KR101695980B1 - Cell-permeable peptide - Google Patents

Abstract

The present invention relates to a cell permeable peptide derived from Lin28 protein, a composition for cell permeation delivery, a composition for tissue regeneration, a composition for promoting stem cell proliferation, a composition for promoting neural stem cell growth or for promoting differentiation of neural stem cells, Lt; / RTI >

Description

Cell-permeable peptide {Cell-permeable peptide}

The present invention relates to a Lin28 protein-derived cell-permeable peptide, a composition for cell-penetrating delivery, a composition for tissue regeneration, a composition for promoting stem cell proliferation, or a composition for promoting the growth of neural stem cells or promoting differentiation of neural stem cells, a cell-penetrating carrier and a drug containing the same. It relates to the carrier.

Cells of higher animals have a plasma membrane that separates the inside and the outside of the cell, and it plays a role in thoroughly discriminating the movement of substances between cells. The cell membrane is composed of a phospholipid bilayer, and due to the hydrophobicity of these lipid bilayers, it is almost impossible for most of the peptides, proteins, nucleotides, and liposomes to move into the cell. Therefore, it can be said that the cell membrane plays a role as an obstacle in the movement of peptides, proteins, or chemical drugs or gene therapy products into cells. In order to overcome this, a method of using cationic lipid or polyethyleneimine (PEI), or using a viral vector or electroporation method has been widely used.

However, these methods can be said to have limitations due to low efficiency, applicable cell limitations, and intracellular toxicity [Non-Patent Documents 1 to 2]. In order to overcome this limitation, many attempts have recently been made to use a cell-penetrating peptide (CPP). CPP is also called a protein transduction domain (PTD). CPP is a part of translocatory protein, and the representative one is the hydrophobic region present in the signal sequence of human fibroblast growth factor 4, the membrane translocating sequence (MTS) and one of the viral proteins of HIV. Tat-PTD (basic amino acid domain) of Tat protein is mentioned [Non-Patent Documents 3-4].

However, in the case of MTS, it is recognized as a good candidate because of its excellent efficiency to enter the cell, but since most of the amino acid sequence is a hydrophobic domain, the inclusion body is formed during the expression process for production. It is likely to be generated and has the disadvantage of having to go through more complex steps for expression and purification, and even a protein that has undergone purification has a disadvantage that its activity may be reduced due to aggregation of proteins due to hydrophobic interaction during storage. There is [Non-Patent Documents 3, 5 to 7]. On the other hand, in the case of a basic domain such as Tat-PTD, where basic amino acids mainly exist [Non-Patent Document 8], since it is hydrophilic, it is unlikely that an inclusion body will be formed during the expression process for production, and can be purified by a relatively easy method in the cytoplasm. And it has the advantage of being able to maintain its activity for a relatively long period of time even in the storage process. In addition to this, numerous PTDs have been reported so far [Non-Patent Document 9].

In the case of penetratin, one of the CPPs, it is currently patented and can be used commercially. Phenatratin is derived from antennapedia protein, and is a kind of homeoprotein as a transcription factor involved in the development of Drosophila and neurogenesis. It was found that it does not use energy, has the property of causing translocation effectively regardless of cell-type, regardless of endocytosis, and 16 amino acids of them play an important role, and this fragment ( fragment) was also found to induce a potential regardless of endocytosis [Non-Patent Document 10].

In this way, researches to find several cell-penetrating peptides and reveal their sequences are being actively conducted worldwide, and both the Tat-PTD series and the MTS series are used as cell-penetrating drugs for direct therapeutic purposes, but other treatment methods such as gene therapy. Compared to them, the effect is insignificant.

Therefore, it is a very important part to secure candidates with good cell-penetrating ability, which are more efficient than previously known cell-penetrating peptides, and to apply them to fusion proteins and to secure an exclusive technical advantage over the amino acid sequence.

Green I, Christison R, Voyce CJ, Bundell KR, Lindsay MA (2003) Protein transduction domains: are they delivering? Trends Pharmacol Sci 24: 213-215 Lindsay MA (2002) Peptide-mediated cell delivery: application in protein target validation. Curr Opin Pharmacol 2: 587-594 Rojas M, Donahue JP, Tan Z, Lin YZ (1998) Genetic engineering of proteins with cell membrane permeability. Nat Biotechnol 16: 370-375 Mann DA, Frankel AD (1991) Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J 10: 1733-1739 Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, Ruley HE (2001) Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase. Nat Biotechnol 19: 929-933 Jo D, Liu D, Yao S, Collins RD, Hawiger J (2005) Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis. Nat Med 11: 892-898 DiGiandomenico A, Wylezinski LS, Hawiger J (2009) Intracellular delivery of a cell-penetrating SOCS1 that targets IFN-gamma signaling. Sci Signal 2: ra37 Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci U S A 97: 13003-13008 Lindgren M, Langel U (2011) Classes and prediction of cell-penetrating peptides. Methods Mol Biol 683: 3-19 Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G, Prochiantz A (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem 271: 18188-18193

Accordingly, the inventors of the present invention believe that it is a very important part to secure candidates with better cell-penetrating ability, which are more efficient than previously known cell-penetrating peptides, and to obtain an exclusive technical advantage for the application to a fusion protein and its amino acid sequence. The amino acid sequence of a cell-penetrating peptide, which is far superior in efficiency than previously known cell-penetrating peptides, was discovered.

An object of the present invention is to provide a composition for cell permeation delivery, a composition for tissue regeneration, a composition for promoting stem cell proliferation, or a composition for promoting neural stem cell growth or promoting differentiation of neural stem cells comprising a cell-permeable peptide derived from Lin28 protein. .

In addition, the present invention has another object to provide a cell-penetrating peptide derived from Lin28 protein.

In addition, the present invention has another object to provide a wound healing agent comprising a cell-penetrating peptide derived from Lin28 protein.

In addition, the present invention has another object to provide a cell therapy comprising a cell-penetrating peptide derived from Lin28 protein.

As a means for solving the above problems, the present invention is a composition for cell permeation delivery comprising a cell-permeable peptide derived from Lin28 protein, a composition for tissue regeneration, a composition for promoting stem cell proliferation, or promoting the growth of neural stem cells or promoting the differentiation of neural stem cells. It provides a composition for use.

As another means for solving the above problems, the present invention provides a cell-penetrating peptide derived from Lin28 protein.

As another means for solving the above problems, the present invention provides a wound healing agent with a cell-penetrating peptide derived from Lin28 protein.

