KR101669203B1 - Novel Cell Penetrating Peptides and Uses Thereof - Google Patents

Abstract

The present invention relates to novel cell permeable peptides and uses thereof. The peptide of the present invention is a human anexin protein-derived amino acid sequence, which not only has excellent cell permeability but also shows a slow degradation rate in serum without cytotoxicity. In addition, the objective cargo (e.g., beta-galactosidase) bound to the peptide of the present invention is stably and effectively transported intracellularly (cytoplasmic). 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

Novel Cell Penetrating Peptides and Uses Thereof < RTI ID = 0.0 >

The present invention relates to novel cell permeable peptides and uses thereof.

As the function of major proteins plays an important role in the development and progression of diseases, low molecular compounds are used as main drugs to control intracellular disease-related proteins. However, It has been known that there is a limitation in controlling the activity of the target protein by binding. To overcome this, attempts have been made to develop peptides and protein-based drugs suitable for controlling the interaction between proteins and proteins. In order to successfully develop a protein drug for the purpose of regulating the activity of a target protein present in a cell, intracellular delivery and transport of the protein are indispensably required. In general, it has been known that intracellular transport of the protein is difficult. To overcome this disadvantage, a protein transduction domain (PTD), a cell penetrating peptide (CPP), is used as a substance that facilitates intracellular permeation of proteins. A new drug delivery system (DDS), which binds PTD or CPP to cells and delivers new drug candidate substances that could not be delivered into cells, has been spotlighted. CPP is the best known so far as a TAT-CPP derived from HIV virus transcription factors in vitro (in vitro) and in vivo (in vivo ) It has been known that CPPs derived from synthetic CPPs or viruses with similar properties and with similar properties are also effective. However, in the case of the above-mentioned CPP, especially CPP having a long amino acid sequence, there is a possibility that a protein is obtained through intracellular expression, and mutation is generated, and mass production is difficult. Therefore, the CPP used in clinical practice is extremely rare, and CPP derived from viruses have problems affecting the immune response, so it is urgent to find a carrier having high delivery efficiency without being derived from virus.

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 sought to develop a novel molecule / substance capable of delivering a cargo of interest into cells and a drug delivery system using the same. As a result, the present inventors identified novel cell permeable peptides from 12 kinds of human annexin proteins, and found that the peptides do not exhibit cytotoxicity but also exist in the serum for a long time without being degraded, , Drug) of the present invention (for example, beta-galactosidase fused with the peptide of the present invention).

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 recombinant vector comprising the above-described nucleotide sequence.

Still another object of the present invention is to provide a drug delivery system.

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.

Another object of the present invention is 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: 6.

According to another aspect of the present invention, the present invention provides a method for producing a cell-permeable peptide comprising: (a) the above-described cell permeable peptide; (b) a cargo of interest bound to the peptide.

According to another aspect of the present invention, the present invention provides a nucleotide sequence encoding the aforementioned cell permeable peptide.

According to another aspect of the present invention, the present invention provides a recombinant expression vector comprising the above-described nucleotide sequence.

The present inventors have sought to develop a novel molecule / substance capable of delivering a target cargo into cells and a drug delivery system using the same. As a result, the present inventors identified novel cell permeable peptides from 12 kinds of human annexin proteins, and found that the peptides do not exhibit cytotoxicity but also exist in the serum for a long time without being degraded, , Drug) into the intracellular delivery of beta-galactosidase (e. G., Beta-galactosidase fused with the peptide of the invention).

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 binding. For most of the cell permeable peptides, interaction with HSPG (heparin sulfate proteoglycans) The present inventors investigated proteins binding to / interacting with the cell membrane and finally selected the peptides of the present invention from annexin proteins likely to have an amino acid sequence having cell penetration function.

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).

The peptide of the present invention includes an amino acid sequence located at the N-terminus of the annexin protein, specifically, an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 12, more specifically, SEQ ID NO: 3 to SEQ ID NO: SEQ ID NO: 11 and SEQ ID NO: 13, and more specifically, the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 5 (see Table 1).

In some embodiments of the present invention, the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 12 are amino acid sequences derived from annexin A1 to annexin A11 and annexin A13, respectively, and more specifically to human annexin proteins Derived amino acid sequence.

