KR102274876B1 - Novel cell penetrating peptides and use thereof - Google Patents

Abstract

The present invention relates to an intracellular delivery technology for delivering a biologically active substance into a cell. More specifically, the present invention relates to a novel cell-penetrating peptide having excellent cell-penetrating ability, and uses thereof. In the present invention, the novel cell-penetrating peptide was synthesized, and excellent cell-penetrating ability thereof was confirmed. Therefore, the cell-penetrating peptide according to the present invention can effectively deliver the biologically active substance into a living body such as cells and tissues, thereby being expected to be usefully used in the field of basic research, diagnosis and treatment of various diseases.

Description

Novel cell penetrating peptides and use thereof

The present invention relates to an intracellular delivery technology for delivering a biologically active substance into a cell, and more particularly, to a novel cell-penetrating peptide having excellent cell-penetrating ability and use thereof.

Until now, studies have been conducted for intracellular delivery of various low-molecular compounds, high molecular substances such as proteins, peptides, RNA, and DNA, and their applications. In particular, enzymes such as SOD, Catalase, SOCS, dnPI3K, ZAP70 mutation Attempts have been made to regulate intracellular functions through the transduction of various proteins, such as inhibitory forms of intracellular signaling proteins, transcription factor proteins such as Foxp3 and RORgt, or DNA binding domains of transcription factors. In addition, attempts to control rejection that occur during organ and cell transplantation by delivering low-molecular compound drugs such as cyclosporin A into cells and tissues, and attempts to control autoimmunity such as psoriasis have been in progress.

However, in general, hydrophilic or large molecular weight substances cannot enter the cell by the barrier called the cell membrane. The cell membrane prevents macromolecules such as peptides, proteins, and nucleic acids from entering the cell, and even if it enters the cell through a physiological mechanism called endocytosis by cell membrane receptors, it is fused with the lysosomal compartment of the cell. As it is eventually degraded, there are many restrictions in the treatment and prevention of diseases using the macromolecules. In addition, in the case of anticancer drugs, in order to deliver the drug into cells, obstacles such as multidrug resistance must be overcome. Accordingly, in order to prevent drug degradation, many methods have been proposed to directly deliver various macromolecules and drug-containing carriers into cells without going through the endocytosis process. These methods include microinjection, electroporation, etc., which have the potential to damage cell membranes. Another method includes a method using a cell-permeable material. However, even if drugs are delivered into cells through this method, there is a problem in that they must be moved to a specific organ in order to exert the drug effect.

Therefore, various drug delivery systems that can overcome the above limitations and increase stability and mass delivery efficiency have been researched and developed, and representative examples include liposomes and micelles. Liposomes are artificially made phospholipid carriers that can encapsulate both lipophilic and hydrophilic drugs, and since they are biocompatible materials, they are nontoxic and protect drugs from external environments. However, absorption is delayed, distribution is limited, metabolic rate is low, and it is captured by cells of the liver or spleen and is rapidly removed from the blood. Although micelles have characteristics that can increase the solubility and bioavailability of drugs, many studies are still needed on the effects of substances on the movement of substances into cells and their basic medical and clinical applicability. Because of these limitations, there is a need for new agents that can effectively deliver biomaterials into the body, have no cytotoxicity, and do not enter through endocytosis in particular.

In this regard, cell penetrating peptides have been attracting attention as a new alternative. The cell-penetrating peptide is a kind of signal peptide and is a peptide that is a combination of a specific amino acid sequence used for the purpose of delivering high molecular substances such as proteins, DNA, RNA, etc. into cells. For example, since the 1990s, when the 11 amino acid sequence present in the TAT protein derived from the HIV virus showed that the intracellular and intracellular delivery of the beta-galactosidase (120 kDa) protein was possible, full-scale since the 1990s. related research was conducted. Antennapedia (Penetratin) derived from Drosophila protein, VP22 derived from HSV-1 virus (Elliott, G. et al., Cell, 88:223, 1997), Simian virus 40 giant antigen T (Simian) Pep-1 derived from Virus 40 large antigen T) is considered to be a representative first-generation cell-penetrating peptide and has been widely used together with TAT. In addition, peptides in which several cationic amino acids such as arginine and lysine are repeatedly linked, such as poly arginine and poly lysine, have also been reported to have excellent cell permeability, and are being applied to various mass transfers. However, most of these cell-penetrating peptides contain the possibility of immunogenicity and toxicity, and are considered to have poor efficacy when delivered to human cells. Therefore, it is necessary to develop a cell-penetrating peptide that does not cause toxicity, has safety in vivo, and can effectively deliver substances.

As a result of research efforts to develop novel cell-permeable peptides having the above-described excellent effects, the present inventors designed and synthesized cell-permeable peptides of various lengths and amino acid sequences according to the present invention and confirmed their excellent cell-permeability. , thereby completing the present invention.

Accordingly, an object of the present invention is to provide a novel cell-penetrating peptide.

Another object of the present invention is to provide a complex comprising the cell-penetrating peptide and a biologically active substance.

In addition, another object of the present invention is to provide a mass transfer composition comprising the complex, and a mass transfer method comprising the step of treating cells with the composition.

Another object of the present invention is to provide a polynucleotide encoding the cell-penetrating peptide.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a cell-penetrating peptide consisting of an amino acid sequence represented by the following [General Formula I].

