KR101647804B1 - Novel Cell Penetrating Peptides and Uses Thereof - Google Patents
Novel Cell Penetrating Peptides and Uses Thereof Download PDFInfo
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- KR101647804B1 KR101647804B1 KR1020150089967A KR20150089967A KR101647804B1 KR 101647804 B1 KR101647804 B1 KR 101647804B1 KR 1020150089967 A KR1020150089967 A KR 1020150089967A KR 20150089967 A KR20150089967 A KR 20150089967A KR 101647804 B1 KR101647804 B1 KR 101647804B1
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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Abstract
The present invention relates to novel cell permeable peptides and uses thereof. The peptide of the present invention is composed of a small number of amino acids (specifically, four amino acids) and has a high specific gravity of hydrophobic amino acids, so that it is not easily degraded by a degrading enzyme and has excellent cell permeability. In addition, the target cargo (for example, siRNA) can be stably and effectively delivered intracellularly (cytoplasmic) by binding to the target cargo at both ends of the peptide of the present invention, particularly at the C-terminus. Accordingly, the drug delivery system comprising the peptide of the present invention, and the method and composition for delivering the target cargo using the peptide of the present invention are useful for the treatment of diseases or diseases, detection of specific cells, diagnosis of diseases (e.g. cancer) In-vivo imaging, and the like.
Description
The present invention relates to novel cell permeable peptides and uses thereof.
Small interfering RNAs (siRNAs) have potential therapeutic potential because they can selectively regulate specific genes. RNA interference (RNAi) is a typical method of inhibiting gene expression. When two siRNAs are administered, an RNA-induced silencing complex is formed and digested from
Various vectors have been proposed for delivering nucleic acid drugs into cells, for example, polymers, liposomes, and virus-derived vectors. Non-viral vectors have the advantage of being free from immunogenicity and virulence of viral vectors, although their ability to penetrate cells is less than viral vectors. Improving the low cellular permeability of non-viral vectors may lead to good drug delivery.
As a new approach to non-viral systems, we have started to use peptides (cell penetrating peptides, CPPs) that permeate various bioactive molecules into cells. CPP consists of 5-30 amino acid sequences, and is easily spotted because it is easy to fuse with the molecule to be permeated, stable, and does not affect the fusion molecule. Therefore, it can be said that it is useful to deliver genes such as siRNA using CPP. Of the CPP sequences, the 48-60 amino acid sequence of the HIV-derived TAT protein was first known to have a transmembrane sequence and was able to deliver various molecules into the cell. Since the intracellular permeation process has been revealed, many cation-rich sequences have been produced to attach to the cell membrane with a negative charge and to be delivered into the cell. Typically, synthetic CPPs have been made using amino acids such as arginine, lysine, and histidine. CPP is composed of 5 short and 30 long amino acid sequences. Long CPPs are often cleaved by proteolytic enzymes or peptide degrading enzymes. Especially, positively charged sequences (arginine, lysine, histidine) It is reported that the more the more the content, the more decomposition occurs.
CPPs known to date can be divided into three broad categories: (i) amphipathic peptides (transportan, pep-1, MPG, pVEC, MAP, CADY); (ii) cationic peptides (polyarginine, TAT, penetratin, P22N); And (iii) hydrophobic peptide (K-FGF, C105Y). Amphiphilic and cationic peptides are well known, while hydrophobic peptides are not artificially synthesized by the synthesis of sequences, so there are very few known peptides. CPP can penetrate cells that biological molecules can not pass through, which can enter the cell via glycosaminoglycan (GAG) in the cell membrane and can also flow through the endocytosis pathway. The binding between CPP and biological materials (e.g., siRNA, nucleic acids, small molecules, proteins, cytotoxic drugs, imaging agents, etc.) An important advantage of CPP over conventional delivery agents (e.g., liposomes, polymers, etc.) is low toxicity and immune rejection response to treatment when delivered intracellularly for therapeutic purposes or diagnostic purposes, And it can be improved by a processing technique.
Accordingly, CPPs used in clinics are extremely rare, and CPPs derived from viruses have problems such as affecting the immune response. Thus, it is urgently required in the art to find a carrier having a high delivery efficiency without being derived from a virus have.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have made efforts to develop a more efficient peptide capable of delivering a cargo of interest into a cell and a drug delivery system using the peptide. As a result, it has been found that a novel peptide comprising four amino acids derived from human annexin protein And the peptide is composed mainly of a hydrophobic amino acid and is efficiently applied to the intracellular delivery of a target transport cargo (for example, a drug such as siRNA) (specifically, siGFP fused with the peptide of the present invention) The present invention has been completed.