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) An amino acid, and a protecting group selected from the group consisting of amino acids. 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 cationic peptides variety of cell-permeable peptides identified / synthesized from (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 are not yet successful. For example, poly-arginine and the conjugate of cyclosporin ^PsorBan? The clinical trial for psoriasis was discontinued at Phase II. 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, cytotoxicity and serum degradation rates are important in cell permeable peptide studies. In particular, the degradation rate of peptides in serum is very important for delivering the target cargo to cells.

Since the peptides of the present invention not only exist in the cytoplasm but also exhibit low cytotoxicity and a slow degradation rate in serum (Reference: Experimental Results 2-3 and 2-4), the possibility of development as a drug delivery system is very high. Accordingly, the present invention relates to (a) a cell permeable peptide as described above; And (b) a cargo of interest bound to the peptide.

In some embodiments of the invention, the peptides of the invention are present in the cytoplasm.

In some embodiments of the invention, the peptides of the invention do not exhibit cytotoxicity. More specifically, when the peptide of the present invention is treated at a concentration of 100 μM, it exhibits about 50-60% cytotoxicity in the cell, but when treated at a concentration of 10 μM it exhibits cytotoxicity of 20% or less , And does not show cytotoxicity when treated at a concentration of 1 μM or less (see FIG. 4).

In some embodiments of the invention, the peptides of the present invention are not degraded by at least 60% until 24 hours upon serum administration (see Figure 5).

Typically, a cell permeable peptide can be constructed by linking a non-covalent or covalent bond with a target cargo. In particular, it is preferred that the cell permeable peptide and the target cargo 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 drug delivery system in which the peptide of the present invention is covalently linked to a beta-galactosidase as a target cargo transmits the beta-galactosidase through the cell membrane in a stable and highly efficient manner : Fig. 9). Considering this, it is expected that the target carbohydrate polypeptide which can be carried in combination with the peptide of the present invention is capable of carrying a polypeptide having a size of 120 kDa or less, but larger than that. In some embodiments of the invention, the size of the target cargo (poly) peptide that can be delivered in combination with the peptide of the invention is less than 120 kDa. 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 bonds and is intended to be transported into cells, for example, Nanoparticles, peptides, polypeptides, antisense oligonucleotides, siRNA, shRNA, miRNA and peptide-nucleic acid (PNA).

In addition, when the target cargo is a polypeptide, the drug delivery system of the present invention may additionally include a linker between the peptide of the present invention and the target cargo. As the linker used in the present invention, various linkers known in the art can be used. The linker may have a length and / or sequence specifically selected to maximize the activity of the peptides of the invention, i. E., The cytotoxic activity. Specifically, the linker is a linker composed of a plurality of amino acid residues. Linkers consisting of amino acid sequences are 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. Suitable linkers for the present invention are composed of hydrophilic amino acids, in particular arginine and glutamine, the length of which may be from 5 to 15 amino acids. In some embodiments of the invention, the linker is composed of a plurality of amino acid residues, more specifically 10-12 arginine and glutamine residues. The linker of the present invention is exemplified by the amino acid sequence of SEQ ID NO: 13 (see Table 3).

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)).

In addition, various labels may be used for the peptide and the target cargo constituting the drug delivery system of the present invention. For example, fluorescent substances such as fluorescein, FITC (fluorescein isothiocyanate), rhodamine 6G Rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino- , a fluorescent protein (fluorescence protein; GFP, RFP, CFP, YFP, BFP, luciferase or variants thereof), a radioactive isotope (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 radioactive isotopes (for example, peptides and nanoparticles of the present invention), single photon emission computed tomography (SPECT) or positron emission tomography (PET) , Positron Emission Tomography) and may be used for tissue imaging.

In some embodiments of the present invention, the drug delivery vehicle of the present invention has FITC bound to the N-terminus of the above-mentioned peptide.

Meanwhile, the present invention provides a nucleotide sequence encoding the above-mentioned cell permeable peptide (SEQ ID NO: 1 to SEQ ID NO: 6), and the nucleotide sequence is not limited as long as it is a nucleotide sequence encoding the above-mentioned peptide, The nucleotide sequence of SEQ ID NO: 7 to SEQ ID NO: 12 for SEQ ID NO: 1 to SEQ ID NO: 6, respectively. More specifically, SEQ ID NO: 1 to SEQ ID NO: 6 are derived from GenBank Accession Nos. NP_005130.1, NP_001145.1, P09525, NP_001146.2, P50995 and P27216, respectively. Specific examples of nucleotide sequences thereof are given in GenBank Accession No. NM_005139. 2, NM_001154.3, NM_001153.3, NM_001155.4, AJ278464.1, and BC125158.