[General Formula I]

X ₁ -X ₂ -ⓧ-X ₃ -X ₄ -X ₅

X ₁ is absent or ESKAVKWSAL;

X ₂ is absent or any one selected from the group consisting of E, AE, HE, HHE, GHHE and AHHE;

ⓧ consists of 3 amino acids of the X _{a -LK structure,}

X _a is Arg(R) or Ala(A),

X ₃ is absent or any one selected from the group consisting of S, SD, SDEWS, ADEWS and CDEWS;

X ₄ is absent or any one selected from the group consisting of V, VT, VTS and VTSG;

X ₅ is absent or any one selected from the group consisting of G, GL, GLIES, and GLIESESAET.

In one embodiment of the present invention, the cell-penetrating peptide may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 36.

In another embodiment of the present invention, the cell is a brain blood barrier endothelial cell (brain endothelial cell), cancer cell, blood cell (blood cell), lymphocyte (lymphocyte), immune cell, stem cell, induced pluripotent stem cell (induced pluripotent stem) cell; iPSC), neural stem cell (NSC), T cell, B cell, natural killer cell (NK cell), macrophage, microglia, neuron ), glial cells, astrocytes, and muscle cells may be selected from the group consisting of.

In addition, the present invention provides a complex comprising the cell-penetrating peptide and a biologically active substance.

In one embodiment of the present invention, the biologically active material is a compound (chemical compound), protein, glycoprotein, peptide, antibody (antibody), enzyme (enzyme), nuclease (nuclease), hormone, cytokine (cytokine), transcription factors, toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots dots), and may be at least one selected from the group consisting of fluorochromes.

In another embodiment of the present invention, the nuclease is CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1 (CxxC-finger protein-1), ZEN (Zinc-finger nucleases) and TALEN ( Transcription activator-like effector nuclease) may be selected from the group consisting of.

In another embodiment of the present invention, the nucleic acid is DNA, RNA, ASO (Antisense oligonucleotide), microRNA (microRNA; miRNA), small interfering RNA (siRNA), aptamer (aptamer), LNA (locked) nucleic acid), peptide nucleic acid (PNA), and morpholino may be selected from the group consisting of.

In addition, the present invention provides a composition for mass delivery comprising the complex.

In addition, the present invention provides a material delivery method comprising the step of treating the cell with the composition.

In addition, the present invention provides a polynucleotide encoding the peptide.

In the present invention, a novel cell-penetrating peptide was synthesized, and its excellent cell-penetrating ability was confirmed. Therefore, the cell-penetrating peptide according to the present invention is expected to be usefully utilized in the field of basic research, diagnosis and treatment of various diseases, etc. because it can effectively deliver a biologically active substance into a living body such as cells and tissues.

1 shows the results of cell permeability analysis of various peptide candidates obtained by cleaving one or more amino acids from the N-terminus and/or C-terminus in a peptide composed of 16 amino acids discovered through the results of previous studies.
2A and 2B show the results of cell permeability analysis of various peptide candidates in which one or more amino acids are cleaved from the C-terminus and the amino acids at positions 1, 5, or 8 are substituted in the peptide consisting of 16 amino acids. will be.
3A and 3B show the results of cell permeability analysis of various peptide candidates in which one or more amino acids are cleaved from the N-terminus and the amino acids at positions 3, 5 or 8 are substituted in the peptide consisting of 16 amino acids. will be.
Figure 4 shows the results of cell permeability analysis for the peptide group consisting of the 16 amino acids in which the length is extended by adding one or more amino acid sequences from the C-terminus, or the amino acid at position 8 is substituted therewith. .
5 shows the results of cell permeability analysis for the peptide group consisting of the 16 amino acids in which the length is extended by adding a specific amino acid sequence from the N-terminus, or the amino acid at position 8 is substituted therewith.
6 shows the results of cell permeability analysis for the peptide group in which the length is extended by adding an amino acid sequence to the N-terminus and the C-terminus in the peptide consisting of 16 amino acids, or the amino acid at position 8 is substituted therewith. it has been shown
7 shows that only the RLK sequence region at positions 5 to 7 from the N-terminus is substituted in the peptide consisting of 16 amino acids, or from both terminals 1 to 4, 8 to 12 and/or 13 to 16 Shows the results of cell permeability analysis for the peptide group in which the amino acid region at position Burn was substituted.
8 shows the results of cell permeability analysis for peptides in which the amino acid region at positions 1 to 5 from the N-terminus is cleaved.

The present invention relates to a cell-permeable peptide that can be usefully utilized in the field of basic research, diagnosis and treatment of various diseases, and the like, and relates to a basic platform peptide structure that can be expanded to an unlimited number of designs.

Hereinafter, the present invention will be described in detail.

The present invention provides a cell-penetrating peptide consisting of an amino acid sequence represented by the following [General Formula I].