Accordingly, an object of the present invention is to provide a cell permeable peptide.
Another object of the present invention is to provide a nucleotide sequence encoding the above-mentioned peptide.
Another object of the present invention is to provide a drug delivery system comprising the above-described cell-penetrating peptide.
It is another object of the present invention to provide a method for intracellular delivery of a target cargo.
It is another object of the present invention to provide a composition for cargo transportation.
It is another object of the present invention to provide a transfection kit of a target cargo.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a cell penetrating peptide (CPP) comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 7.
According to another aspect of the present invention, the present invention provides a nucleotide sequence encoding the above-described cell-penetrating peptide.
According to another aspect of the present invention, the present invention provides a method for producing a cell-penetrating peptide comprising: (a) the above-described cell permeable peptide; (b) a cargo of interest bound to the peptide.
As a result of efforts to develop a more efficient peptide capable of delivering a target cargo into a cell and a drug delivery system using the same, the present inventors have identified a novel cell permeable peptide composed of four amino acids derived from human annexin protein, (SiGFP fused with the peptide of the present invention) to an intracellular delivery of a desired transport cargo (for example, a drug such as siRNA) composed of a hydrophobic amino acid.
When the cell-permeable peptide is delivered into the cell, the first step is to exchange the proteoglycans with the proteoglycans on the cell surface to perform the electrostatic bonding. In most of the cell-permeable peptides, interaction with HSPG (heparin sulfate proteoglycans) The present inventors irradiated proteins previously bound / interacted with the cell membrane to isolate / identify a plurality of cell permeable peptides from the annexin proteins likely to have an amino acid sequence having a cell penetrating function. Based on this, the peptides of the present invention composed of shorter amino acids were finally selected.
Annexin binds to the phospholipid of the cell membrane by calcium ion, which regulates the ion concentration through the cell membrane and is involved in the transport of the ion into and out of the cell membrane. It binds to F-actin, a protein involved in cell structure While controlling the dynamics of the cell structure. Anexin is classified into five major groups (A to E groups) (ie, humans, insects, fungi, plants and protists), depending on the organism. In addition, it has been reported by Gerke V et al ( Nature , 2005) that anexin binds to cell membranes and has the ability to move in and out of cells through endocytosis and exocytosis Review Mol Cell Biol . , 6: 449-461, 2005).
Serine and trypsin were very important for cell permeability in the annexin-derived peptide consisting of the 10 amino acid sequences previously identified by the present inventors (see FIG. 1). Therefore, the present inventors prepared various peptides composed of four amino acid sequences based on the amino acid residues.
More specifically, the peptide of the present invention comprises an amino acid sequence located at the N-terminus of the annexin protein. Generally, the cell-penetrating peptide of the present invention is a peptide represented by any one of the following general formulas 1 to 4
, And peptides of SEQ ID NO: 5 to SEQ ID NO: 7.
In the above formula 1 to formula 4, wherein X aa1, X aa3 and X aa4 is any one of amino acids of the hydrophobic amino acid other than the tryptophan (Trp), wherein X aa2 is any one of amino acids of the polar amino acid, X aa5 Is glycine (Gly), alanine (Ala), cysteine (Cys) or proline (Pro), and Xaa6 may be an amino acid of any one of basic amino acids. More specifically, the X aa1, X aa3 and X aa4 is alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met) or tyrosine (Tyr), the X aa2 is serine (Ser), threonine (Thr), asparagine (Asn) or glutamine (Gln), Xaa5 is glycine or alanine and Xaa6 is histidine (His) or lysine (Lys). Still more particularly, the X aa1, X aa3 and aa4 X is alanine, valine, and isoleucine or leucine, wherein X aa2 is serine, X aa5 is a glycine or alanine, X aa6 is a histidine.
In some embodiments of the present invention, the peptide of the present invention is a peptide represented by any one of the following formulas 3 to 6:
, And peptides of SEQ ID NO: 5 to SEQ ID NO: 7.
