Nucleolus transport carrier peptide fragment and its use

Abstract

To provide a technique of efficiently introducing a target foreign substance from outside an eukaryotic cell into at least a cytoplasm of the cell (and furthermore into a nucleolus).SOLUTION: A method for introducing a target foreign substance from outside an eukaryotic cell into at least a cytoplasm of the cell includes: (1) a process of preparing a structure for foreign substance introduction having a carrier peptide fragment made up of any amino acid sequence of KKRTLRKKKRKKR, KKRTLRKRRRKKR, KKRTLRKRKRKKR and KKRTLRKKRRKKR, and a foreign substance bound to N-terminal side and / or C-terminal side of the carrier peptide fragment; (2) a process of supplying the structure for foreign substance introduction into a sample including a target eukaryotic cell; and (3) a process of incubating the sample into which the structure for foreign substance introduction has been supplied, and introducing the structure into an eukaryotic cell of the sample.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] The present invention relates to a method for introducing (transporting) a foreign substance from the outside of a eukaryotic cell into the cytoplasm (or even into the nucleolus) of the cell, and a construct for introducing a foreign substance comprising a carrier peptide fragment used in the method. [Background technology]

[0002] Conventionally, foreign substances such as polypeptides, particularly physiologically active substances, have been introduced into cells (eukaryotic cells) of humans and other mammals, etc., to transform the characteristics of the cells (and even tissues and organs made of the cells) or to improve or enhance the functions of the cells. One example of this technology uses cell penetrating peptides (CPPs).

[0003] For example, Patent Document 1 discloses a construct for introducing a foreign substance, which contains a foreign substance of interest and the amino acid sequence (carrier peptide fragment) described in SEQ ID NO: 5, known as the nucleolar localization signal (hereinafter referred to as "NoLS") of LIM kinase 2 described in Non-Patent Document 1 and Non-Patent Document 2. Since the construct includes a carrier peptide fragment having cell membrane permeability, the foreign substance can be introduced into the cytoplasm of a eukaryotic cell from the outside of the cell. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2011 / 013700 [Non-patent literature]

[0005] [Non-Patent Document 1] JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 281, No. 35, 2006, pp. 25223-25230 [Non-Patent Document 2] Protein & Peptide Letters, Vol. 17, No. 12, 2010, pp. 1480-1488 Summary of the Invention [Problem to be solved by the invention]

[0006] In recent years, interest in cell membrane-permeable peptides such as the above-mentioned NoLS has been growing from the viewpoint of medical care, and there is a demand for the development of a technology for more efficiently introducing foreign substances into target cells. In addition, in recent years, the relationship between cancer and various diseases (e.g., amyotrophic lateral sclerosis (ALS), etc.) and abnormalities in nucleoli has been reported, and interest in nucleoli is increasing. Therefore, there is a demand for the development of new nucleolus markers and means for transporting foreign substances to nucleoli.

[0007] The present invention has been created to meet such demands, and aims to provide a method for efficiently introducing a foreign substance of interest into at least the cytoplasm (and further into the nucleolus) of a eukaryotic cell from the outside of the cell. Another aim of the present invention is to provide a construct having a carrier peptide fragment and a foreign substance, which can efficiently introduce a foreign substance of interest from the outside of a eukaryotic cell into the inside of the cell. [Means for solving the problem]

[0008] In order to improve the cell membrane permeability of NoLS of LIM kinase 2 disclosed in the above-mentioned Patent Document 1, the present inventors tried to substitute various amino acid residues in the amino acid sequence of NoLS shown in SEQ ID NO: 5. As a result, it was found that a mutant in which the 8th asparagine residue and the 9th aspartic acid residue counting from the N-terminus of the amino acid sequence of NoLS shown in SEQ ID NO: 5 were substituted with basic amino acid residues (arginine residue or lysine residue), respectively, improved cell membrane permeability and further improved nucleolar migration (localization). Surprisingly, these substitutions were not so-called conservative substitutions (for example, a sequence in which a basic amino acid residue is substituted with another basic amino acid residue), but substitutions that change the charge of the peptide. That is, the construct disclosed herein was created and completed through extensive trial and error by the present inventors.