[General Formula I]

X ₁ -X ₂ -ⓧ-X ₃ -X ₄ -X ₅

X ₁ is absent or ESKAVKWSAL;

X ₂ is absent or any one selected from the group consisting of E, AE, HE, HHE, GHHE and AHHE;

ⓧ consists of 3 amino acids of the X _{a -LK structure,}

X _a is Arg(R) or Ala(A),

X ₃ is absent or any one selected from the group consisting of S, SD, SDEWS, ADEWS and CDEWS;

X ₄ is absent or any one selected from the group consisting of V, VT, VTS and VTSG;

X ₅ is absent or any one selected from the group consisting of G, GL, GLIES, and GLIESESAET.

The amino acid sequence used in the present invention is abbreviated as follows according to the IUPAC-IUB nomenclature.

As used herein, the term “cell permeability” refers to the ability or property of a peptide to penetrate a cell (membrane) and penetrate into the cell.

In the present invention, a “peptide” is a polymer of amino acids, usually a form in which a few amino acids are linked is called a peptide, and when many amino acids are linked, it is called a protein. In these peptide and protein structures, the linkage between amino acids consists of an amide bond or a peptide bond. A peptide bond is a bond between a carboxyl group (-COOH) and an amino group (-NH ₂ ) through which water (H ₂ O) escapes and forms -CO-NH-.

The novel peptide of [General Formula I] according to the present invention may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 36, but is not limited thereto. In this case, the cell-penetrating peptide is 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 91%, 92%, 93 of the amino acid sequence shown in SEQ ID NOs: 1 to 36, respectively. %, 94%, 95%, 96%, 97%, 98%, 99% or more of an amino acid sequence having sequence homology.

In the present invention, various amino acids capable of enhancing the effect as the cell-permeable peptide may be additionally added or deleted to the N-terminus and C-terminus of the cell-permeable peptide represented by the [General Formula I].

In the present invention, the types of cells permeable to the cell-penetrating peptide include, but are not limited to, brain endothelial cells, cancer cells, blood cells, lymphocytes, and immune cells. Cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells (NSCs), T cells, B cells, natural killer cells (NK cells), macrophages (macrophage) ), microglia, nerve cells (neuron), glial cells (glial cells), astrocytes (astrocyte) and may be any one selected from the group consisting of muscle cells (muscle cell).

The peptide of the present invention can be manufactured so that the purity of each peptide is 90% or more through a conventional peptide synthesis method or manufacturing method known to those skilled in the art, for example, it can be synthesized directly or purchased after requesting production from a peptide manufacturer. . The peptide can be used by producing both D-form or L-form, a peptide composed of only a part of the D-form or L-form sequence, or a racemate form thereof through a conventional peptide synthesis method or manufacturing method known to those skilled in the art. can In addition, in order to increase the stability of the peptide, other conventional modifications known in the art are possible. In the present invention, the peptide was preferably synthesized using a solid state peptide synthesis method, but as described above, the peptide synthesis method and conditions are not limited thereto.

In addition, the present invention provides a polynucleotide encoding the peptide.

In the present invention, 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 the degeneracy or redundancy of the genetic code.

The polynucleotides of the present invention may also include variants of the polynucleotides described above, wherein the variants of the polynucleotide are either naturally occurring allelic variants of the polynucleotide or non-naturally occurring variants of the polynucleotide. can be Allelic variants are alternate forms of a polynucleotide sequence that may have substitutions, deletions, or additions of one or more nucleotides that do not substantially alter the function of the polynucleotide being encoded (encoded). 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 present inventors have discovered cell-penetrating peptides with excellent cell-penetrating ability through previous prior studies, and in order to additionally discover peptides with excellent cell-penetrating ability, the length based on 16 amino acids based on the sequence and structure of the discovered peptide Various peptide candidate groups were designed and synthesized by modifying or modifying amino acids together, and various cell-penetrating peptides having excellent cell-penetrating ability according to the present invention were discovered by analyzing their cell permeability.

More specifically, in this example, L and K at positions 6 and 7 from the N-terminus are fixed and i) one or more amino acids are cleaved from the N-terminus and/or C-terminus, or ii) from the C-terminus At the same time as cleaving one or more amino acids, the amino acid at position 1, 5 or 8 is substituted with Ala(A); the amino acid at position 8 is substituted with Ala (A), or iv) one or more amino acids are added from the N-terminus and/or C-terminus to extend the length, or at the same time the amino acid at position 8 from the N-terminus A group of peptide candidates induced to substitute for the substitution was designed and synthesized. In addition, when L and K at positions 6 and 7 are modified, i) only the RLK sequence region at positions 5 to 7 is substituted alone or together with positions 1 to 4, 8 to 12 and / Alternatively, a group of candidate peptides including a case in which the amino acid region at positions 13 to 16 was substituted, and ii) a case in which the amino acid region 1 to 5 was cleaved from the N-terminus without amino acid substitution was designed and synthesized. Afterwards, in vitro cell permeability analysis was performed on the candidates, and a total of 36 cell permeability peptides were finally discovered. As a result, the amino acid regions at positions 5 to 7 from the N-terminus are preserved, and numbers 6 and 7 It was found that the maintenance of L and K at burn positions is important for attracting cell permeability (see Examples 1 and 2).

From the results of the above examples, it was found that the peptide according to the present invention has excellent cell permeability, and based on this, it can be used as a carrier that can effectively introduce any substance bound to the peptide into the cell.

Accordingly, as another aspect of the present invention, the present invention provides a complex comprising the cell-penetrating peptide and a biologically active substance.