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 aforementioned cell permeable 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: 7 to SEQ ID NO: 12 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 still another aspect of the present invention, there is provided an intracellular delivery method of a target cargo comprising the steps of contacting the above-mentioned drug carrier with a cell or injecting it into blood.

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 permeable peptide and its drug carrier 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 formulations include, but are not limited to, 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 with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person skilled 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. Until now, the main transfection kits include calcium phosphate method, lipid conjugate method, and dextran conjugate method, but their efficiency is about 1/10 ⁶ to 1/10 ² and they are dependent on the kind of cells There is a limit. 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 fusion construct of the cell permeable peptide of the present invention with 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 a human anexin protein-derived amino acid sequence, which not only has excellent cell permeability but also shows a slow degradation rate in serum without cytotoxicity.

(c) In addition, the objective cargo (for example, beta-galactosidase) bound to the peptide of the present invention is stably and effectively transported intracellularly (cytoplasmic).

(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.

Fig. 1 shows FACS results of measuring the cell permeability of 12 kinds of annexin-derived peptides according to the treatment concentration. X-axis, Green Fluorescence; And the Y axis, the number of cells (count).
FIG. 2 shows the results of comparing the cell permeation efficiency between the peptides (AA3H and AA5H) of the present invention and the TAT-CPP peptide. X-axis, green fluorescence; And Y axis, cell number.
Figure 3 shows the results showing the concentration-dependent cellular permeability of the peptides of the invention (AA3H and AA5H). X-axis, green fluorescence; And Y axis, cell number.
Fig. 4 shows the results of comparing the cytotoxicity of the peptides (AA3H and AA5H) of the present invention with the TAT-CPP peptide.
FIG. 5 shows the results of measuring the resolution of the peptide AA3H of the present invention in serum.
Figure 6 shows the results of confirming the cell permeation pathway of the peptide AA3H-CPP of the present invention through an endocytosis inhibitor.
FIG. 7 shows the results of confirming intracellular permeation sites of the peptide AA3H-CPP of the present invention using intracellular organelle marker proteins.
Figure 8 shows the intracellular permeation of Gal bound to the peptide AA3H of the present invention. X-axis, green fluorescence; And Y axis, cell number.
FIG. 9 shows the result of staining showing intracellular delivery of? -Galactosidase using the peptide AA3H-CPP of the present invention.

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 methods and experimental results

The present inventors selected twelve kinds of annexin proteins found in humans and analyzed the characteristics of each sequence, and structurally translated post-translationally modified positions (for example, myristolylation) and cell membrane and cell membrane proteins The 10 amino acid sequences from the N-terminal which are important for binding to the ssDNA were selected from each kind of annexin and the relationship between them and the cell permeability was examined. The 12 kinds of annexin proteins are known to be located in various organelles of cells. Locations (subcellular localization database) were confirmed and they were reconstituted in Table 1 (Table 1).

name N-terminal sequence Isoelectric point (pI) Majesty Size (Da) Scavenger location Anexin A1 MAMVSEFLKQ 5.61 0 1685.99 Cytoplasm, cell membrane, ER, melanosome Annexin A2 MSTVHEILCK 8.86 One 1765.07 Cytoplasmic membrane-bound vesicles, ER, cell membrane, nucleus, cytoplasm Annexin A3
(SEQ ID NO: 1) MASIWVGHRG 10.06 2 1615.84 cytoplasm Annexin A4
(SEQ ID NO: 3) MATKGGTVKA 10.80 2 1465.70 Cytoplasm, Golgi, nuclear Annexin A5
(SEQ ID NO: 2) MAQVLRGTVT 10.06 One 1577.83 ER, cytoplasm, cell membrane, extracellular region, nuclear Annexin A6
(SEQ ID NO: 4) MAKPAQGAKY 10.23 2 1566.81 Cell membrane, ER, cytoplasm Annexin A7 MSYPGYPPTG 5.28 0 1571.73 Cytoplasmic membrane-associated vesicles, ER, cytoplasm, nucleus, cytoplasm Annexin A8 MAWWKSWIEQ 5.61 0 1867.12 Cell membrane Annexin A9 MSVTGGKMAP 8.86 One 1480.73 cytoplasm Annexin A10 MFCGDYVQGT 3.75 -One 1705.87 Mitochondria Annexin A11
(SEQ ID NO: 5) MSYPGYPPPP 5.28 0 1607.81 Cytoplasmic, nuclear Annexin A13
(SEQ ID NO: 6) MGNRHAKASS 11.66 3 1560.72 Cell membrane, cell membrane TAT-CPP YGRKKRRQRRR 12.81 8 2062.38

Sequence in the annexin subfamily.