Wherein in the general formula 3 to formula 6, wherein X aa1, X aa3 and X aa4 is any one of amino acids of hydrophobic amino acids, except tryptophan, X aa5 is a glycine, alanine or cysteine, X aa6 is either of a basic amino acid Lt; / RTI > More specifically, the X aa1, X aa3 and aa4 X is alanine, valine, and isoleucine, leucine or methionine, X aa5 is a glycine or alanine, X aa6 is a histidine or lysine. Still more particularly, the X aa1, X aa3 and aa4 X is alanine, valine, isoleucine or leucine, and Shin, X aa5 is a glycine or alanine, X aa6 is a histidine.
In some embodiments of the invention, the peptides of the invention comprise the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 7, more specifically the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, 1 and SEQ ID NO: 3, and most specifically, the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 (see Table 2 and Fig. 2A).
In some embodiments of the invention, the peptides of the invention are permeable to the cytoplasm.
In some embodiments of the invention, the peptides of the invention have excellent cellular permeability at a concentration of 1 [mu] M or more, more specifically at a concentration of 5 [mu] M or more, and more particularly at a concentration of 10 [mu] M or more.
As used herein, the term "peptide" refers to a linear molecule formed by peptide bonds and amino acid residues joined together. The peptides of the present invention can be prepared according to chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem . Soc . 85: 2149-54 (1963) ; Stewart, et al., Solid Phase Peptide Synthesis , 2nd. ed., Pierce Chem. Co .: Rockford, 111 (1984)).
The peptide of the present invention is much more stable than natural annexin itself, but the stability can be further improved by modification of the amino acid.
In some embodiments of the present invention, the N-terminal or C-terminal of the peptide is an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a polyethylene glycol (PEG) A protecting group selected from the group consisting of amino acids can be additionally combined. In addition, the N-terminal of the peptide of the present invention may be modified into a hydroxyl group (-OH) or an amino group (-NH 2 ) to increase the stability.
The above-mentioned protecting group acts to protect the peptide of the present invention from attack of a protein cleaving enzyme in vivo. In addition, the modified peptide exhibits excellent thermal stability due to a protecting group, and is excellent in stability against physicochemical factors such as acid and alkali. Therefore, the peptide of the present invention can be advantageously applied to products requiring long-term storage such as medicines, quasi-drugs, cosmetics, and oral care products because they can be highly modified for long-term preservation. The above-mentioned modification of the amino acid acts to greatly improve the peptide stability of the present invention, and the term "stability" as referred to herein also means storage stability (for example, room temperature storage stability) as well as in vivo stability.
Generally, the protein transport domain (PTD) mainly contains basic amino acid residues such as lysine / arginine, and functions to penetrate the cell membrane and permeate into the cell. The protein transport domain (PTD) can be selected from the group consisting of HIV-1 Tat protein, the homeom domain of Drosophila Anapadia (Phenetradine), the HSV VP22 transcriptional regulatory protein, the vFGF-derived MTS peptide, the transposon or the Pep- Derived sequences, and the like. In this way, the virus or a cationic peptide with a variety of cell identification / synthetic peptide from the transmission (e.g., TAT 48 -60, page net Latin (pAntp) 43 -58, polyarginine, Pep-1, trans-formyl carbon, etc. ) Has an activity capable of internalizing cells for mediating the movement of biologically active substances, drug delivery vectors. However, the clinical results of drugs using conventional cell permeable peptides have not been successful yet. For example, PsorBan ® , a conjugate of polyarginine and cyclosporine, was discontinued at Phase II for the treatment of psoriasis. In addition, clinical Irl using the first cell permeable peptide, TATp, is currently under development (ISS P-002; Istituto Superiore di Sanita and Novartis). In addition, a study on the PTD (TAT-DRBD (double stranded RNA binding domain)) for cancer therapy is being conducted by Traversa, Inc. The above studies have been terminated or are currently in progress but have not yet been clinically approved. In this respect, important points in cell permeable peptide studies are cytotoxicity and degradation rate in serum. In particular, the degradation rate of peptides in serum is very important for delivering the target cargo to cells.
The peptide of the present invention is composed of four amino acid sequences containing a hydrophobic amino acid, and is predicted to have a very low degradation rate in serum. Thus, the possibility of development as a drug delivery system is very high. Thus, the present invention provides a method for the manufacture of a medicament comprising (a) the above-described cell permeable peptide; And (b) a cargo of interest bound to the peptide.
As described above, the peptide of the present invention can effectively transfer the target cargo bound thereto to the cytoplasm due to excellent cell permeability.