In the present invention, the complex includes all of a compound formed by simply mixing a peptide and a substance, a compound formed by mixing a peptide and a substance, or a compound formed by connecting or conjugating them by a chemical bond. In addition, the complex may be linked by a physical bond, a chemical bond, a covalent bond, a non-covalent bond, self-assembly, or may be linked in an integrated or fused form using a mediator.

In addition, the complex may be a complex formed by expressing the peptide and the biologically active material in a fusion state. For example, when a gene expressing the peptide and a biologically active substance is 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 biologically active substance are fusion proteins (fusion protein) can be expressed. When expressed as a fusion protein, any linker may be included between the peptide and the biologically active material.

In addition, in the complex according to the present invention, the cell-penetrating peptide may include both single or multiple combined forms in order to efficiently deliver a biologically active substance into a cell, and the cell-penetrating peptide may be cell-permeable depending on the biologically active substance to be delivered. The number of peptide bonds can be easily selected or adjusted by those skilled in the art.

In the present invention, a biologically active substance capable of forming a complex by binding to a cell-penetrating peptide preferably means 'a substance having biological or pharmaceutical activity', which penetrates into the cell (in the cytoplasm or nucleus) and is physiologically It refers to a substance that is involved in the regulation of activity or can express a pharmacological effect, or has biological activity in various parts of the body, such as intracellular, tissue, intercellular, and blood, which must be transported and acted upon. For example, but not limited to, a chemical compound, a protein, a glycoprotein, a peptide antibody, an enzyme, a nuclease, a hormone, a cytokine, a transcription factor ), toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorescence It may be one or more selected from the group consisting of dyes (fluorochromes).

The nucleases are CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, CxxC-finger protein-1 (Cfp1), Zinc-finger nucleases (ZEN) and transcription activator-like effector nuclease (TALEN). It may be selected from the group consisting of, but is not limited thereto.

The nucleic acid is DNA, RNA, antisense oligonucleotide (ASO), microRNA (miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid) ), and may be selected from the group consisting of morpholino, and may additionally include decoy DNA, plasmid, shRNA, antisense RNA, oligoribonucleotide, or transfer RNA, but is not limited thereto. does not

The drug (drug) is a compound drug (chemical drug), bio drug (bio drug), nucleic acid drug (nucleic acid drug), peptide drug (peptide drug), protein drug (protein drug), natural product drug (natural product drug), It may be selected from the group consisting of a hormone, a contrast agent, and an antibody, but is not limited thereto.

The “bio drug” refers to various biologics such as (original) biologics, biogenerics, biobetters, and biosuperiors. The bio-drug means any drug manufactured, secreted or semi-synthesized from a biological origin, and includes, but is not limited to, vaccines, blood products, antigens, cell products, gene therapy products, stem cells, and the like.

The nanoparticles may be selected from the group consisting of iron oxide, gold, carbon nanotubes, and magnetic beads, but is not limited thereto.

As another aspect of the present invention, the present invention provides a composition for mass transfer comprising the complex as an active ingredient.

The composition for mass delivery may be used to deliver a biologically active material to a living tissue or blood or to promote cell permeation. The composition may be delivered through cells constituting a living tissue or cell-to-cell junctions, but there is no limitation in the delivery method.

The biological tissue refers to one or more epithelial tissue, muscle tissue, nervous tissue, and connective tissue, and each organ may consist of one or more tissues, so mucosa, skin, brain, lung, liver, kidney, spleen, lung, heart, stomach , large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genitalia, muscle, blood, blood vessels, lymphatic vessels, lymph nodes, thymus, pancreas, adrenal gland, thyroid gland, parathyroid gland, larynx, tonsils, bronchi, alveoli However, it is not limited thereto.

When the complex is to be delivered to a specific cell, tissue or organ, the material having the biological activity is an extracellular partial protein of a ligand capable of selectively binding to a receptor specifically expressed in a specific cell, tissue or organ, or A complex can be formed by binding to monoclonal antibodies (mAbs) capable of specifically binding these receptors or ligands and modified forms. The binding between the peptide and the biologically active substance is either by indirect linking by cloning technique using an expression vector at the nucleotide level, or by direct linking by chemical or physical covalent or non-covalent binding between the peptide and the biologically active substance. can do.

In the present invention, when the composition including the complex is used as a pharmaceutical composition, the composition may include the active ingredient in an amount of 0.0001 to 50% by weight based on the total weight of the composition.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and may act specifically on target organs. A target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's literature.

The composition comprising the complex as an active ingredient is intravenous (intravenous), intraperitoneal (intraperitoneal), intramuscular (intramuscular), intrathecal (intrathecal), intracerebroventricular (intracerebroventricular), subcutaneous (subcutaneous), intradermal , intranasal (nasal), intramucosal (mucosal), inhalation (inhalation), it can be delivered into the living body by injecting orally (oral). The dosage range varies according to the subject's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

As another aspect of the present invention, the present invention provides a method for transferring a mass into a cell, comprising the step of treating the cell with the composition for mass transfer.