After labeling the selected peptide sequence with a fluorescent substance, it was tried to confirm whether or not these peptides were transported well into cells by various experiments. As a control group, well-known TAT-CPP was selected and compared. Information on the sequence, isoelectric point and size of 12 kinds of annexin and control TAT-CPP are shown in Table 1 above.

In the past, the peptide was synthesized by selecting a virus or a cationic peptide, or a sequence superior in cell membrane permeability. On the other hand, the present invention developed a novel CPP by identifying a sequence having a transporter function from a protein existing in a human.

2-1. Annexin  From protein Derived  Cell-mediated amino acid sequence synthesis

Each amino acid sequence was synthesized using a solid phase peptide synthesis method, and each amino acid was linked from the C-terminus using a link-amide methylbenzylhydrylamine resin (Novabiochem, # 855003) FITC isomer I (fluorescein isothiocyanate isomer I; Sigma, # F7250). 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 5-80% concentration of water containing 0.1% TFA and acetonitrile (Fisher scientific, # 955-1). Molecular size of each peptide was confirmed by mass spectroscopy (MALDI-TOF).

2-2. Annexin  From protein Derived  Cell permeation Of peptide  Intracellular transport effect

In order to confirm the intracellular transport capacity of the synthesized 12 kinds of Annexin-CPP candidate substances, human cervical cancer cell line (HeLa) was treated at a concentration of 0, 0.1, 1, 10 μM for 4 hours and then analyzed using a flow cytometer (Guava easyCyte , Milipore, USA) (Fig. 1). FIG AA1 through AA11 and AA13 displayed on the first respectively annexin A1 to A11 and AA13 represents the human form (Human form) Ah has been indicated by annexin AA1 through AA11 and AA13, of which AA3H (A nnexin A 3 H uman form) - CPP or AA5H-CPP candidates at 1 [mu] M had better transport capacity than TAT-CPP (Fig. 2). Five concentrations (0, 0.625, 1.25, 2.5, 5, and 10 μM) of human cancer cells were treated with human cervical cancer cells to determine whether the candidate substances were permeated in cells in a concentration- Dependent manner into the cells (Fig. 3).

2-3. In human uterine cancer cells AA3H - CPP  And AA5H - CPP Of cytotoxicity

Cell permeability was determined in the concentration-dependent manner of AA3H-CPP and AA5H-CPP in human uterine cancer cells. Cell permeability was determined by using cell counting kit (CCK-8; Dojindo, Japan) Were measured. Human uterine cancer cells were cultured in a 96-well plate at a confluence of about 80%, and the comparative groups TAT-CPP, AA3H-CPP and AA5H-CPP were treated at concentrations of 0.01, 0.1, 1, Lt; / RTI > 10 μl of CCK-8 solution was added to the cells treated with each CPP, reacted for 2 hours, and measured at 450 nm absorbance. As a result, AA3H-CPP, AA5H-CPP and TAT-CPP showed cytotoxicity at a concentration of 100 μM, while AA3H-CPP and AA5H-CPP showed about 50% and 60% The comparative group TAT-CPP showed about 70% toxicity. In addition, TAT-CPP showed more than 20% toxicity when treated at 10 μM concentration, whereas AA5H-CPP showed about 24% cytotoxicity at 10 μM concentration, but relatively less cytotoxicity at 100 μM concentration , And AA3H-CPP showed no cytotoxicity at 10 μM and 100 μM concentrations (FIG. 4).