Cell-permeable fusion proteins with specific functionality are applied to cell therapy using antiviral proteins, anticancer proteins, anti-inflammatory proteins, stem cells, etc., which have good effects by using the peptides with high efficiency cell permeability according to the present invention. If it is created and its effectiveness is proven, it is expected that it can be used in many fields as a promising drug delivery system. In addition, since it can be applied to epitopes that induce immune responses, it is expected to contribute to vaccine development.

1 is a comparison of the cell permeation efficiency of Lin28 protein-derived cell-penetrating peptides (Lin-pep and Lin-half) in mesenchymal stem cells (MSC), (a) is a Lin28-derived cell-penetrating peptide (Lin -Pep and Lin-half) are FACS data comparing the cell permeation efficiency with Tat-PTD. (b) is a graph showing the intracellular permeation efficiency of the cell-penetrating peptide in terms of %. Each peptide was labeled with FITC to measure cell permeability by fluorescence. In a medium containing serum, after treatment at a concentration of 1 μM for 1 hour, the fluorescent peptide attached to the cell surface was removed using the Heparin washing method, and the cells were removed by treatment with trypsin, followed by FACS analysis. Was performed. In the case of Lin-pep, it was confirmed that it has the ability to permeate almost all cells in 1 hour.
2 is a comparison of the cell permeation efficiency for Lin-pep in neural stem cells (NSC) for 1 hour, and the neural stem cells are cultured in a medium without serum, at this time, similar to the previous paper report, Tat- In the case of PTD, it can be seen that the permeation efficiency is high in the absence of serum, and on the contrary, it can be seen that the cell permeation efficiency of Lin-pep is very high regardless of the presence or absence of serum. In addition, in the case of Tat-PTD, it is said that there are many differences in permeation efficiency for each cell type, but it is thought that the primary cell or MSC shows very low cell permeation efficiency.
3 is a diagram showing a transduction efficiency experiment to determine the cell permeation efficiency of Lin-pep for a relatively long time in human MSC. (a) is the result of FACS analysis after treatment with Lin-pep for 5 hours. Compared to the Tat-PTD used as a control, human MSC showed remarkably higher permeation efficiency. In (b), it is an indicator that the amount of peptide entering per unit cell can be known. The higher the index value than the control (No treatment), the greater the number of peptides per unit cell can be introduced into the cell. Compared to Tat-PTD, it has been shown that a much greater number of peptides per unit cell can be introduced into the cell. As can be seen in the micrograph, the fluorescence intensity was high enough to be confirmed with the naked eye.
FIG. 4 is a confocal microscope image showing translocation in cells 18 hours after Lin-pep (5 μM) is permeated to cells in human MSCs. It can be seen that the cell-permeated peptide is stably present in the cell even after a day has passed. (a) is a confocal fluorescence image taken after treatment with Lin-pep while MSC is attached and growing, and (b) is a cell attached to the bottom with trypsin to more clearly show that it exists in the cell. Was removed, and an immunofluorescence experiment was performed with an additional membrane marker. As can be seen, it can be seen that FITC fluorescence is visible inside the membrane marker, indicating that it is present in the cell, and that it is partially present in the nucleus as well as the cytoplasm.
5 is a result of examining the cell permeation efficiency in other cells in addition to stem cells. (a) is the cell permeation efficiency in primary cells derived from mouse lymph nodes, and it can be seen that the permeation efficiency at 5 μM concentration of Lin-pep is higher than that at the concentration of Tat-PTD or MTS 5 μM. (b) is the result of the cell permeation efficiency of mouse-derived fibroblasts, and it can be seen that the permeation efficiency of Lin-pep is significantly higher than that of WT1-pTj or Tat-PTD at a concentration of 1 μM.
6 is a schematic diagram showing the role of the Lin28 protein in the cell, and a series of differentiation and self-renewal of stem cells such as inhibiting the role by directly binding the Lin28 protein to let-7, a kind of micro RNA, in the cell It describes its role as a transcription factor that promotes (self-renewal).
FIG. 7 is a diagram illustrating a pET11 vector by inserting a human Lin28 gene to make a vector so that the Lin28 gene is expressed in E. coli, and a large amount of Lin28 protein was expressed using IPTG in E. coli BL21 expression strain, and purified using His tag. to be.
Figure 8 is a view showing that the proliferation effect of the Lin28A protein in NIH3T3 cells has a proliferative effect that promotes cell proliferation compared to the microscopic photograph observed on the 4th day after culture and the control.
9 is a diagram showing that Lin-pep has an effect of promoting cell proliferation in mesenchymal stem cells and neural stem cells. Neural stem cells showed a higher cell proliferation effect than mesenchymal stem cells.
10 is a diagram showing the effect of promoting migration by Lin28 and Lin-pep in MSC cell lines. (a) shows the migration promoting effect of Lin28 on mouse MSCs, and it can be seen that the His-Lin28 group promotes cell migration more than the untreated group and His-GFP used as a control group. Proteins were treated every 6 hours and photographed under a microscope after 12 hours. (b) shows the effect of promoting migration of Lin-pep and His-Lin28 on human MSC. In this case, this is a photomicrograph after staining the cells after 24 hours.
11 is a result showing that in human MSCs, Lin-pep or His-Lin28 also has the effect of increasing cells having the ability to form cell colonies. It was shown that there was an increase of about 42% and 56% in the Lin-pep or His-Lin28 treatment group, respectively, than in the untreated group or the Tat-PTD treatment group.
12 shows that in addition to the effect of promoting cell proliferation of Lin-pep in neural stem cells (FIG. 10B), it is possible to promote differentiation from neural stem cells into neural stem cells. (a) shows the morphology of adherent cells extending axons in the Lin-pep treatment group, unlike the untreated group, as well as such morphological changes, as shown in (b) It was found that the cell differentiation marker βⅢ-Tubulin (Tuj1) protein was expressed, and as shown in (c), immunofluorescence (Immunofluoroscence) also showed that βⅢ-Tubulin (Tuj1) protein was expressed in the cytoplasm. have.

As described above, in the case of a general stem cell therapeutic agent, in order to exert protein drug efficacy by introducing the protein itself into the stem cell, it is difficult to penetrate the cell membrane and move into the cell. Has played a role.

In the case of Lin28, a study has been reported on confirming the proliferation effect of stem cells by introducing the gene of Lin28 into stem cells and penetrating the cell membrane. However, the study of introducing the Lin28 protein itself into stem cells has not been revealed yet.