Since the cell-penetrating peptide having a mass transfer function according to the present invention is a very small peptide, it is possible to minimize any biological interference with the active material that may occur.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Cell-permeable peptide candidate group design and synthesis

1-1. Cell-penetrating peptide candidate group design

The present inventors discovered a peptide consisting of 16 amino acids having excellent cell permeability as a result of previous studies (N-GHHERLKSDEWSVTSG-C). Furthermore, in order to additionally discover peptides having excellent cell-penetrating ability, the sequence and structure of the discovered peptides are analyzed and the amino acids at positions 5-7 from the N-terminus based on 16 amino acids in consideration of stability and cell permeability, etc. The region was preserved, but Leu (L) and Lys (K) of Nos. 6 and 7 were fixed, the length of the peptide was modified or some amino acids were substituted along with the length, and a candidate group in which L and K was modified was also designed. did.

More specifically, when L and K at positions 6 and 7 are fixed, i) one or more amino acids are cleaved from the N-terminus and/or C-terminus in the peptide consisting of 16 amino acids, or ii) the C-terminus At the same time as cleaving one or more amino acids from the amino acid at position 1, 5, or 8, substituted with Ala (A), or iii) cleaving at least one amino acid from the N-terminus and simultaneously cleaving one or more amino acids 3, 5 or the amino acid at position 8 is substituted with Ala(A), or iv) one or more amino acids are added from the N-terminus and/or C-terminus to extend the length, or at the same time from the N-terminus to the position 8 A group of peptide candidates induced to change amino acid substitution was designed and synthesized.

Conversely, when L and K at positions 6 and 7 are modified, i) only the RLK sequence region at positions 5 to 7 is substituted alone or together with positions 1 to 4, 8 to 12 and / Alternatively, a group of peptide candidates including a case in which the amino acid region at positions 13 to 16 was substituted, and ii) a case in which the amino acid region 1 to 5 was cut from the N-terminus without amino acid substitution was designed and synthesized.

1-2. Synthesis, separation and purification of peptide candidates

The present inventors used a solid state peptide synthesis (SPPS) method to synthesize each of the peptides described in Example 1-1. The method is an organic synthesis method in which the C-terminus of an amino acid whose N-terminus is protected with F-moc is joined to the N-terminus of the resin one by one. The solvent for all reactions was N,N -dimethylformamide ( N,N -dimethylformamide; DMF), and the amino acid coupling condition was 0.5M DIC ( N,N ′-Diisopropylcarbodiimide; DIC) in a 2M concentration amino acid solution. 1ml, 0.5ml of 1M Ethyl cyanohydroxyiminoacetate (Oxyma) was mixed and reacted in a microwave synthesizer. In addition, amino acids were prepared by varying the reaction time, temperature, or microwave voltage for each amino acid sequence. In order to synthesize the next amino acid, the F-moc of the previous amino acid must be removed. For this, deprotect the F-moc protecting group twice at 80 ° C for 2 minutes using 80% DMF and 20% piperidine solution ( deprotecting). Between all the coupling processes and the deprotecting process, a process of washing three times with DMF and methylene chloride (dichloromethane; DCM) was alternately performed.

For the peptide synthesized through the above method, a fluorescent substance including a carboxyl group (-COOH) at the N-terminus of the peptide may be linked by a chemical bonding method in order to later observe and quantify cell permeability. In this case, the fluorescent material that can be used is 5-FAM (5-carboxyfluorescein), FITC (Fluorescein-5-isothiocyanate), cyanine 3 carboxylic acid, cyanine 5 carboxylic acid, cyanine 7 carboxylic acid ( Cyanine 7 carboxylic acid), and the like, and one or more of the above fluorescent materials may be used.

To this end, the present inventors first synthesized the last amino acid of the peptide synthesized in a solid resin, and then mixed well with 5-FAM: DIC: Oxyma: resin in a 2: 2.5: 4: 1 ratio, and then reacted in the resin. It proceeded at room temperature for 2 hours, but a magnetic stirrer was used. Next, when the color of the resin changed from yellow to dark yellow or orange during the synthesis process, a process of washing DMF and methylene chloride three times was alternately performed. Then, in order to separate the peptide/5-FAM from the solid resin, the synthesis was completed in a cleavage solution in which trifluoroacetic acid (TFA): triisopropylsilane (TIS): distilled water was mixed in a ratio of 95: 2.5: 2.5. After the resin was reacted for 2 hours using a stirrer, the resin was filtered out with an asbestos filter. From the filtered solution, the solution was evaporated under nitrogen gas, and when a precipitate was formed, it was precipitated with diethyl ether stored cold. The precipitated peptide/5-FAM was dried under vacuum and then dissolved in distilled water and freeze-dried.

The lyophilized peptide was dissolved in distilled water or acetonitrile (ACN), etc., and separated and purified using reverse phase high-performance liquid chromatography. At this time, solvent A (distilled water 99.9%, TFA 0.1%) and solvent B (distilled water 9.9%, acetonitrile 90%, TFA 0.1%) were used as mobile phase solvents of the HPLC. The HPLC mobile phase started with Solvent A 90% and Solvent B 10%, and the separation proceeded while increasing the solvent B to 1%/min gradient. Thereafter, the separated peptide solution was lyophilized to remove the solvent and then dissolved in a desired solvent to conduct the experiment.

Example 2. in vitro Cell permeability assay

2-1. Experimental method

In order to evaluate the cell permeability of the peptide candidates synthesized in Example 1, the present inventors performed an in vitro permeability analysis on the blood-brain barrier cells.