2-4. In mouse serum AA3H - CPP  Decomposition evaluation

Since AA3H-CPP among AA3H-CPP and AA5H-CPP showed better cell membrane permeation efficiency, AA3H-CPP was mainly used in later studies. CPP is used as a drug delivery agent in various disease models. When CPP is delivered to tissues or animals, the CPP is degraded by the serum and the efficiency is lowered. In order to confirm the resolution of AA3H-CPP in serum, 10 μM AA3H-CPP was added to 10% mouse serum and the degree of degradation was determined by HPLC over time (0.25, 2, 4, 8, 10 and 24 hours) (Fig. 5). FIG. 5A shows the HPLC results of AA3H-CPP in the serum according to time, and FIG. 5B shows the HPLC values as a percentage of the time. AA3H-CPP degraded about 20% when reacted with mouse serum for 2 hours, decomposed by about 30% at 8 hours, and about 40% when reacted for 24 hours. Therefore, it was confirmed that the AA3H-CPP of the present invention was not easily degraded in serum because about 60% or more of AA3H-CPP remained even when the mouse AA3H-CPP was reacted in the serum for 24 hours. In addition, the reaction between serum and AA3H-CPP was only degraded by about 20% within 1 hour, and then it showed almost no degradation rate from 2 hours to 24 hours, indicating that the degradation rate in serum was very slow.

2-5. In human uterine cancer cells AA3H - CPP Of intracellular permeation pathways and location of

The mechanism by which CPP enters the cell is mainly endocytosis, but it has been reported that the intracellular transport path varies depending on the nature of CPP and the transporter, and recently, it also translocates to direct translocation. Endocytosis may be caused by macropinocytosis, clathrin-mediated pathway, caveolae / lipid-raft-mediated pathway, or clathrin, The path is divided into two. In order to investigate the pathway of AA3H-CPP through these pathways, pretreatment of each pathway-specific inhibitor (Table 2) in human cervical cancer cells was carried out for 30 minutes and then the intracellular permeation change was observed.

type Inhibitor company
(Catalog number) Clathrin-mediated endocytosis
Macropinocytosis
Caveolae / lipid-raft-mediated endocytosis
Endosomes / destruction of lysosomes
Actin destruction Chlorpromazine (CPZ)
5- (N-ethyl-nisopropyl) amiloride (EPIA)
Methyl-beta-cyclodextrin (M? CD),
Nystatin
Chloroquine (CQ)
Cytochalasin D (CYD) Sigma (C8138)
Sigma (A3085)
Sigma (C4555, N6261)

Sigma (C6628)
Sigma (C8273)

Specific inhibitors depending on endocytosis type.

Through the flow cytometry analysis, AA3H-CPP appears to enter the cell through two pathways. As can be seen in FIG. 6, it was observed that it penetrated mainly through macropinocytosis, and partly through the caveolae / lipid-raft into the cells. In the flow cytometry graph, the X-axis represents the fluorescence of FITC and the Y-axis represents the number of cells. The AA3H-CPP is attached to the N-terminus of AA3H-CPP. In addition, in order to remove AA3H-CPP attached to the cell membrane, it was treated with 0.01% trypsin for 10 minutes to inhibit nonspecific activity of cell membrane permeation.

Next, confocal microscopy (LSM700, GER) was used to confirm the location of AA3H-CPP in the organs when intracellularly transported, using markers from each organ (Table 3).

Scavenger type Marker company
(Catalog number) Lysosome
nucleus
Golgi
ER
Actin Litho tracker
Hoechst
Celite Golgi - RFP Backing 2.0
Cell Light ER-RFP Backing 2.0
Celite Actin-RFP Backing 2.0 Invitrogen (L12492)
Invitrogen (33342)
Invitrogen (C10593)
Invitrogen (C10591)
Invitrogen (C10502)

Markers of each organelle.

Cells were treated with AA3H-CPP for 4 hours. The cells were stained with markers of each organelle and confocal microscopy confirmed that they were not located in lysosomes, nuclei, corpus, ER, and actin. From the above results, it can be confirmed that AA3H-CPP is not decomposed by the lysosome and is not located in the nucleus. In addition, it can be expected that it is highly likely to be present in the cytoplasm in that it is not observed in other organelles such as Golgi and ER (FIG. 7).

2-6. AA3H - CPP For the application of Intracellular  beta- Galactosidase ( 가alat 시드세제 ) relay

In order to use AA3H-CPP as a carrier, beta-galactosidase was selected as an intracellular transporter protein. Since beta-galactosidase is a protein with a size of more than 120 kDa, if beta-galactosidase can be delivered into a cell, it may be possible to transport a substance with a size below 120 kDa. It is a protein that has been widely used for. To confirm that AA3H-CPP can deliver beta-galactosidase into cells, beta-galactosidase fusion peptide was synthesized by linking to AA3H-CPP. Sequences that can be fused with beta-galactosidase are described by Kristopher M, et al. ( Scientific reports . 1661 (3): 1-7, 2013). The synthesis sequence, size and isoelectric point of the beta-galactosidase fusion peptide are described in Table 4 below.

name Peptides  order Isoelectric point pI ) size( Da ) Gal-linking
AA3H-gal RRQQQQQQRRR
FITC-MASIWVGHRG-RRRQQQQQQRRR 13.20
13.28 2225.56
3434.06

Peptide sequence for fusion between beta-galactosidase and AA3H-CPP.