The present inventors revealed for the first time that the Lin28 protein was expressed and purified, and then directly introduced into stem cells to confirm the stem cell proliferation effect, thereby allowing the protein to be introduced into the cell itself, not in the form of a gene.

Therefore, since it can be used as a cell-permeable peptide derived from Lin28 protein, a composition for cell permeation delivery containing the peptide, a composition for promoting stem cell proliferation, a composition for promoting neural stem cell growth or promoting differentiation of neural stem cells, or a composition for tissue regeneration, The present invention includes a wound healing agent comprising the composition. In addition, the present invention may include a wound healing pharmaceutical composition comprising a Lin28 protein-derived cell-permeable peptide, a cell therapeutic agent, a drug delivery system, or a cell-permeable delivery system.

Lin28 protein directly binds to let-7, a kind of micro RNA, inhibits its role, and acts as a transcription factor that promotes a series of differentiation and self-renewal of stem cells. There are two types of Lin28a and Lin28b, and their amino acid sequence may be represented by any one of SEQ ID NOs: 1 to 4 as follows, but is not limited thereto.

The present invention relates to a peptide having cell penetrating activity derived from Lin28 protein. The peptide having cell penetrating activity may be represented by the following sequence (any one of SEQ ID NOs: 5 to 12), but is not limited thereto.

The composition for cell-penetrating delivery comprising the cell-penetrating peptide of the present invention is a composition used to pass or permeate the cell membrane to deliver into the cell or to promote or stimulate migration into the cell, including a reagent composition and/or a pharmaceutical composition. do. In the present specification, the reagent composition is also expressed as a reagent, and the pharmaceutical composition is also expressed as a medicine, medicine, or pharmaceutical composition.

The reagent composition used to stimulate cell migration of the present invention can be used as a reagent necessary for basic research and clinical research for regenerative medicine, regenerative induction medicine development, for example. For example, the reagent composition can be used to mobilize cells to a living tissue of an experimental animal to examine the level of tissue repair and tissue function reconstruction. Further, for example, by using the reagent composition, a study inducing tissue regeneration by mobilizing cells in vitro can be conducted.

The pharmaceutical composition used to stimulate cell migration of the present invention can be used, for example, as a medicine for regenerative medicine and induction medicine for regenerative medicine. For example, tissue can be regenerated by using the pharmaceutical composition. Further, for example, the pharmaceutical composition can be used as a so-called prophylactic drug to prevent deterioration of tissue and organ function due to the decrease of tissue stem cells, or alternatively, as an anti-aging drug, to delay the progression of aging changes.

In the present specification, the composition for cell-penetrating delivery is also expressed as a cell-permeable carrier to pass through or permeate the cell membrane to deliver into the cell, and is used to promote or stimulate cell migration, a cell migration stimulator, a cell migration promoter, and a cell migration agent. It is also expressed as a composition used to induce, an agent used to induce cell migration, a cell migration inducing agent, or a cell inducing agent.

In the present invention, cell-penetrating activity refers to an activity that passes through or permeates a cell (membrane) and delivers it into a cell, and the cell migration promoting activity refers to an activity that promotes or stimulates cell migration. In the present specification, the cell migration promoting activity is also expressed as a cell migration inducing activity or a cell induction activity.

The composition for tissue regeneration of the present invention is also expressed as a composition used to induce or promote tissue regeneration, an agent used to induce or promote tissue regeneration, a tissue regeneration inducing agent or a tissue regeneration accelerator. In addition, tissue regeneration includes tissue repair.

The composition for tissue regeneration of the present invention does not select the site of administration or addition. That is, the composition can exert its effect even if it is administered to any tissue such as a tissue in need of regeneration, a tissue different from a tissue in need of regeneration, or in blood. For example, by administering or adding the composition, tissue cells at or near the site of administration or addition are mobilized to induce or promote tissue regeneration. Further, for example, by administering and adding the composition to a damaged tissue site or its vicinity, the damaged tissue cells are mobilized and tissue regeneration is induced or promoted.

In addition, the present invention may include a wound healing agent or a wound healing accelerator or cell therapy agent including the cell penetrating peptide.

Tissues in need of regeneration include, for example, damaged tissues, necrotic tissues, tissues after surgery, tissues with reduced function, fibrous tissues, aged tissues, diseased tissues, and the like. Examples of such tissues include biological skin tissue and tissues (brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, testicles, blood, etc.) obtained by biopsy (surgery) in the body. The tissues of the present invention include, for example, skin tissue, bone tissue, cartilage tissue, muscle tissue, adipose tissue, myocardial tissue, nervous system tissue, lung tissue, digestive tract tissue, liver, phlegm, pancreatic tissue, urinary system, genital organs, etc. It includes all tissues in the living body. In addition, functional tissue regeneration in tissues requiring regeneration such as cerebral infarction, myocardial infarction, fracture, pulmonary infarction, gastric ulcer, enteritis, as well as skin diseases such as intractable skin ulcers, skin wounds, blisters, alopecia, etc. Inducing treatment is possible. The animal species to which the above composition is administered is not particularly limited, and includes mammals, birds, fish, and the like. Mammals include humans or non-human animals, and include, but are not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales.

Nervous tissue includes, but is not limited to, central nervous tissue. In addition, the composition used to regenerate nerve tissue can be used for treatment of, for example, a cerebral infarction, a cerebral hemorrhage, and a brain strain, but is not limited thereto. In addition, the composition used to regenerate bone tissue may be used for, for example, fracture treatment, but is not limited thereto. In addition, the composition used to regenerate skin tissue can be used for treatment of, for example, skin ulcers, suture failure of surgical wounds, burns, wounds, bruises, skin sores, and abrasions, but is not limited thereto.

In addition, the present invention provides a composition for promoting stem cell proliferation comprising Lin28 protein as an active ingredient.