Specifically, hCMEC/D3 cells, which are human blood-brain barrier cells, are seeded in a 96-well plate, and the epithelial cell growth medium is EGM (endothelial cell growth medium) until the cells reach 80-90% of the plate area at 37°C. , and incubated overnight under _{CO 2 conditions.} Then, the FAM-linked candidate peptides prepared in Example 1-2 and the negative control FAM alone were diluted in the medium supplemented with the growth factor at 4uM to prepare an amount to be treated by 100 μL per well. Then, the culture solution was removed from the cell culture plate by suction, and the peptide was diluted and treated with a solution prepared in advance, and then cultured at _{37° C. and CO 2 conditions for 2 hours.} Next, all the peptide-treated solutions from each well were removed with an inhaler, 100 μL of EGM was added, tapping 5-6 times, and the removal process was repeated twice. After that, Hoechst 33342 was diluted 1:5000 in EGM, 100 μL was added to each well, and incubated for 30 minutes at 37° C., CO ₂ conditions, and washed twice. Next, after focusing based on DAPI with Cytation 5 equipment, the cells are partitioned with a size of 30 μM centered on DAPI, and only cells of this size are selected for image reading of FAM fluorescence, and then image processing is performed to mean -FAM value was obtained and a graph was created in which the FAM experimental group was corrected with a negative control group, and the blood-brain barrier cell permeability was calculated. Statistics were performed by ONE-WAY ANOVA analysis, and multiple comparisons were obtained using the FAM value as a control and corrected by the Bonferroni method (95% confidence) (mean±SEM, *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001).

2-2. Cell permeability analysis of the peptide candidate group in which amino acids at positions 6 and 7 are fixed from the N-terminus

2-2-1. Cell permeability assays following N-terminal and/or C-terminal cleavage

As designed in Example 1-1, various peptide candidates were designed in which one or more amino acids were cleaved from the N-terminus and/or C-terminus in a peptide composed of 16 amino acids discovered through previous prior research results, and specific Sequence information is shown in Table 1 below.

number designation order SEQ ID NO: One #One GHHERLKSD One 2 #2 GHHERLKS 2 3 #3 GHHERLK 3 4 #4 ERLKSDEWSVTSG 4 5 #5 HHERLKSDEWS 5 6 #6 ERLKSDEWSV 6 7 #7 RLKSDEWSV 7 8 #8 ERLKSDEWSVTS 8 9 #9 RLKSDEWS 9 10 #10 ERLKSDEWSVT 10 11 #11 RLKSDEWSVT 11

As a result of confirming cell permeability in the peptide group, As shown in FIG. 1 , it was confirmed that the peptides of sequences #1 to #11 in Table 1 showed an excellent effect of influx into the blood-brain barrier cells.

From the above results, if the region of the RLK sequence at positions 5-7 from the N-terminus is not cleaved and is conserved, excellent cell permeability is maintained even when one or more amino acids are cleaved from the N-terminus, C-terminus, or both ends. knew that it could be

2-2-2. Cell permeability analysis according to C-terminal cleavage and amino acid substitution

Next, in a peptide consisting of 16 amino acids discovered through the results of previous studies, one or more amino acids were cleaved from the C-terminus and at the same time the amino acids at positions 1, 5, or 8 were substituted with Ala(A). Peptide candidates were designed, and specific sequence information is shown in Table 2 below.

number designation order SEQ ID NO: One #12 AHHERLKSDEWSVT 12 2 #13 AHHERLKSDEWSV 13 3 #14 AHHERLKSDEWSVTS 14 4 #15 GHHEALKSDEWSVT 15 5 #16 GHHEALKSDEWSVTS 16 6 #17 GHHERLKADEWSVTS 17 7 #18 GHHERLKADEWSVT 18

As a result of confirming cell permeability in the peptide group, As shown in FIGS. 2A and 2B , the peptides of sequences #12 to #18 in Table 2 showed an excellent effect on influx into the blood-brain barrier cells, especially #17 and #18, although there was a difference in degree. It was confirmed that the cell permeability of

From the above results, at least one of 1 to 3 amino acids from the C-terminus (positions 14 to 16 from the N-terminus) is cleaved and the amino acids at positions 1, 5 or 8 from the N-terminus are replaced with Ala (A) It was found that excellent cell permeability of the peptide can be maintained even when substituted with .

2-2-3. Cell permeability analysis according to N-terminal cleavage and amino acid substitution

Next, in a peptide composed of 16 amino acids discovered through the results of previous studies, one or more amino acids were cut from the N-terminus and at the same time, amino acids at positions 3, 5, or 8 were substituted with Ala(A). Peptide candidates were designed, and specific sequence information is shown in Table 3 below.

number designation order SEQ ID NO: One #19 AERLKSDEWSVTSG 19 2 #20 ALKSDEWSVTSG 20 3 #21 HHERLKADEWSVTSG 21 4 #22 HERLKADEWSVTSG 22 5 #23 ERLKADEWSVTSG 23 6 #24 RLKADEWSVTSG 24

As a result of confirming cell permeability in the peptide group, As shown in FIGS. 3A and 3B , the peptides of the sequences #19 to #24 of Table 3 showed an excellent effect on influx into the blood-brain barrier cells, especially #23 and #24, although there was a difference in degree. It was confirmed that the cell permeability of

From the above results, at least one of 1 to 4 amino acids from the N-terminus (positions 1 to 4 from the N-terminus) is cleaved and the amino acids at positions 3, 5 or 8 from the N-terminus are replaced with Ala (A) It was found that excellent cell permeability of the peptide can be maintained even when substituted with .