In some embodiments of the invention, the peptides of the present invention were treated at a concentration of 10 [mu] M and showed similar cellular permeability to a control group (AA3H) consisting of 10 amino acid sequences (see Figures 2a and 2b). Also, since the control group (AA3H) was stably maintained for at least 24 hours in terms of peptide degradation rate in the serum, it was predicted that the peptides of the present invention would also exhibit excellent serum stability (no result).
Typically, a cell permeable peptide can be constructed by linking a non-covalent or covalent bond with a target cargo. For example, a cell-permeable peptide and a target cargo may form a covalent bond conjugate through chemical cross-linking (Zatsepin, TS, et al . , Curr . Pharm . Des . , ≪ / RTI > 11: 3639-3654 (2005)). Therefore, the peptide of the present invention may function as a covalent bond or a non-covalent bond with the target cargo and deliver the same into cells or tissues. According to the present invention, the peptide of the present invention, as a target cargo, is linked to siRNA via a fluorescent moiety and transmits the siRNA through the cell membrane in a stable and highly efficient manner (see FIGS. 4A and 4B).
The term "cargo of interest" as used herein refers to a substance that is conjugated with the peptide of the present invention through covalent or non-covalent bonding to be delivered into a cell, for example, But are not limited to, peptides, peptides, polypeptides, antisense oligonucleotides, siRNA, shRNA, miRNA and peptide-nucleic acid (PNA).
The drug delivery system of the present invention may further include a linker between the peptide of the present invention and the target cargo. The linker used in the present invention may use various linkers known in the art unless they affect cell permeability. For example, a fluorescent substance (such as fluorescein, FITC (fluorescein isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine) (4,6-diamidino-2-phenylindole) and coumarin], fluorescence proteins (GFP, RFP, CFP, YFP, BFP, luciferase or mutants thereof), Cy-5, Texas Red, Alexa Fluor, ), radioactive isotopes (e.g., 14 C, 125 I, 32 P And S 35 ), chemicals (e.g., biotin), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). When the drug delivery system of the present invention comprises a radioactive isotope (i. E. Isotope labeled peptide and siRNA complex of the invention), single photon emission computed tomography (SPECT) or positron emission It can be applied to PET (Positron Emission Tomography) and used for tissue imaging.
In some embodiments of the invention, the subject cargo of the drug delivery vehicle of the invention is bound to the N- or C-terminus, more specifically the C-terminus, of the peptide described above, and the bond may be covalent or noncovalent .
In some embodiments of the invention, the linkage between the peptide and the target cargo is through the linker, and more specifically, the fluorescence moiety (e.g., FITC, biotin, streptomycin, Etc.).
The linker may be a linker consisting of a length and / or sequence, specifically a plurality of amino acid residues, selected specifically to maximize the cytotoxic activity of the peptide of the present invention. The linker consisting of the amino acid sequence is described in Huston, et al. , Methods in Enzymology , 203: 46-88 (1991), and Whitlow, et al . , Protein Eng. , 6: 989 (1993), which is incorporated herein by reference.
In addition, the drug delivery system of the present invention can be used for various purposes. Specifically, the drug delivery system of the present invention can be used for transportation of substances such as chemicals, nucleic acids, nanoparticles, etc., and the nucleic acid includes antisense oligonucleotides, siRNA, shRNA, miRNA and PNA It is not. In addition, the drug delivery system of the present invention can be used for treatment of diseases or diseases, detection of specific cells, diagnosis of diseases (for example, cancer), tracking of specific cells and in vivo imaging according to cartoons to be delivered . As used herein, the term "nucleic acid molecule" is meant to encompass both DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is the basic building block in the nucleic acid molecule, includes not only natural nucleotides, But may also include modified analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).
Meanwhile, the present invention provides a nucleotide sequence encoding the above-described cell-penetrating peptide (SEQ ID NO: 1 to SEQ ID NO: 7), and the nucleotide sequence is not limited as long as it is a nucleotide sequence encoding the above-mentioned peptide. But are not limited to, the nucleotide sequences of SEQ ID NO: 8 to SEQ ID NO: 14 for SEQ ID NO: 1 to SEQ ID NO: 7, respectively.
As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).