The beta-galactosidase fusion amino acid sequence and the AA3H-CPP sequence were linked by a linker (6- (fmoc-amino) caproic acid; Fluka, # 04067) and named AA3H-gal. After the FITC fluorescent substance was ligated to the N-terminal of AA3H-CPP and the β-galactosidase was fused, permeability of the cell membrane was confirmed by flow cytometry (FIG. 8). As a result, it was found that even when the beta-galactosidase fusion peptide was ligated, the beta-galactosidase fusion peptide was well permeated into the cell. On the other hand, only the beta-galactosidase fusion peptide was transported into cells I could see the phenomenon. In order to confirm whether intracellular transport of the above-mentioned fusion peptide itself also occurs when beta-galactosidase is fused, a method of staining only beta-galactosidase-expressing cells when beta-galactosidase is delivered into cells Respectively.

(50 mM Tris-HCl pH 7.4, 5 mM CaCl ₂ , 1 mM DTT) was added to fusions of AA3H-gal and beta-galactosidase (Sigma, # GALS) in a fusion buffer (transgultaminase; Sigma, # T5398) and incubated at room temperature for one hour. The cells were treated with human cervical cancer cells for 3 hours. Then, the cells were washed twice with PBS, and the beta-galactosidase transferred to the cells was stained with X-gal (5-bromo-4-chloro- -3-indolyl-beta-D-galactopyranoside) substrate was treated and stained and observed with a microscope (nuance microscopy; Thermo Fisher Scientific). When beta-galactosidase is transferred into the cell and expressed, it reacts with X-gal to display a blue color, otherwise it does not appear. In this experiment, beta-galactosidase-treated cells and Gal-linking-conjugated beta-galactosidase (hereinafter referred to as Gal-linking / beta-galactosidase) (AA3H-gal / beta-galactosidase) fused with AA3H-gal and beta-galactosidase (FIG. 9). AA3H-gal / beta-galactosidase showed a deep blue color when beta-galactosidase was well transferred into cells and stained with X-gal. In the case of Gal-linking / beta-galactosidase as a control, beta-galactosidase was also transferred into cells and stained blue. Similar to the flow cytometry analysis shown in FIG. 8, when beta-galactosidase and Gal-linking peptide were fused, beta-galactosidase was delivered into the cells. This is thought to have affected the intracellular permeability due to the presence of a positive arginine sequence in the Gal-linking peptide. However, it was confirmed that the beta-galactosidase transfer ability by AA3H-CPP was much superior to that of the control (beta-galactosidase or Gal-linking / beta-galactosidase) (FIG.

In the case of siRNA, which has been spotlighted as a transporter in recent years, when it is permeated into a cell, it is condensed in the nucleus or condensed in the endosome ) Can not escape from the cytoplasm is not able to play a role in the disadvantage. AA3H-CPP derived from the N-terminus of human annexin protein is highly resistant to serum and is not toxic and has a differentiability from conventional carriers. In addition, AA3H-CPP has the advantage that it has low resolution by serum and is not located in nucleus or lysosome, so that it can overcome the above limitations.

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

Oliver L, et al . , Novel organization and properties of anexin-2-membrane complexes. J. Biol . Chem . 279 (12): 10872-10882, 2004.

Gerke V and Moss SE , Annexins: From structure to fuction. Physiol . Rev. 82: 331-371, 2001.

Holton TA , et al ., CPPpred: prediction of cell penetrating peptides. Bioinformatics . 1-3, 2013.

Menke M, et al ., The molecular arrangement of the membrane-bound annexin A2-S100A10 tetramer as revealed by scanning force microscopy. ChemBioChem . 5, 1003-1006, 2004.

Brian S, et al ., Annexin A2 binds to endosomes following organelle destabilization by particulate wear debris. Nat . com . 755 (3): 1-10, 2012.