2-2-4. Cell permeability analysis according to N-terminal and/or C-terminal extension

Next, various peptide candidates whose length was extended by adding one or more amino acids from the N-terminus and/or C-terminus were designed in a peptide consisting of 16 amino acids discovered through the results of previous prior research, and detailed sequence information is provided below. Table 4 shows. In this case, the following peptide candidate group included not only the length extension without amino acid substitution, but also the length extension and substitution of the amino acid at position 8 with Cys (C).

number designation order SEQ ID NO: One #25 GHHERLKSDEWSVTSGGL 25 2 #26 GHHERLKSDEWSVTSGG 26 3 #27 GHHERLKSDEWSVTSGGLIES 27 4 #28 GHHERLKCDEWSVTSGG 28 5 #29 GHHERLKSDEWSVTSGGLIESESAET 29 6 #30 GHHERLKCDEWSVTSGGLIECESAET 30 7 #31 ESKAVKWSALGHHERLKSDEWSVTSG 31 8 #32 ESKAVKWSALGHHERLKCDEWSVTSG 32 9 #33 ESKAVKWSALGHHERLKSDEWSVTSGGLIES 33 10 #34 ESKAVKWSALGHHERLKSDEWSVTSGGLIESESAET 34 11 #35 ESKAVKWSALGHHERLKCDEWSVTSGGL 35 12 #36 ESKAVKWSALGHHERLKSDEWSVTSGGL 36

First, as a result of confirming cell permeability in the peptide group extending the length by adding the G, GL, GLIES or GLIESESAET amino acid sequence from the C-terminus, As shown in FIG. 4, it was confirmed that all of the peptides of the sequence corresponding to #25 to #30 of Table 4 showed excellent influx effect into the blood-brain barrier cells regardless of the substitution of C at the 8th position from the N-terminus. did.

In addition, in the case of a peptide whose length is extended by adding an ESKAVKWSAL sequence from the N-terminus, as shown in FIG. 5, the peptide having the sequences corresponding to #31 and #32 in Table 4 is located at position 8 from the N-terminus. It was confirmed that excellent cell permeability was exhibited regardless of the substitution of C.

Furthermore, as a result of analyzing cell permeability for the peptide candidate group whose length was extended by adding the amino acid sequences from the C-terminus and the N-terminus simultaneously, as can be seen in FIG. 6, in #33 to #36 of Table 4, It was confirmed that the peptide of the corresponding sequence exhibited excellent cell permeability regardless of the substitution of C at position 8 from the N-terminus.

Through the above results, without replacing the LK amino acids at positions 6 and 7 from the N-terminus, at least one end of both ends of the peptide is cleaved or extended, or the amino acid at positions 3, 5, or 8 is substituted with A at the same time It was found that the cell permeability of the peptide was excellently maintained even in various cases where the peptide was released, and the LK sequence was expected to have an important effect on maintaining cell permeability.

2-3. Cell permeability analysis of the peptide candidate group in which the amino acid at position 6 or 7 is substituted from the N-terminus

From the results of Example 2-2, the present inventors designed several peptide candidates and evaluated their cell permeability to determine whether LK amino acids 6 and 7 from the N-terminus are important for the cell permeability of the peptide, and the designed peptide Specific sequence information for the candidates is shown in Table 5 below.

number designation order SEQ ID NO: One #37 SHLERTKSDEWSIISE 37 2 #38 SHLERTKCDEWSIISE 38 3 #39 GHHEGKSDEWSVTSG 39 4 #40 GHHERLGGDEWSVTSG 40 5 #41 LKSDEWSVTSG 41

Specifically, a peptide in which only the RLK sequence region at positions 5-7 from the N-terminus corresponding to #39 in Table 5 is substituted alone, L, the amino acid at position 6 corresponding to #37, is Thr(T) Peptide in which the amino acid region at positions 1 to 4 and 13 to 16 from both terminals is substituted with Thr (T), and the amino acid at position 6 corresponding to #38 is substituted with Thr (T). A peptide in which the amino acid region at positions 4, 8 to 12, and 13 to 16 is substituted, and K at 7 corresponding to #40 is substituted with Gly (G) and the amino acid region at positions 8 to 12 is substituted Cell permeability was confirmed for the peptide group.

As a result, as shown in FIG. 7, there was no low or significant difference in fluorescence intensity compared to the negative control FAM alone, and through this, in the case of a peptide comprising a sequence in which L or K was substituted, there was no cell permeability. confirmed that.

In addition, as a result of evaluating cell permeability for the peptide corresponding to #41 of Table 5, in which the amino acid region at positions 1-5 from the N-terminus was cleaved, as can be seen in FIG. 8 , a level almost similar to the negative control group It was confirmed that cell permeability was not maintained.