In the present invention, “stem cells” refer to undifferentiated cells having multipotency derived from mammals, including humans, preferably adult cells, for example, bone marrow, blood, brain, skin, fat (ie, fat Tissue or adipocyte), umbilical cord blood, and Wharton's jelly. Stem cells may include both embryonic stem cells, which are pluripotent stem cells, and adult stem cells, which are multipotent stem cells, preferably adult stem cells, More preferably, it may be a hematopoietic stem cell. The "adult stem cell" refers to an undifferentiated cell that is capable of self-reproduction as a stem cell generated from a differentiated tissue and can be differentiated into all cell types of the derived tissue. Specifically, the adult stem cells include umbilical cord blood (umbilical cord blood), bone marrow, spleen, ovaries, testis, peripheral blood, amniotic fluid, brain, blood vessels, skeletal muscles, epithelium of the skin or gastrointestinal tract, cornea, dimensions of teeth, retina, It can be extracted from the liver or pancreas, and refers to the primitive cells just before differentiation into cells of specific organs such as bone, liver, and blood. For example, it is reported that the spinal cord has hematopoietic stem cells (HSC) and mesenchymal stem cells, and neural stem cells, which are stem cells derived from the brain, are subventricular regions ( It is known to be separated from the subventricular zone, the ventricular zone, and the hippocampus of the central nervous system (CNS). In general, adult stem cells are difficult to proliferate but are known to have a strong tendency to differentiate easily, so not only can the organ regeneration required in actual medicine using various types of adult stem cells, but also the characteristics of each organ after transplantation. It has the characteristics that can be differentiated accordingly. In addition, the “hematopoietic stem cell” is a parent cell of lymphocytes that form various blood cells and immune systems, and can develop into, for example, lymphocytes such as B cells and T cells, granulocytes, platelets, and red blood cells. It refers to stem cells. As essential cells for bone marrow transplantation, about 1% of cells (CD34 positive cells) with the ability to make all blood cells exist in the bone marrow blood of a normal person, which are called hematopoietic stem cells. It is found in the whole body, including umbilical cord blood (umbilical cord blood) and spleen, meaning that it is a mother cell that makes blood, but it is produced in large quantities, especially in the bone marrow, and has a self-replicating function that can create the same self. Peripheral blood hematopoietic stem cells are derived from the bone marrow and are hematopoietic stem cells that circulate in the bloodstream and have the property of self-replicating and differentiating into mature cells.

Accordingly, the present invention can provide a composition containing as an active ingredient Lin28 protein having an effect of promoting the proliferation or differentiation of hematopoietic stem cells derived from umbilical cord blood, bone marrow, spleen, blood, tissue or body fluid. More preferably, hematopoietic stem cells derived from bone marrow, spleen or umbilical cord blood may be targeted.

In addition, the present invention can provide a composition for promoting the growth and differentiation of neural stem cells comprising Lin28 protein as an active ingredient.

Therefore, the composition according to the present invention promotes their growth and/or differentiation by treating neural stem cells isolated from the body in order to secure the number of cells for cell transplantation in cell therapy for treating various diseases related to neural cell deficiency. It can be used as a cell culture agent.

In another embodiment of the present invention, the composition for promoting growth or differentiation of neural stem cells is dementia, Parkinson's disease, Alzheimer's disease, Peak's disease, Huntington's disease, epilepsy, stroke, stroke, ischemic brain disease, degenerative brain disease, memory loss, Traumatic central nervous system disease, spinal cord injury, peripheral nerve injury, amyotrophic axonal sclerosis, peripheral nerve disease, behavioral disorders, developmental disorders, mental retardation, Down syndrome or schizophrenia. Can be.

In addition, the present invention includes a peptide having cell penetrating activity consisting of or including a part of the Lin28 protein.

The peptide containing a part of the Lin28 protein may be a fusion protein fused with another protein or another protein delivery domain, and the other protein may include all existing proteins requiring cell penetration, and the protein delivery domain (PTD ) Is a trans-activating transcriptional activator (TAT) derived from human immunodeficiency virus type I (HIV-1); Antp (Antennapedia or penetratin) peptide, which is an Antennapedia Homeodomain of Drosophila; Mph-1 (Mutator phenotype protein 1) of murine transcription factors, VP22 (viral protein 22) of HSV-1 (Herpes simplex virus); Herring protamine HP4 (human protamine P4); Transportan, MAP (Model amphipathic peptide), TP10 (Transportan-10), CTP (Cardiac targeting peptide), K5-FGF (K5-fibroblast growth factor, AAVALLPAVLLALLP), HAP-1 (Huntingtin-associated protein 1, SFHQFARATLAS), 293P-1 (SNNNVRPIHIWP), CADY (cysteamidation PTD, Ac-GLWRALWRLLRSLWRLLWRA-Cya [SEQ ID NO: 14]), PF6 (PepFect6, Stearyl-AGYLLGK(εNH)INLKALAALAKKIL-NH ₂ ), RXR (arginine rich peptide), Poly-arginine (Rn (n = 6-12)), poly-lysine, or a modified peptide thereof (e.g., a peptide modified from the 47-57 amino acid residues of the TAT protein, etc.) One selected from the group consisting of is preferable, but any one that can deliver proteins into cells can be used.

In addition, the present invention provides a DNA encoding the peptide, a vector into which the DNA is inserted, and a transformed cell into which the vector is introduced. DNA encoding the peptide of the present invention, a vector into which the DNA is inserted, and a transformed cell into which such a vector is introduced are produced using a known technique. The DNA may be, for example, artificially synthesized DNA (eg, degenerate mutants) as long as it encodes the peptide of the present invention.

The peptide of the present invention also provides a peptide prepared using cells or an artificially synthesized peptide. The peptides of the present invention may be obtained as recombinants or artificially synthesized by putting DNA encoding the peptide into an appropriate expression system. In order to obtain the peptide of the present invention according to genetic engineering techniques, the DNA encoding the peptide may be put into an appropriate expression system for expression.

The method of administering the composition of the present invention includes oral administration and parenteral administration. Specifically, the parenteral administration method includes, but is not limited to, injection administration, nasal administration, transpulmonary administration, transdermal administration, and the like. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc., by using the composition of the present invention systemically or locally (for example, subcutaneous, intradermal, skin surface, eye or eyelid conjunctiva, nasal mucosa, oral cavity It can be administered to the mucous membrane of the internal and digestive tract, the mucous membrane of the vagina and the uterus, the damaged area, etc.