In addition, the present invention can provide a recombinant expression vector comprising a nucleotide sequence encoding the above-mentioned cell-penetrating peptide. More specifically, the recombinant expression vector of the present invention comprises (a) a promoter; And (b) a nucleotide sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 14 operatively linked to the promoter. Such vector systems can be constructed through a variety of methods known in the art, and specific methods for this can be found in Sambrook et < RTI ID = 0.0 > al ., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference. The term "promoter" as used herein refers to a DNA sequence that regulates the expression of an encoding sequence or a functional RNA. As used herein, the term "operatively linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter sequences, signal sequences, or transcription factor binding sites) , Whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequences.
According to another aspect of the present invention, there is provided an intracellular delivery method of a target cargo comprising the step of contacting the above-mentioned drug carrier with a cell.
According to still another aspect of the present invention, there is provided a composition for carrying a cargo comprising the above-mentioned drug carrier.
According to another aspect of the present invention, there is provided a transfection kit comprising the above-mentioned drug delivery vehicle.
Since the methods, compositions and kits of the present invention include the above-described cell-penetrating peptides and drug carriers containing the same as active ingredients, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.
The compositions of the present invention may be administered with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Suitable pharmaceutically acceptable carriers and preparations are described in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
The composition of the present invention is preferably administered parenterally. In the case of parenteral administration, it can be administered by intravenous injection, intradermal injection, intralesional injection, intramuscular injection, intraperitoneal injection, and the like. The appropriate dosage of the composition of the present invention may be variously determined by such factors as the formulation method, the manner of administration, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, It may generally range from 0.0001-100 mg / kg.
The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
In some embodiments of the invention, the methods and compositions of the present invention are administered to a mammal, more particularly a human, a mouse, a rat, a guinea pig, a rabbit, a monkey, a pig, a horse, But is not limited thereto.
Meanwhile, the transfection kit of the present invention is a system optimized for facilitating the introduction of external DNA / RNA into mammalian cells. To date, transfection kits for DNA / RNA are mainly calcium phosphate method, lipid conjugate method, and dextran conjugate method, but their efficiency is about 1/10 6 to 1/10 2 , and cell type There is a limit to dependence on. To overcome this, a transfection kit using a cell-permeable peptide / protein can be used.
The transfection kit of the present invention may further comprise a linker / binding factor between the peptide and the target cargo. The binding factor includes all or a portion of a protein capable of binding a target cargo such as a specific DNA / RNA sequence or protein including a transcription factor or a viral protein. For example, Gal4 is a DNA binding factor, a transcription factor widely used in eukaryotic, prokaryotic, and viruses. DNA / RNA binding factor may be used by applying in vivo, protein expression vectors capable of producing a fusion protein and the PTD in vitro. Fusion between DNA / RNA binding factors and PTDs can also be achieved by chemical bonding, physical covalent bonding, or noncovalent bonding.
By constructing a fused construct of the cell-penetrating peptide of the present invention and DNA / RNA and treating it outside the cell, it is possible to overcome the limitations depending on the efficiency and the cell type. It is possible to efficiently introduce DNA / RNA into cytoplasm of various cells in In vivo and in vitro using the peptide of the present invention and a DNA / RNA binding factor. For example, the delivery method may be through a variety of methods including intramuscular, intraperitoneal, intravenous, oral, subcutaneous, intradermal, intranasal, and inhalation. Therefore, the transfection kit of the present invention can provide a novel method for gene therapy and DNA / RNA vaccine by the method of the present invention, and can be transiently or permanently expressed and used for DNA / RNA vaccines and gene therapy It can be used in clinical applications as well as in basic research applications.
The features and advantages of the present invention are summarized as follows:
(a) The present invention relates to novel cell permeable peptides and uses thereof.
(b) The peptide of the present invention is composed of a small number of amino acids (specifically, four amino acids) and has a high specific gravity of hydrophobic amino acid, and is not well degraded by a degrading enzyme and has excellent cell permeability.
(c) Furthermore, the target cargo (for example, siRNA) can be stably and effectively delivered intracellularly (cytoplasmically) by binding to the target cargo at both ends of the peptide of the present invention, particularly at the C-terminus.
(d) Therefore, the drug delivery system comprising the peptide of the present invention, and the method and composition for delivering the target cargo using the peptide of the present invention are useful for the treatment of diseases or diseases, the detection of specific cells, the diagnosis of diseases Location tracking and in-beam imaging, and the like.
Fig. 1 shows the results of confirming the cell permeability of AA3H-CPP through alanine substitution.