[0009] The method disclosed herein is a method for introducing (transporting) a foreign substance of interest from the outside (i.e., the outside of the cell membrane) of a eukaryotic cell (particularly various animal cells not having a cell wall, such as human cells and other mammalian cells) into at least the cytoplasm (and even the nucleolus) of the cell. That is, the method for introducing a foreign substance disclosed herein is as follows: (1) the amino acid sequence: KKRTLRKKKRKKR (SEQ ID NO:1); KKRTLRKRRRKKR (SEQ ID NO:2); KKRTLRKRKRKKR (SEQ ID NO:3); KKRTLRKKRRKKR (SEQ ID NO:4); and a foreign substance of interest bound to the N-terminus and / or C-terminus of the carrier peptide fragment; (2) providing the construct for introducing a foreign substance into a sample containing a target eukaryotic cell; (3) incubating the sample to which the construct for introducing an exogenous substance has been supplied, thereby introducing the construct into eukaryotic cells in the sample. Here, the term "foreign substance" refers to an inorganic or organic compound that can be bound directly or indirectly via a suitable linker to the N-terminus or C-terminus of the carrier peptide fragment, and that has a molecular size and chemical properties that allow it to be introduced into eukaryotic cells.

[0010] According to the method for introducing an exogenous substance having the above-mentioned configuration, a construct for introducing an exogenous substance is constructed by binding the desired exogenous substance (typically an organic compound such as a polypeptide, nucleic acid, dye, drug, etc.) directly or indirectly via a suitable linker to the N-terminus and / or C-terminus of the carrier peptide fragment, and is then supplied to a sample containing the target eukaryotic cell (e.g., a culture containing the cell) (i.e., added to a living eukaryotic cell), whereby the desired exogenous substance can be introduced with high efficiency from the outside of the eukaryotic cell (outside the cell membrane) through the cell membrane into the cytoplasm (and even through the nuclear membrane into the nucleolus).

[0011] In a preferred embodiment of the method for introducing a foreign substance disclosed herein, the foreign substance is an organic compound selected from the group consisting of a polypeptide, a nucleic acid, a dye, and a drug. A construct prepared to contain this type of organic compound is efficiently introduced into at least the cytoplasm of a target cell. Here, "polypeptide" refers to a polymer having a structure in which multiple amino acids are bound by peptide bonds. Polypeptides are not limited by the number of peptide bonds (i.e., the number of amino acid residues). In other words, polypeptides include those generally called peptides, which have 10 or more but less than 300 amino acid residues, and those generally called proteins (polymeric compounds typically consisting of 300 or more amino acid residues). In this field, there is no strict distinction between polypeptides and proteins. In this specification, polymers (including oligomers) consisting of multiple amino acid residues are collectively referred to as polypeptides. In addition, the term "nucleic acid" refers to a polymer of nucleotides, and includes DNA and RNA. "Nucleic acid" is not limited by the number of bases.

[0012] In another preferred embodiment of the method for introducing a foreign substance disclosed herein, the foreign substance is bound to the C-terminus of the carrier peptide fragment, and the α-amino group of the lysine at the N-terminus of the carrier peptide fragment is acetylated. With this configuration, the stability of the construct in cells is increased, and the foreign substance can be more stably retained in the cytoplasm and nucleolus.

[0013] In another preferred embodiment of the method for introducing an exogenous substance disclosed herein, the eukaryotic cells into which the construct for introducing an exogenous substance is introduced are human or non-human mammalian cells. The methods disclosed herein allow for highly efficient introduction of foreign substances into the cytoplasm, and even into the nucleolus, of human or non-human mammalian cells.

[0014] Furthermore, in order to achieve the above-mentioned objective, the present disclosure provides an artificially constructed construct for introducing (transporting) a desired foreign substance from the outside (i.e., outside the cell membrane) of a eukaryotic cell (particularly various animal cells such as humans and other mammals that do not have a cell wall) into at least the cytoplasm (and further into the nucleolus) of the cell. That is, the construct for introducing a foreign substance disclosed herein has the following amino acid sequence: KKRTLRKKKRKKR (SEQ ID NO:1); KKRTLRKRRRKKR (SEQ ID NO:2); KKRTLRKRKRKKR (SEQ ID NO:3); KKRTLRKKRRKKR (SEQ ID NO:4); and the above-mentioned foreign substance of interest bound to the N-terminus and / or C-terminus of the carrier peptide fragment. Such a construct has a carrier peptide fragment that has high cell membrane permeability, and therefore allows efficient transfer of foreign substances into the cytoplasm (and even into the nucleolus) of eukaryotic cells.