Gerke V, Creutz CE and Moss SE , Annexins: linking Ca2 + signaling to membrane dynamics. Nat. Rev. Mol . Cell Biol . 6, 449-461, 2005.

Kristopher M, et al ., Xentry, a new class of cell-penetrating peptides that are uniquely provided for delivery of drugs. Scientific reports . 1661 (3): 1-7, 2013.

Paulo F, et al ., Allosterism in membrane binding: A common motif of the annexins ?. Biochemistry . 44, 10905-10913, 2005.

Lizarbe MA , et al ., Annexin-phospholipid interactions. Functional implications. Int . J. Mol. Sci . 14, 2652-2683, 2013.

Feihu W, et al . , Recent progress of cell-penetrating peptides as new carriers for intracellular cargo delivery. J. Control . Release . 174, 126-136, 2014.

Hoyer J and Neundorf I, Peptide vectors for the nonviral delivery of nucleic acids. Accounts . chem . res . 45 (7), 1048-1056, 2012.

<110> Korea Institute of Science and Technology <120> Nove Cell Penetrating Peptides and Uses Thereof <130> F07151 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA3H <400> 1 Met Ala Ser Ile Trp Val Gly His Arg Gly   1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA5H <400> 2 Met Ala Gln Val Leu Arg Gly Thr Val Thr   1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA4H <400> 3 Met Ala Thr Lys Gly   1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA6H <400> 4 Met Ala Lys Pro Ala Gln Gly Ala Lys Tyr   1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA11H <400> 5 Met Ser Tyr Pro Gly Tyr Pro Pro Pro Pro   1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA13H <400> 6 Met Gly Asn Arg His Ala Lys Ala Ser Ser   1 5 10 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA3H_NT <400> 7 atggcatcta tctgggttgg acaccgagga 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA5H_NT <400> 8 atggcacagg ttctcagagg cactgtgact 30 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA4H_NT <400> 9 atggccatgg caaccaaagg aggtactgtc 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA6H_NT <400> 10 atggccaaac cagcacaggg tgccaagtac 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA11H_NT <400> 11 atgagctacc ctggctatcc cccgccccca 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA13H_NT <400> 12 atgggcaatc gtcatgctaa agcgagcagt 30

Claims (22)

Cell penetrating peptide (CPP) consisting of the amino acid sequence of SEQ ID NO: 1. 2. The peptide of claim 1, wherein the amino acid sequence is derived from human annexin protein. 2. The peptide of claim 1, wherein the peptide is permeable to the cytoplasm. 2. The peptide of claim 1, wherein the peptide does not exhibit cytotoxicity. The peptide of claim 1, wherein the peptide is not degraded by at least 60% by 24 hours upon serum administration. The peptide of claim 1, wherein the peptide carries a target cargo of interest linked by covalent or non-covalent bonds into a cell or tissue. 7. The peptide of claim 6, wherein said carbohydrate polypeptide is less than 120 kDa in size. (a) a cell permeable peptide according to any one of claims 1 to 7; (b) a drug delivery system comprising a target cargo of interest bound to the peptide. 9. The drug delivery vehicle of claim 8, wherein the drug delivery vehicle additionally comprises a linker between the peptide and the target cargo. 10. The drug delivery system according to claim 9, wherein the linker is a linker consisting of a plurality of amino acid residues. 9. The drug delivery system of claim 8, wherein the cargo is a chemical, nanoparticle, peptide, polypeptide, antisense oligonucleotide, siRNA, shRNA, miRNA or PNA. 9. The drug delivery system according to claim 8, wherein the size of the target cargo polypeptide is 120 kDa or less. The drug delivery system according to claim 8, wherein the binding is a covalent bond or a non-covalent bond. 9. The peptide according to claim 8, wherein the peptide is a peptide wherein a protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group and a polyethylene glycol (PEG) Lt; / RTI &gt; 15. The drug delivery vehicle according to claim 14, wherein the protecting group is a methyl group. A nucleotide sequence encoding the peptide of claim 1. 17. The nucleotide sequence according to claim 16, wherein the nucleotide sequence is SEQ ID NO: 7 to SEQ ID NO: 12. A recombinant vector comprising the nucleotide sequence of claim 16. delete 9. A composition for cargo transportation comprising the drug carrier of claim 8. 9. A transfection kit comprising the drug delivery system of claim 8. 22. The transfection kit of claim 21, wherein the cargo in the drug delivery vehicle is a nucleic acid molecule.

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