2A and 2B show the results of confirming the cell permeability of the peptide of the present invention. FIG. 2A shows the cell permeability (left panel) and its numerical value graph (right panel) through flow cytometry, and FIG. 2B is a photograph showing fluorescence thereof.
FIG. 3A and FIG. 3B are the results showing the cell permeability of the construct, which is protein-fused at each end of the peptide of the present invention. FIG. 3A shows a comparison (upper panel) and a numerical result (lower panel) of comparison of cell permeation efficiency after protein fusion of N- and C-terminal of CPP through flow cytometry analysis. FIG. 3B shows the result of confirming the cell permeability of the protein fused to the C-terminal of CPP by fluorescence microscopy.
4A and 4B show results of siRNA delivery using the peptide CPP of the present invention. FIG. 4A is a flow cytometer showing the decrease in the expression of GFP through the fusion of CPP and siGFP, and the graphical result thereof (upper panel) and the graphical result thereof (bottom panel) confirming that siGFP has entered the cell. Fig. 4B shows the result of confirming the decrease in the expression level of GFP in CPP-siGFP by fluorescence microscopy.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Experimental Method
2-1. Peptides synthesis
Using a solid phase peptide synthesis was synthesized AA3H (A nnexin A3 H uman form) the 10 amino acid sequence, derived therefrom modified amino acids of the N- terminal sequence and a 4-mer CPP, link-amide MBHA resin (rink-amide methylbenzylhydrylamine resin (Novabiochem, # 855003), and the fluorescent substance, fluorescein isothiocyanate isomer I (Sigma, # F7250) was attached to the N-terminus. In the final stage 95% TFA (trifluoroacetic acid; Sigma , # 302031) /2.5% triisopropylsilane (triisopropylsilane; Sigma, # 233781) /2.5% H 2 O reaction mix at room temperature for 2 hours, diethyl ether (Sigma # 309966). The synthesized peptides were extracted on a reversed phase HPLC C18 column and each peptide was obtained at a concentration of 5-80% in water containing 0.1% TFA and acetonitrile (Fisher scientific # 955-1). Also, biotin (NHS, biotin (Lys) -ε amino) was attached to each peptide N- or C-terminus. Molecular size of each peptide was confirmed by mass spectroscopy (MALDI-TOF).
2-2. Of peptide Cell permeation efficacy analysis
To determine the cell permeability of each peptide, human cervical cancer cell line (HeLa) was treated with 1 μM or 10 μM of the peptide for 4 hours. After treatment, the cells were washed twice with CBS (Culbecco's phosphate buffered saline) and treated with 0.01% trypsin / EDTA for 10 minutes. Single cells were suspended in DPBS. The resulting cells were pelleted at 1,500 x g for 2 minutes using a centrifuge, washed twice with the same procedure, and then checked for cell permeability using a flow cytometer (Guava easyCyte, Milipore, USA). After the peptide treatment and washing twice, the cells were observed under a fluorescence microscope.
2-3. Fusion of biotin-labeled peptide with FITC-labeled streptavidin
(B-CPPs, CPPs-B) and streptavidin-FITC (STV-FITC) labeled with each N- or C-terminal were reacted at a ratio of 4: 1 for 1 hour at room temperature. Respectively. The peptides in the cells showed green fluorescence and were confirmed by flow cytometry and again confirmed by fluorescence microscopy under the same conditions.
2-4. Cell permeation Peptides and siGFP Comparison of Fusion and Efficacy
In order to fuse the siGFP with the cell permeable peptide, maleimide was connected to the peptide and a thiol group was attached to the C-terminal of the sense of siGFP. For the disulfide bond, the thiol group-attached siGFP was activated by treatment with DTT for 15 minutes and the remaining DTT was removed with a 7K zeba column (Cat # 89883; Thermo scientific, USA). The activated thiol siGFP was reacted overnight with the peptide at low temperature (4 ° C). The siGFP fused with the peptide was identified on a 12% native gel (results not shown).
The fused construct (CPPs-siGFP; 100 nM on siGFP) was reacted with HeLa-GFP cells for 48 hours. HeLa-GFP cells were cultured in the presence of 1 μg / mL of puromycin antibiotics. The decrease in the expression level of HeLa-GFP was confirmed by flow cytometry and fluorescence microscopy. Sequence information of siGFP used is shown in Table 1 below.