[0015] In a preferred embodiment of the construct for introducing an exogenous substance disclosed herein, as described above, the exogenous substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes and drugs. Also, preferably, the foreign substance is bound to the C-terminus of the carrier peptide fragment, and the α-amino group of lysine at the N-terminus of the carrier peptide fragment is acetylated. [Brief description of the drawings]

[0016] [Figure 1] This is a graph showing the relative MFI values ​​(Example 6 is taken as 1.0) obtained by analyzing the cultured cells using a flow cytometer in tests (Examples 1 to 4) in which a construct for introducing an exogenous substance according to one embodiment was added to the culture medium of HeLa cells, a test (Example 5) in which a construct comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO:5 was added, and a test (Example 6) in which FAM was added. [Figure 2A] 1 is an image observed by a confocal laser microscope, showing the intracellular localization of a construct having a carrier peptide fragment consisting of the amino acid sequence shown in SEQ ID NO:1. [Figure 2B] 1 is an image observed by a confocal laser microscope showing the intracellular localization of a construct having a carrier peptide fragment consisting of the amino acid sequence shown in SEQ ID NO:2. [Figure 2C] 1 is an image observed by a confocal laser microscope, showing the intracellular localization of a construct having a carrier peptide fragment consisting of the amino acid sequence shown in SEQ ID NO:3. [Figure 2D] 1 is an image observed by a confocal laser microscope showing the intracellular localization of a construct having a carrier peptide fragment consisting of the amino acid sequence shown in SEQ ID NO:4. [Figure 2E] 1 is an image observed by a confocal laser microscope showing the intracellular localization of a construct having a carrier peptide fragment consisting of the amino acid sequence shown in SEQ ID NO:5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Preferred embodiments of the technology disclosed herein are described below. Matters other than those specifically mentioned in this specification that are necessary for implementation (e.g., general matters such as methods for chemically synthesizing peptides, cell culture techniques, and preparation of compositions containing peptides or nucleic acids as components) can be understood as design matters for those skilled in the art based on conventional techniques in the fields of cell engineering, physiology, medicine, pharmacology, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc. The technology disclosed herein can be implemented based on the contents disclosed in this specification and the common general technical knowledge in the field. In the following explanation, amino acids are sometimes represented by one-letter symbols (but three-letter symbols in the sequence listing) in accordance with the nomenclature for amino acids set forth in the IUPAC-IUB guidelines. In this specification, the term "amino acid residue" includes the N-terminal amino acid and the C-terminal amino acid of a peptide chain, unless otherwise specified.

[0018] In addition, as used herein, the term "synthetic peptide" refers to a peptide fragment whose peptide chain does not exist stably in nature independently, but is produced by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and can exist stably in a given composition. Here, "peptide" is a term that refers to an amino acid polymer having multiple peptide bonds, and is not limited by the number of amino acid residues. In addition, in the amino acid sequences described in this specification, the left side always represents the N-terminus and the right side represents the C-terminus.

[0019] The construct for introducing a foreign substance disclosed herein comprises a carrier peptide fragment and a foreign substance bound to the N-terminus and / or C-terminus of the carrier peptide fragment. The carrier peptide fragment disclosed herein has the following amino acid sequence: KKRTLRKKKRKKR (SEQ ID NO:1); KKRTLRKRRRKKR (SEQ ID NO:2); KKRTLRKRKRKKR (SEQ ID NO:3); KKRTLRKKRRKKR (SEQ ID NO:4); and is an amino acid sequence that exerts cell membrane permeability and further nucleolus transport ability in eukaryotic cells.

[0020] The amino acid sequences shown in SEQ ID NOs: 1 to 4 are mutants in which the 8th and 9th amino acids from the N-terminus of the amino acid sequence known as NoLS of LIM kinase 2 shown in SEQ ID NO: 5 have been mutated to basic amino acids. LIM kinase 2 is a type of protein kinase involved in intracellular signal transduction present in human endothelial cells, and the amino acid sequence from the 491st to 503rd amino acids is known to function as a nucleolar localization signal (NoLS) (see Non-Patent Documents 1 and 2).