From the above results, it was found that the amino acid region of positions 5-7 from the N-terminus is conserved, and that L and K of positions 6 and 7 are maintained is important for maintaining cell permeability.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> IMNEWRUN BIOSCIENCES, INC. RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY <120> Novel cell penetrating peptides and use thereof <130> PD20-453 <160> 41 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> #1 <400> 1 Gly His His Glu Arg Leu Lys Ser Asp 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> #2 <400> 2 Gly His His Glu Arg Leu Lys Ser 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> #3 <400> 3 Gly His His Glu Arg Leu Lys 1 5 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> #4 <400> 4 Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> #5 <400> 5 His His Glu Arg Leu Lys Ser Asp Glu Trp Ser 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> #6 <400> 6 Glu Arg Leu Lys Ser Asp Glu Trp Ser Val 1 5 10 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> #7 <400> 7 Arg Leu Lys Ser Asp Glu Trp Ser Val 1 5 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> #8 <400> 8 Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser 1 5 10 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> #9 <400> 9 Arg Leu Lys Ser Asp Glu Trp Ser 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> #10 <400> 10 Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> #11 <400> 11 Arg Leu Lys Ser Asp Glu Trp Ser Val Thr 1 5 10 <210> 12 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #12 <400> 12 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr 1 5 10 <210> 13 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> #13 <400> 13 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val 1 5 10 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> #14 <400> 14 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser 1 5 10 15 <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #15 <400> 15 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr 1 5 10 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> #16 <400> 16 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser 1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> #17 <400> 17 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser 1 5 10 15 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #18 <400> 18 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr 1 5 10 <210> 19 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #19 <400> 19 Ala Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> #20 <400> 20 Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 <210> 21 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> #21 <400> 21 His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #22 <400> 22 His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 <210> 23 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> #23 <400> 23 Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> #24 <400> 24 Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 <210> 25 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #25 <400> 25 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #26 <400> 26 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 27 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #27 <400> 27 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #28 <400> 28 Gly His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 29 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #29 <400> 29 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 30 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #30 <400> 30 Gly His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Cys Glu Ser Ala Glu Thr 20 25 <210> 31 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #31 <400> 31 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 32 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #32 <400> 32 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 33 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #33 <400> 33 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 34 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #34 <400> 34 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 35 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #35 <400> 35 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 36 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #36 <400> 36 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 37 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #37 <400> 37 Ser His Leu Glu Arg Thr Lys Ser Asp Glu Trp Ser Ile Ile Ser Glu 1 5 10 15 <210> 38 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #38 <400> 38 Ser His Leu Glu Arg Thr Lys Cys Asp Glu Trp Ser Ile Ile Ser Glu 1 5 10 15 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #39 <400> 39 Gly His His Glu Gly Gly Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 40 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #40 <400> 40 Gly His His Glu Arg Leu Gly Gly Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> #41 <400> 41 Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10

Claims (11)

As a cell-permeable peptide consisting of amino acids represented by the following [general formula I],
[General Formula I]
X ₁ -X ₂ -ⓧ-X ₃ -X ₄ -X ₅
X ₁ is absent or ESKAVKWSAL;
X ₂ is absent or any one selected from the group consisting of E, AE, HE, HHE, GHHE and AHHE;
ⓧ consists of 3 amino acids of the X _{a -LK structure,}
X _a is Arg(R) or Ala(A),
X ₃ is absent or any one selected from the group consisting of S, SD, SDEWS, ADEWS and CDEWS;
X ₄ is absent or any one selected from the group consisting of V, VT, VTS and VTSG;
X ₅ is absent or any one selected from the group consisting of G, GL, GLIES, and GLIESESAET,
The cell-permeable peptide is characterized in that it consists of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 36, cell-permeable peptide.
delete According to claim 1,
The cells include brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells ( neural stem cells (NSC), T cells, B cells, natural killer cells (NK cells), macrophages, microglia, neurons, glial cells , Astrocytes (astrocyte) and muscle cells (muscle cell), characterized in that selected from the group consisting of, cell-penetrating peptide.
A complex comprising the cell penetrating peptide of claim 1 or 3 and a biologically active substance.
5. The method of claim 4,
Biologically active substances include chemical compounds, proteins, glycoproteins, peptides, antibodies, enzymes, nucleases, hormones, cytokines, transcription factors, toxins , nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorochromes ), characterized in that at least one selected from the group consisting of, the complex.
6. The method of claim 5,
The nucleases are CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, CxxC-finger protein-1 (Cfp1), Zinc-finger nucleases (ZEN) and transcription activator-like effector nuclease (TALEN). Characterized in that selected from the group consisting of, the complex.
6. The method of claim 5,
The nucleic acid is DNA, RNA, antisense oligonucleotide (ASO), microRNA (miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid) ), and morpholino (morpholino), characterized in that selected from the group consisting of, the complex.
A composition for mass transfer, comprising the complex of claim 4.
A material delivery method comprising the step of treating cells of a mammal other than a human with the composition of claim 8.
10. The method of claim 9,
The cells include brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells ( neural stem cells (NSC), T cells, B cells, natural killer cells (NK cells), macrophages, microglia, neurons, glial cells , Astrocytes (astrocyte) and muscle cells (muscle cell), characterized in that selected from the group consisting of, the mass delivery method.
A polynucleotide encoding the peptide according to claim 1 .

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