The siGFP sequence used.
Experiment result
3-1. Identification of major amino acid sequences by alanine scanning
Ten amino acid sequences were replaced with alanine to identify the major sequences. Alanine is a popular method for identifying the important role of sequence without secondary structure and no effect on other amino acids. The sequence, size and information of the sequence substituted with alanine are shown in Table 2. As can be seen in FIG. 1, serine (S3A) and tryptophan (W5A) among 10 amino acids were found to be important residues for cell permeation. Cell permeability was reduced by 31% when serine was substituted with alanine, and by 71% when tryptophan was substituted by alanine (Fig. 1). Therefore, it was found that serine and tryptophan are important residues in the cell permeable peptide sequence. The cell permeability was confirmed by dividing the peptide into serine and tryptophan, which are the major residues of cell permeation, into seven amino acids.
name
Information on peptide type, sequence, size isoelectric point, charge.
3-2. By Major Residue parted Of peptide Comparison of cell penetration efficiency
When the total of 7 peptides (MASI, ASIW, SIWV, IWVG, WVGH, VGHR and GHRG) identified in FIG. 1 were treated with low concentration (1 μM) and high concentration (10 μM) for 4 hours, CPP and cell permeability were compared. At low concentrations, SIWV more than twice the cell permeability of the original sequence, AA3H-CPP, and ASIW was as good as AA3H-CPP. At high concentrations, the cell permeability of AA3H-CPP was good, but among the other seven peptides, the SIWV peptide showed the best effect (Fig. 2a). The fluorescence microscope also showed the same results. At 1 μM, the SIWV cell permeability was found to be low (20 ×) and high (100 ×) (FIG. 2b). From the above results, four types of peptides (ASIW, SIWV, IWVG and WVGH) with good permeation efficiency were selected as the final candidates for delivery of biological materials in that four amino acid sequences alone had excellent cell permeation efficiency.
In addition, four amino acid sequences proved to be effective for cell permeability, and four types of peptides were selected in consideration of efficiency and importance of amino acid sequence. Based on this paper, it is shown that tryptophan is an important amino acid to exchange with the cell membrane through the existing paper. Based on this paper, it is shown that 4 kinds of efficient peptides (ASIW, SIWV, IWVG, WVGH) Were selected as candidates.
3-3. Through N- or C-terminal protein fusion CPP Comparison of Mass Transfer Efficiency
Biotin was attached to the N- or C-terminus of each CPP and cell permeability was compared via streptavidin fusion. A schematic diagram of streptavidin (STV) fused with biotin (B) is shown in the upper panel of Fig. 3a. STV-FITC fused with B-CPPs and CPPs-B showed fluorescence when penetrated into cells, and thus could be confirmed by flow cytometry. The fused CPPs-FITC and FITC-CPPs were compared to human cervical cancer cells (HeLa), which showed better efficiency when fusing the transmitter to the C-terminus of CPP. In particular, in the case of ASIW-CPP, it was confirmed that the permeation efficiency increased by 15 times or more when fused to the C-terminal than when the substance was fused to the N-terminal. In addition, SIWV-CPP was found to be 5 times more efficient when fused to the C-terminal. IWVG-CPP increased 4-fold when fused to the C-terminus and more than 2-fold increased to WVGH-CPP (FIG. When the permeability of CPPs-FITC was confirmed by fluorescence microscopy, ASIW-CPP showed the best efficiency (Fig. 3B). According to the results shown in FIG. 3, the cell permeability varies from 2 to 15 times depending on the location of the transducer, and thus it can be estimated that the efficiency differs depending on where the transduction substance is fused.
3-4. At the C-terminus siGFP Fused CPPs Comparison of efficiency
Based on the results shown in Fig. 3, the C-terminus of CPPs was fused with a transmitter to be delivered into cells. HeLa-GFP cells, in which GFP is expressed continuously, were treated with siGFP fused to each of four peptides and their efficiency was confirmed. Fused siGFP was treated with 100 nM for 48 h, and the results were confirmed by flow cytometry. As shown in Fig. 4a, GFP expression was decreased. The efficiency of reducing the expression level of GFP in each CPPs-siGFP was about 48-53%. Cy5.5 was attached to the sense strand of siGFP, and it was confirmed by flow cytometry that siGFP was introduced into the cell by CPP (Fig. 4A, lower panel). Intracellular delivery of siGFP showed similar efficiencies for all four CPPs. In addition, we confirmed that the expression level of GFP was decreased by fluorescence microscopy, showed the morphology of the cells with DIC, and fluorescence images of GFP were fluorescent cells. In addition, nuclear staining showed that the cells were normal, and each image was overlapped to determine the amount of GFP expression in which cells were reduced (FIG. 4B).