In addition, the administration method of the composition of the present invention includes, for example, intravascular administration (intraarterial administration, intravenous administration, etc.), intravascular administration, intramuscular administration, subcutaneous administration, intradermal administration, and intraperitoneal administration. It is not limited to things. In addition, the administration site is not limited and may include, for example, a tissue site requiring regeneration or a location near it, a location different from the tissue requiring regeneration, or a site distal to and different from the tissue requiring regeneration. Examples include, but are not limited to, blood (intraarterial, intravenous, etc.), muscle, subcutaneous, intradermal, abdominal cavity, and the like. In addition, an appropriate administration method can be selected according to the patient's age and symptoms. When administering the protein or peptide of the present invention, for example, the one-time dose of the protein may be selected in the range of 0.0000001 mg to 1000 mg per 1 kg of the patient's body weight. Alternatively, for example, a dosage may be selected in the range of 0.00001 to 100000 mg/body per patient. In the case of administering a vector for gene therapy into which a cell secreting a protein or peptide of the present invention or a DNA encoding the peptide is inserted, it may be administered so that the amount of the protein or peptide falls within the above range. However, the pharmaceutical composition of the present invention is not limited to such dosage.

The pharmaceutical composition of the present invention may be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA), and may contain a pharmaceutically acceptable carrier or additive together. . Examples include, but are not limited to, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrating agents, lubricants, fluidity accelerators, sweetening agents, etc. Can be used appropriately. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinyl acetaldiethylamino acetate, polyvinyl pyrroly. Pigs, gelatin, triglycerides of medium-chain fatty acids, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, and the like.

Hereinafter, the present invention is described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

Example: Experimental process and results

(1) Confirmation of cell-penetrating peptides that are far superior to cell-penetrating peptides than previously known cell-penetrating peptides

The peptides having the amino acid sequence described in the present invention are the cell-penetrating peptides (Lin-pep) [SEQ ID NO: 5] and Lin-half [SEQ ID NO: 12] with cell-penetrating ability derived from Lin28. Lin-half corresponds to the peptide sequence at the front half of the Lin-pep sequence. To compare the penetration efficiency of Tat-PTD, a previously well-known cell-penetrating peptide, and WT1-pTj, a cell-penetrating peptide reported in a recent paper, an experiment was conducted by including it as a control.

Each of the cell-penetrating peptides was synthesized by LifeTein (USA) in the form of attaching FITC, and the Heparin & Trypsin washing method used in the Keplan group was applied to remove each peptide that may be attached to the cell membrane surface to measure the cell permeation efficiency. [Kaplan IM, Wadia JS, Dowdy SF (2005) Cationic TAT peptide transduction domain enters cells by macropinocytosis. J Controlled release 102: 247-253]. In summary, each peptide was added to a culture medium at a concentration of 1 μM, incubated at 37° C. for 1 hour, and then the cells were washed 3 times with Heparin, and the cells were removed by trypsin for FACS analysis. I did it.

The cell-penetrating peptide (Lin-pep) derived from Lin28 shows an efficiency of more than 90% of permeation into the cells in just 1 hour at a concentration of 1 μM for mesenchymal stem cells (MSC), and has a very high cell permeability. It can be seen that it is a peptide. In the case of Lin-half, it was found that the cell permeability was 50-70% more efficient than that of Lin-pep. In addition, in the case of Tat-PTD, when serum is present in the medium, its permeation efficiency is significantly lowered, and when treated at a concentration of 1 μM for 1 hour, it exhibits a very low level of permeation efficiency (FIG. 1).

Unlike mesenchymal stem cells, when measuring the cell permeation efficiency in neural stem cells (NSCs) grown in serum-free medium conditions, Tat-PTD was also found to flow into the cells at a level of 70-80%. It was found (Fig. 2). Therefore, it can be seen that the permeation efficiency of Tat-PTD is greatly affected by the type of cells and the presence of serum, and the cell permeation efficiency of Tat-PTD to mesenchymal stem cells is poor in the presence of serum. I could see that. On the other hand, the cell-penetrating peptide derived from Lin28 showed very high permeation efficiency regardless of the presence of serum.

And, as shown in Fig. 3a, when treated for 5 hours, it could be observed that it clearly flowed into the cells with the naked eye using a fluorescence microscope. In addition, the average fluorescence intensity was compared to determine whether the amount of the cell-penetrating peptide introduced per unit cell increased. As a result, it was found that as the treatment time increased (time-dependent) and the cell-penetrating peptide concentration increased (dose-dependent), the number of peptides introduced into the cell increased (FIG. 3B ).

In addition, to determine whether the Lin28-derived cell-penetrating peptide (Lin-pep) actually exists in the nucleus or cytoplasm of the cell, it was analyzed using confocal microscopy after Lin-pep treatment. As a result of checking the microscope after 18 hours elapsed after the peptide treatment, it was confirmed that most of Lin-pep was present in the cytoplasm (FIG. 4A). As an additional experiment, a membrane marker (FM-64) was used to determine whether it actually exists inside the cell membrane, and to allow the reagent to be well inserted into the cell membrane, the cells were removed with trypsin, and the reagent was treated and analyzed. Even when doing so, it could be said that most of the peptides were present in the cytoplasm inside the cell membrane. In addition, it was found that some peptides were also present in the nucleus (Fig. 4b).

Cell-penetrating peptides such as Tat-PTD are known to have low permeation efficiency in primary cells, so we examined the permeation efficiency of Lin-pep in primary cells derived from mouse lymph nodes. Compared to Tat-PTD or MTS peptide, Lin-pep had a high efficiency cell permeation ability (FIG. 5A). Likewise, it was found that fibroblasts were almost permeated into most of the cells at a low concentration of 1 uM (Fig. 5b).

(2) Efficacy of Lin-pep derived from Lin28

Lin28 protein is considered to be one of the pluripotency factor and transcription factor, and when it is mass-expressed by introducing a gene expression system into a cell, it has been reported to increase growth or quickly heal injured areas. It is a protein that is receiving (Fig. 6). Therefore, if such a protein is a protein that has the property of being able to enter the cell by itself, only the purified protein of Lin28A is not used, without using a gene expression system (gene therapy is subject to a lot of caution and limitation in human treatment). Treatment alone has the advantage of being able to be used as a factor of the above-described therapeutic effect or dedifferentiation.

For mass expression and purification of the human Lin28A gene, construction was performed on an E. coli expression vector called pET11. In order to facilitate purification, the protein was prepared so that the six histidine-attached His tags could be expressed together (FIG. 7A). In order to confirm the expression of Lin28 protein, the expression strain was selected by transforming it into the E. coli BL21 strain, which is an expression strain, and when the strain was grown to an OD of 0.5, induction was performed at a concentration of 0.4 mM IPTG and incubated for 6 hours. . It was confirmed by SDS-PAGE that the expression was stably around 20 kDa compared to the strain not induction by IPTG (Fig. 7B). In addition, since the His tag was present, affinity chromatography was used, and in order to maintain the activity of the protein to be purified, it was purified by a native condition purification method that does not use urea (Fig. 7C). In an experiment with Lin28 cells, to use as a control, a vector was constructed so that GFP can also be expressed in E. coli by attaching His tag to it, and it was confirmed that it is well expressed in E. coli. In addition, it was purified using the His tag purification method in the same manner as the Lin28 protein.