[0021] The amino acid sequence shown in SEQ ID NO:1 is a sequence in which the 8th asparagine residue and the 9th aspartic acid residue of NoLS of LIM kinase 2 are substituted with lysine residues. The amino acid sequence shown in SEQ ID NO:2 is a sequence in which the 8th asparagine residue and the 9th aspartic acid residue of NoLS of LIM kinase 2 are substituted with arginine residues. The amino acid sequence shown in SEQ ID NO:3 is a sequence in which the eighth asparagine residue of NoLS of LIM kinase 2 is substituted with an arginine residue, and the ninth aspartic acid residue is substituted with a lysine residue. The amino acid sequence shown in SEQ ID NO:4 is a sequence in which the 8th asparagine residue of NoLS of LIM kinase 2 is substituted with a lysine residue and the 9th aspartic acid residue is substituted with an arginine residue.

[0022] The carrier peptide fragment disclosed herein typically has the same amino acid sequence as any of the amino acid sequences shown in SEQ ID NOs: 1 to 4, but includes modified sequences of these amino acid sequences as long as the cell membrane permeability and nucleolus transportability are not impaired. Here, the "modified sequence" refers to an amino acid sequence (modified amino acid sequence) formed by substituting, deleting and / or adding (inserting) one or several (typically two or three) amino acid residues. Such slightly modified sequences can be easily utilized by those skilled in the art based on the information disclosed herein, and are therefore included in the "carrier peptide fragment" as a technical idea disclosed herein. Typical examples of modified sequences herein include sequences resulting from conservative substitution of one, two, or three amino acid residues, so-called conservative amino acid replacement, and sequences in which one, two, or three amino acid residues are added (inserted) or deleted from a given amino acid sequence. Typical examples of conservative substitutions include sequences in which a basic amino acid residue is replaced with another basic amino acid residue (e.g., mutual replacement of a lysine residue with an arginine residue) and sequences in which a hydrophobic amino acid residue is replaced with another hydrophobic amino acid residue (e.g., mutual replacement of a leucine residue, an isoleucine residue, and a valine residue).

[0023] In the amino acid sequences shown in SEQ ID NOs: 1 to 4, 11 of the 13 amino acid residues are basic amino acids (arginine, lysine). Conventionally, as a peptide having cell membrane permeability, for example, octaarginine (hereinafter also referred to as "R8") consisting of only arginine residues shown in SEQ ID NO: 6 is known, but the inventors have confirmed that R8 has relatively high cytotoxicity. On the other hand, the inventors have confirmed that a carrier peptide fragment consisting of any of the amino acid sequences shown in SEQ ID NOs: 1 to 4 has almost no cytotoxicity even at a concentration of 100 μM, and has a much lower cytotoxicity than R8. That is, the carrier peptide fragment disclosed here has high cell membrane permeability and nucleolus migration, and has a significantly lower cytotoxicity. Although the details of the mechanism are not clear, it is presumed that the carrier peptide fragment disclosed here has a neutral amino acid threonine (threonine) residue and a leucine residue at the fourth and fifth positions, respectively, in addition to basic amino acids, which contribute to the reduction of cytotoxicity.

[0024] A construct for introducing an exogenous substance can be designed and constructed by binding (linking) a desired exogenous substance directly or indirectly via a suitable linker to the N-terminus and / or C-terminus of the carrier fragment. The linker is not particularly limited, but may be a peptidic linker or a non-peptidic linker. Although not particularly limited, the amino acid sequence constituting the peptidic linker is preferably an amino acid sequence that does not cause steric hindrance and is flexible. The peptidic linker may be, for example, a linker consisting of 10 or less amino acid residues (more preferably 1 to 5, for example, 1, 2, 3, 4, or 5 amino acid residues) containing one or more amino acid residues selected from glycine, alanine, serine, etc. In addition, β-alanine may be used as such a linker. As the non-peptidic linker, although not particularly limited, for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminohexanoyl spacer, etc. may be used.

