CO 2 .
  • the cell-penetrating peptide newly discovered by the present inventors has a remarkably excellent cell-penetrating ability to transport biologically active molecules into cells, and the biologically active molecules transported into cells can effectively maintain their activity in cells. Confirmed. That is, substances exhibiting various activities can be bound to the cell-permeable peptide of the present invention and effectively delivered into target cells, and the cell-permeable peptide according to the present invention can be applied to various fields such as beauty, diagnosis, vaccine, and treatment. I was able to confirm.
  • the cell-permeable peptide according to the present invention can be deleted even when up to 4 amino acids are deleted from the N-terminus (Amp.96-4) or up to 7 amino acids from the C-terminus are deleted (Amp.96-8). It was confirmed that the cell penetrating ability can be maintained at least 10 times higher than that of the cell penetrating peptide.
  • the 7 amino acids (KIITILI) inside the cell penetrating peptide according to the present invention are the minimally active sequence, and the peptides including the minimally active sequence have excellent cell penetrating ability and biological substance delivery ability.
  • the cell-permeable peptide according to the present invention is a novel cell-permeable peptide capable of effectively transporting biologically active molecules into cells through cell membranes even when biologically active molecules are bound thereto.
  • the cell permeability that can be transported into the cell is remarkably excellent, and the transported biologically active molecules can effectively maintain their activity in the cell. Therefore, the cell-permeable peptide of the present invention can be very usefully used in various fields such as beauty, diagnosis, drug delivery system, recombinant protein vaccine, DNA/RNA therapy, and gene and protein therapy.

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Abstract

Un peptide de pénétration cellulaire selon la présente invention est un nouveau peptide de pénétration cellulaire capable de pénétrer dans une membrane cellulaire et d'administrer efficacement une molécule biologiquement active dans des cellules même s'il est lié aux molécules biologiquement actives, le peptide de pénétration cellulaire ayant une capacité de pénétration cellulaire, permettant d'administrer des molécules biologiquement actives dans des cellules, qui est remarquablement meilleure que celle d'un peptide de pénétration cellulaire connu de manière classique et qui peut conserver efficacement l'activité des molécules biologiquement actives administrées dans des cellules. Par conséquent, le peptide de pénétration cellulaire selon la présente invention peut être utilisé très efficacement dans divers domaines tels que ceux des produits cosmétiques, des diagnostics, des systèmes d'administration de médicament, des vaccins protéiques recombinants, des agents thérapeutiques à ADN/ARN, et de la thérapie génique et protéique.
PCT/KR2022/004938 2021-07-28 2022-04-06 Peptide amphipathique de pénétration cellulaire et son utilisation Ceased WO2023008683A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160063349A (ko) * 2013-10-17 2016-06-03 서울대학교산학협력단 알파나선형 세포 투과 펩타이드 다합체，이의 제조방법 및 그 용도
US20170112760A1 (en) * 2014-06-10 2017-04-27 The Queen's University Of Belfast Cell delivery system and method
US20190091344A1 (en) * 2012-12-07 2019-03-28 The Queen's University Of Belfast Amphipathic peptide
KR102274999B1 (ko) * 2020-04-23 2021-07-08 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도

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Publication number Priority date Publication date Assignee Title
US20190091344A1 (en) * 2012-12-07 2019-03-28 The Queen's University Of Belfast Amphipathic peptide
KR20160063349A (ko) * 2013-10-17 2016-06-03 서울대학교산학협력단 알파나선형 세포 투과 펩타이드 다합체，이의 제조방법 및 그 용도
US20170112760A1 (en) * 2014-06-10 2017-04-27 The Queen's University Of Belfast Cell delivery system and method
KR102274999B1 (ko) * 2020-04-23 2021-07-08 주식회사 아임뉴런바이오사이언스 신규한 세포 투과성 펩타이드 및 이의 용도

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OHGITA TAKASHI; TAKECHI-HARAYA YUKI; NADAI RYO; KOTANI MANA; TAMURA YUKI; NISHIKIORI KARIN; NISHITSUJI KAZUCHIKA; UCHIMURA KENJI; : "A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E", BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER, AMSTERDAM, NL, vol. 1861, no. 3, 1 January 1900 (1900-01-01), AMSTERDAM, NL , pages 541 - 549, XP085584325, ISSN: 0005-2736, DOI: 10.1016/j.bbamem.2018.12.010 *

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