In order to examine the effect of the purified Lin28 protein, it was confirmed by a proliferation assay method using NIH3T3. A GFP protein was used as a control, and a vector was prepared, expressed, and purified in the same manner as Lin28. The cells were treated with a concentration of 0.5 and 2.0 μM of purified GFP and Lin28 proteins, and treated twice on the first and second days. Upon follow-up observation, it was confirmed that there was a difference in the cell proliferation rate so that it could be distinguished with the naked eye from the third day, and on the fourth day, the cells were detached with trypsin and the number of cells was measured. In the case where Lin28 protein (SEQ ID NO: 1) was treated at a concentration of 0.5 and 2.0 μM, compared with the case where it was not treated and the case where the GFP protein was treated, results of promoting proliferation were obtained. When treated at a concentration of 0.5 μM, growth promotion of 32.7% was exhibited compared to the control group, and when treatment at a concentration of 2.0 μM showed a growth promoting effect of 56.0% (FIG. 8). In particular, when treated with a concentration of 2.0 μM, it was confirmed that the cells clump together and grow.

In order to find out whether the effect of promoting cell growth by Lin28-derived cell-penetrating peptide (Lin-pep) appears in stem cells, the number of cells after 2 days after treatment with Lin-pep 5 μM concentration in human MSCs. Was measured. As shown in FIG. 9A, there was no difference in cell growth between untreated cells and Tat-PTD used as a control, but a cell growth promoting effect of about 61% was observed in the Lin-pep-treated group. Another stem cell, a mouse neural stem cell (mNSC), was treated with a concentration of 3 μM Lin-pep, and then the number of cells was measured 1 day later. 9b).

In addition, the Lin28 protein was subjected to migration analysis for MSCs derived from mice. When the Lin28 protein expressed and purified in E. coli is added to the medium in which MSC is growing, it is expected that if it is automatically introduced into the cell due to the cell penetrating ability of Lin28 itself, the phenomenon that promotes the MSC migration ability can be confirmed.

One day before Lin28 treatment, MSCs were grown in culture plates. The next day, the first time that Lin28 was treated was set to 0 hours, and the phenomenon of promoting the movement of MSC by Lin28 was observed with a microscope. Treatment was performed twice every 6 hours, and observed under a microscope at 6 and 12 hours after the initial treatment. When compared with the case without treatment and the case where the GFP protein was treated, it was confirmed that migration was markedly promoted in the Lin28 treatment group (FIG. 10A). This time, in order to find out the effect of Lin-pep as well, we examined the effect of promoting migration in human-derived mesenchymal stem cells. When a cell migration assay kit was used and Lin-pep at a concentration of 5 μM was treated, it exhibited a distinct effect of promoting cell migration compared to the untreated cell group (FIG. 10B). As described above, it was demonstrated that not only the composition purely purified Lin28 protein, but also the Lin28-derived cell-penetrating peptide (Lin-pep) has the effect of promoting the ability to move mesenchymal stem cells. When damaged tissues or wounded areas occur, nearby MSCs receive a signal and move to the wound area and play a role in promoting the regeneration of the damaged area or wound area. Lin28-derived cell-penetrating peptide (Lin-pep) It has been suggested that MSC's ability to move to a higher level can be enhanced to promote regeneration.

In addition, an experiment was conducted to confirm whether colony-forming unit-fibroblasts (CFU-F), which can be an important indicator for the regenerative ability of stem cells, are accompanied by an increase. To determine the significance of the difference, the Tat-PTD peptide was also used as a control. The Tat-PTD and Lin-pep peptides were used at a concentration of 5 μM, and for His-Lin28, a protein having a relatively larger size than the peptide, a concentration of 0.5 μM was used. After incubation for 18 hours after treatment, 500 cells were cultured with a medium in a 10 cm plate, and the number of CFU-F was observed after 3 weeks. There was no significant difference between the untreated cell group and the Tat-PTD-treated cell group, but the number of CFU-F increased by about 42% in the case of Lin-pep and 56% in the case of His-Lin28. Results were shown (Fig. 11).

On the other hand, as shown in Fig. 9, since it was found that the cell growth promoting effect of Lin-pep has a higher growth effect in neural stem cells (NSC, Fig. 9b) than in mesenchymal stem cells (MSC, Fig. 9a), other effects To see if there are any, I first observed the change in shape. As shown in FIG. 12A, the morphology of adherent cells extending axons was very well observed in the cell group treated with Lin-pep at a concentration of 2 μM compared to the cell group not treated with the form of NSC. It was judged that this form has the potential to induce differentiation into neuronal cells. Therefore, Western blot analysis was performed to determine whether beta-III tubulin (Tuj1), which is well known as an early marker of differentiated neurons in subsequent experiments, was induction. As shown in FIG. 12B, beta-III tubulin marker was not observed in the untreated control group, whereas initial neuronal differentiation marker was observed in the Lin-pep treatment group. In neural stem cells, it was observed that the initial marker expression of neurons appeared within one day of Lin-pep treatment, and it was observed that this initial marker was expressed in the cytoplasm even in immunofluorescence (Immunofluorocence) (FIG. 12C). In immunofluorescence, this is the result of an experiment 3 days after Lin-pep treatment.