4. View
The present invention suggests that even four amino acid sequences are capable of cell penetration and can deliver proteins and genes. In addition, since the efficiency varies depending on the location of the transfer material, it will be a basic data suggesting an effective method for mass transfer using CPP. The synthesis of short amino acid sequences can be produced at a lower cost than the synthesis and preparation of conventional CPPs, and has the advantage of transferring the substance through a simple fusion method, and is expected to be useful as a carrier.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
references
1. Samad Mussa Farkhani et al ., Cell penetrating peptides: Efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules, Peptides , 57, 2014, 78-94.
2. Yuki Takahashi et al . , Nonviral vector-mediated RNA interference: RNAi-based gene therapy, Advanced Drug delivery reviews , 61 (9), 2009, 760-766.
3. Jan Hoyer et al ., Peptide vectors for the nonviral delivery of nucleic acid, Accounts of chenical research, 45 (7), 2012, 1048-1056.
4. Tamaki endoh et al., Cellular siRNA delivery using cell-penetrating peptides modifed for endosomal escape, Advanced drug delivery reviews , 61, 2009, 704-709.
5. Daniel Knappe et al., Easy strategy to protect antimicrobial peptides from fast degradation in serum, Antimicrobial agents and chemotherapy, 54 (9), 2010 , 4003-4005.
6. WS Eum et al . , Enhanced transduction of Cu, Zn-superoxide dismutase with HIV-1 Tat protein transduction domains at both termini, Mol . Cells , 19, 2005, 191-197.
7. Cherine Bechara et al ., Tryptophan within basic peptide sequence triggers glycosaminoglycan-dependent endocytosis, The FASEB Journal , 27, 2013, 738-749.
<110> Korea Institute of Science and Technology <120> Novel Cell Penetrating Peptides and Uses Thereof <130> K-07794 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AA3H_01 <400> 1 Ser Ile Trp Val One <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AA3H_02 <400> 2 Ala Ser Ile Trp One <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AA3H_03 <400> 3 Trp Val Gly His One <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AA3H_04 <400> 4 Ile Trp Val Gly One <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AA3H_05 <400> 5 Val Gly His Arg One <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AA3H_06 <400> 6 Gly His Arg Gly One <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> AA3H_07 <400> 7 Met Ala Ser Ile One <210> 8 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> AA3H_01_NT <400> 8 tcgatttggg tt 12 <210> 9 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> AA3H_02_NT <400> 9 gcttctatat gg 12 <210> 10 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> AA3H_03_NT <400> 10 tgggtgggcc ac 12 <210> 11 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> AA3H_04_NT <400> 11 atctgggtgg gg 12 <210> 12 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> AA3H_05_NT <400> 12 gttggccata ga 12 <210> 13 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> AA3H_06_NT <400> 13 ggtcacagag gg 12 <210> 14 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> AA3H_07_NT <400> 14 atggcgtcca ta 12
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| CN107987129A (en) * | 2017-12-25 | 2018-05-04 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
| KR20190078409A (en) * | 2017-12-26 | 2019-07-04 | 한국화학연구원 | Cancer microenvironment-sensitive combinatorial drug delivery system containing a new lipid-peptide-polymer conjugate as a substrate of cancer-overexpressing enzyme |
| KR20190141368A (en) | 2018-06-14 | 2019-12-24 | 한국과학기술연구원 | Novel cell penetrating peptide comprising beta-defensin dimer and uses thereof |
| KR102267519B1 (en) * | 2020-01-08 | 2021-06-21 | 경희대학교 산학협력단 | Nanoparticles for drug delivery surface-modified with brain tumor cell targeting peptide, preparation method thereof and use thereof |
| KR102274999B1 (en) * | 2020-04-23 | 2021-07-08 | 주식회사 아임뉴런바이오사이언스 | Novel cell penetrating peptides and use thereof |
| KR102274876B1 (en) * | 2020-12-24 | 2021-07-08 | 주식회사 아임뉴런바이오사이언스 | Novel cell penetrating peptides and use thereof |
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