In conclusion, we suggest that the Lin-pep domain, which has the ability to introduce Lin28 protein into cells, is very important. After it was first reported in 1988 that a protein called Tat, a protein derived from the HIV-1 virus, has the ability to automatically enter the cell, it was found that the Tat-PTD domain at the intermediate position leads the role. Since then, hundreds of cell-penetrating peptides have been reported. The amino acid sequence for which the patent is being applied is a cell-penetrating peptide capable of introducing Lin28 protein into cells. The cell-penetrating efficiency is significantly higher than that of the Tat-PTD peptide or the previously reported WT1-pTj peptide with a similar site. It was revealed that it possesses not only cell permeation efficiency but also actual functional properties, which not only enables the development of new drugs targeting Lin28 protein, but also enables development as a protein capable of cell permeation targeting other promising proteins with excellent functionality. It is believed to be.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Cell-permeable peptide <130> P16U11C1730 <150> KR 10-2014-0050751 <151> 2014-04-28 <160> 12 <170> KopatentIn 2.0 <210> 1 <211> 209 <212> PRT <213> Human Lin28a amino acid sequence <400> 1 Met Gly Ser Val Ser Asn Gln Gln Phe Ala Gly Gly Cys Ala Lys Ala 1 5 10 15 Ala Glu Glu Ala Pro Glu Glu Ala Pro Glu Asp Ala Ala Arg Ala Ala 20 25 30 Asp Glu Pro Gln Leu Leu His Gly Ala Gly Ile Cys Lys Trp Phe Asn 35 40 45 Val Arg Met Gly Phe Gly Phe Leu Ser Met Thr Ala Arg Ala Gly Val 50 55 60 Ala Leu Asp Pro Pro Val Asp Val Phe Val His Gln Ser Lys Leu His 65 70 75 80 Met Glu Gly Phe Arg Ser Leu Lys Glu Gly Glu Ala Val Glu Phe Thr 85 90 95 Phe Lys Lys Ser Ala Lys Gly Leu Glu Ser Ile Arg Val Thr Gly Pro 100 105 110 Gly Gly Val Phe Cys Ile Gly Ser Glu Arg Arg Pro Lys Gly Lys Ser 115 120 125 Met Gln Lys Arg Arg Ser Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly 130 135 140 Leu Asp His His Ala Lys Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys 145 150 155 160 Cys His Phe Cys Gln Ser Ile Ser His Met Val Ala Ser Cys Pro Leu 165 170 175 Lys Ala Gln Gln Gly Pro Ser Ala Gln Gly Lys Pro Thr Tyr Phe Arg 180 185 190 Glu Glu Glu Glu Glu Ile His Ser Pro Thr Leu Leu Pro Glu Ala Gln 195 200 205 Asn <210> 2 <211> 250 <212> PRT <213> Human Lin28b amino acid sequence <400> 2 Met Ala Glu Gly Gly Ala Ser Lys Gly Gly Gly Glu Glu Pro Gly Lys 1 5 10 15 Leu Pro Glu Pro Ala Glu Glu Glu Ser Gln Val Leu Arg Gly Thr Gly 20 25 30 His Cys Lys Trp Phe Asn Val Arg Met Gly Phe Gly Phe Ile Ser Met 35 40 45 Ile Asn Arg Glu Gly Ser Pro Leu Asp Ile Pro Val Asp Val Phe Val 50 55 60 His Gln Ser Lys Leu Phe Met Glu Gly Phe Arg Ser Leu Lys Glu Gly 65 70 75 80 Glu Pro Val Glu Phe Thr Phe Lys Lys Ser Ser Lys Gly Leu Glu Ser 85 90 95 Ile Arg Val Thr Gly Pro Gly Gly Ser Pro Cys Leu Gly Ser Glu Arg 100 105 110 Arg Pro Lys Gly Lys Thr Leu Gln Lys Arg Lys Pro Lys Gly Asp Arg 115 120 125 Cys Tyr Asn Cys Gly Gly Leu Asp His His Ala Lys Glu Cys Ser Leu 130 135 140 Pro Pro Gln Pro Lys Lys Cys His Tyr Cys Gln Ser Ile Met His Met 145 150 155 160 Val Ala Asn Cys Pro His Lys Asn Val Ala Gln Pro Pro Ala Ser Ser 165 170 175 Gln Gly Arg Gln Glu Ala Glu Ser Gln Pro Cys Thr Ser Thr Leu Pro 180 185 190 Arg Glu Val Gly Gly Gly His Gly Cys Thr Ser Pro Pro Phe Pro Gln 195 200 205 Glu Ala Arg Ala Glu Ile Ser Glu Arg Ser Gly Arg Ser Pro Gln Glu 210 215 220 Ala Ser Ser Thr Lys Ser Ser Ile Ala Pro Glu Glu Gln Ser Lys Lys 225 230 235 240 Gly Pro Ser Val Gln Lys Arg Lys Lys Thr 245 250 <210> 3 <211> 209 <212> PRT <213> Mouse (Mus musculus) Lin28a amino acid sequence <400> 3 Met Gly Ser Val Ser Asn Gln Gln Phe Ala Gly Gly Cys Ala Lys Ala 1 5 10 15 Ala Glu Lys Ala Pro Glu Glu Ala Pro Pro Asp Ala Ala Arg Ala Ala 20 25 30 Asp Glu Pro Gln Leu Leu His Gly Ala Gly Ile Cys Lys Trp Phe Asn 35 40 45 Val Arg Met Gly Phe Gly Phe Leu Ser Met Thr Ala Arg Ala Gly Val 50 55 60 Ala Leu Asp Pro Pro Val Asp Val Phe Val His Gln Ser Lys Leu His 65 70 75 80 Met Glu Gly Phe Arg Ser Leu Lys Glu Gly Glu Ala Val Glu Phe Thr 85 90 95 Phe Lys Lys Ser Ala Lys Gly Leu Glu Ser Ile Arg Val Thr Gly Pro 100 105 110 Gly Gly Val Phe Cys Ile Gly Ser Glu Arg Arg Pro Lys Gly Lys Asn 115 120 125 Met Gln Lys Arg Arg Ser Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly 130 135 140 Leu Asp His His Ala Lys Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys 145 150 155 160 Cys His Phe Cys Gln Ser Ile Asn His Met Val Ala Ser Cys Pro Leu 165 170 175 Lys Ala Gln Gln Gly Pro Ser Ser Gln Gly Lys Pro Ala Tyr Phe Arg 180 185 190 Glu Glu Glu Glu Glu Ile His Ser Pro Ala Leu Leu Pro Glu Ala Gln 195 200 205 Asn <210> 4 <211> 271 <212> PRT <213> Mouse (Mus musculus) Lin28b amino acid sequence <400> 4 Met Ala Glu Gly Gly Ala Ser Lys Gly Glu Glu Pro Glu Lys Leu Pro 1 5 10 15 Gly Leu Ala Glu Asp Glu Pro Gln Val Leu His Gly Thr Gly His Cys 20 25 30 Lys Trp Phe Asn Val Arg Met Gly Phe Gly Phe Ile Ser Met Ile Ser 35 40 45 Arg Glu Gly Asn Pro Leu Asp Ile Pro Val Asp Val Phe Val His Gln 50 55 60 Ser Lys Leu Phe Met Glu Gly Phe Arg Ser Leu Lys Glu Gly Glu Pro 65 70 75 80 Val Glu Phe Thr Phe Lys Lys Ser Pro Lys Gly Leu Glu Ser Ile Arg 85 90 95 Val Thr Gly Pro Gly Gly Ser Pro Cys Leu Gly Ser Glu Arg Arg Pro 100 105 110 Lys Gly Lys Thr Leu Gln Lys Arg Lys Pro Lys Gly Asp Arg Trp Arg 115 120 125 Arg Gln Asp Leu Leu Met Asp Gln Met Trp Thr Val Arg Glu Glu Glu 130 135 140 Ser Arg Met Ile Pro Arg Cys Tyr Asn Cys Gly Gly Leu Asp His His 145 150 155 160 Ala Lys Glu Cys Ser Leu Pro Pro Gln Pro Lys Lys Cys His Tyr Cys 165 170 175 Gln Ser Ile Met His Met Val Ala Asn Cys Pro His Lys Leu Ala Ala 180 185 190 Gln Leu Pro Ala Ser Ser Gln Gly Arg Gln Glu Ala Glu Ser Gln Pro 195 200 205 Cys Ser Ser Ala Ala Pro Arg Glu Val Gly Gly Gly His Gly Cys Thr 210 215 220 Val Leu Phe Pro Gln Glu Val Lys Ser Glu Met Ala Glu His Ser Asp 225 230 235 240 Arg Ser Pro Gln Glu Val Ser Ser Thr Lys Ala Phe Ala Ala Ile Gly 245 250 255 Glu Gln Asn Lys Lys Gly Pro Leu Ile Gln Lys Arg Lys Lys Thr 260 265 270 <210> 5 <211> 62 <212> PRT <213> H. sapiens (human) Lin28a <400> 5 Gly Ser Glu Arg Arg Pro Lys Gly Lys Ser Met Gln Lys Arg Arg Ser 1 5 10 15 Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly Leu Asp His His Ala Lys 20 25 30 Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys Cys His Phe Cys Gln Ser 35 40 45 Ile Ser His Met Val Ala Ser Cys Pro Leu Lys Ala Gln Gln 50 55 60 <210> 6 <211> 64 <212> PRT <213> H. sapiens (Human) Lin28b <400> 6 Gly Ser Glu Arg Arg Pro Lys Gly Lys Thr Leu Gln Lys Arg Lys Pro 1 5 10 15 Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly Leu Asp His His Ala Lys 20 25 30 Glu Cys Ser Leu Pro Pro Gln Pro Lys Lys Cys His Tyr Cys Gln Ser 35 40 45 Ile Met His Met Val Ala Asn Cys Pro His Lys Asn Val Ala Gln Pro 50 55 60 <210> 7 <211> 62 <212> PRT <213> Mus musculus (mouse) Lin28a <400> 7 Gly Ser Glu Arg Arg Pro Lys Gly Lys Asn Met Gln Lys Arg Arg Ser 1 5 10 15 Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly Leu Asp His His Ala Lys 20 25 30 Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys Cys His Phe Cys Gln Ser 35 40 45 Ile Asn His Met Val Ala Ser Cys Pro Leu Lys Ala Gln Gln 50 55 60 <210> 8 <211> 89 <212> PRT <213> Mus musculus (mouse) Lin28b <400> 8 Gly Ser Glu Arg Arg Pro Lys Gly Lys Thr Leu Gln Lys Arg Lys Pro 1 5 10 15 Lys Gly Asp Arg Trp Arg Arg Gln Asp Leu Leu Met Asp Gln Met Trp 20 25 30 Thr Val Arg Glu Glu Glu Ser Arg Met Ile Pro Arg Cys Tyr Asn Cys 35 40 45 Gly Gly Leu Asp His His Ala Lys Glu Cys Ser Leu Pro Pro Gln Pro 50 55 60 Lys Lys Cys His Tyr Cys Gln Ser Ile Met His Met Val Ala Asn Cys 65 70 75 80 Pro His Lys Leu Ala Ala Gln Leu Pro 85 <210> 9 <211> 62 <212> PRT <213> Xenopus laevis Lin28a <400> 9 Gly Ser Glu Arg Arg Pro Lys Val Lys Gly Gln Gln Lys Arg Arg Gln 1 5 10 15 Arg Gly Asp Arg Cys Tyr Asn Cys Gly Gly Leu Asp His His Ala Lys 20 25 30 Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys Cys His Phe Cys Gln Asn 35 40 45 Pro Asn His Met Val Ala Gln Cys Pro Glu Lys Ala Met Gln 50 55 60 <210> 10 <211> 60 <212> PRT <213> Drosophila melanogaster Lin28a <400> 10 Gly Ser Thr Tyr Arg Pro Arg Ile Asn Arg Arg Thr Arg Arg Met Arg 1 5 10 15 Cys Tyr Asn Cys Gly Glu Phe Ala Asn His Ile Ala Ser Glu Cys Ala 20 25 30 Leu Gly Pro Gln Pro Lys Arg Cys His Arg Cys Arg Gly Glu Asp His 35 40 45 Leu His Ala Asp Cys Pro His Lys Asn Val Thr Gln 50 55 60 <210> 11 <211> 67 <212> PRT <213> C. elegans Lin28 <400> 11 Gly Ser Arg Ile His Pro Leu Gly Arg Lys Lys Ala Val Ser Leu Arg 1 5 10 15 Cys Phe Arg Cys Gly Lys Phe Ala Thr His Lys Ala Lys Ser Cys Pro 20 25 30 Asn Val Lys Thr Asp Ala Lys Val Cys Tyr Thr Cys Gly Ser Glu Glu 35 40 45 His Val Ser Ser Ile Cys Pro Glu Arg Arg Arg Lys His Arg Pro Glu 50 55 60 Gln Val Ala 65 <210> 12 <211> 30 <212> PRT <213> H. sapiens (human) Lin28a <400> 12 Lys Arg Arg Ser Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly Leu Asp 1 5 10 15 His His Ala Lys Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys 20 25 30

Claims (2)

A cell-permeable carrier comprising a cell-permeable peptide represented by the amino acid sequence of SEQ ID NO: 5 or 12.
A cell permeable peptide represented by the amino acid sequence of SEQ ID NO: 5 or 12;

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