[0025] The foreign substance contained in the construct for introducing a foreign substance disclosed herein is typically an organic compound such as a polypeptide, a nucleic acid, a dye, or a drug. When the foreign substance is a polypeptide, a peptide chain is designed to include an amino acid sequence constituting the polypeptide and an amino acid sequence constituting the carrier peptide fragment, and the peptide chain is biosynthesized or chemically synthesized to produce a desired construct for introducing a foreign substance. In addition, various nucleic acids such as DNA or RNA, dyes (e.g., various fluorescent dye compounds such as FAM and FITC), or organic compounds that function as drugs (e.g., antitumor agents including nucleic acid-based antitumor agents such as 5-fluorouracil (5FU) and antiviral agents such as azidothymidine (AZT)) can be directly or indirectly bound to the N-terminus and / or C-terminus of the above-mentioned carrier peptide fragment by various scientific techniques known in the art to construct a construct for introducing a foreign substance. Although not particularly limited, the function of the foreign substance can be, for example, promotion of stem cell differentiation induction (stem cell differentiation induction activity), inhibition of tumor cell proliferation (antitumor activity), inhibition of virus-infected cell proliferation (antiviral activity), control of proteins and nucleic acids in nucleoli, etc.

[0026] In the construct for introducing a foreign substance, the number of foreign substances bound to the carrier peptide fragment is not particularly limited. That is, one or more foreign substances may be bound to one carrier peptide fragment. Although not particularly limited, for example, a polypeptide, a nucleic acid, a drug, etc. may be bound to the N-terminus of one carrier peptide fragment, and a dye may be bound to the C-terminus. By binding a dye to the carrier peptide fragment, it is preferable because it becomes easier to evaluate the introduction efficiency of the construct for introducing a foreign substance into a eukaryotic cell and its localization within the cell.

[0027] Since the construct for introducing an exogenous substance disclosed herein has a high degree of migration to the nucleolus, for example, a construct for introducing an exogenous substance having a dye can be used as a nucleolus marker. Such a construct can be introduced into a eukaryotic cell by being added to the culture solution thereof, and can be highly efficiently transferred to the nucleolus. As an example of a conventional technique, a method of analyzing the position of a nucleolus is widely known in which a nucleolus staining solution is added after cell membrane permeabilization (e.g., treatment with a surfactant such as TritonX-100). However, the construct disclosed herein can be used as a nucleolus marker by a simple method of adding the construct to cultured cells and incubating them, without the need for cell membrane permeabilization.

[0028] In addition, when the foreign substance is a polypeptide, the polypeptide (amino acid sequence) to be adopted is not particularly limited. For example, a foreign substance having a relatively large number of amino acid residues, such as a polypeptide or protein having about 100 to 1000 amino acid residues, can also be adopted. Typically, the total number of amino acid residues constituting a synthetic peptide prepared as a construct for introducing a foreign substance is several to several tens (e.g., 10 or more), and is suitably 1000 or less, preferably 600 or less, more preferably 500 or less, and particularly preferably 300 or less (e.g., 10 to 300). Polypeptides of such a length are easy to synthesize (biosynthesize or chemically synthesize) and easy to use.

[0029] Preferred foreign substances are mature or precursor (including pro and prepro forms) of polypeptides involved in functions such as the development, differentiation, proliferation, canceration, homeostasis, and metabolic regulation of various cells and tissues (organs). The foreign substance introduction method disclosed herein can also be carried out to introduce into cells a polypeptide whose function has not been previously known, in order to elucidate the function of the polypeptide within the cell (within a living tissue). For example, when the eukaryotic cells to which a foreign substance is introduced are human or other mammalian stem cells, it is preferable to use mature polypeptides or their precursors having various physiological activities involved in the differentiation induction of the stem cells. Note that "stem cells" include somatic stem cells, embryonic stem cells, and induced pluripotent stem cells (hereinafter referred to as iPS cells). When the eukaryotic cells to which a foreign substance is introduced are cancer cells (tumor cells), it is preferable to use various polypeptides involved in the induction of apoptosis of the cancer cells (tumor cells). Alternatively, in this case, it is preferable to use polypeptides that can inhibit the cancer cells (tumor cells) from suppressing the function of the immune surveillance mechanism. Furthermore, when the eukaryotic cells to which a foreign substance is introduced are bacteria-infected cells or virus-infected cells, it is preferable to use various polypeptides involved in the induction of apoptosis of the infected cells, polypeptides that can suppress the proliferation of bacteria or viruses in the infected cells, and polypeptides that can suppress the spread of bacterial or viral infection from the infected cells. As with the carrier peptide fragment, the foreign polypeptide may contain a modified amino acid sequence formed by the substitution, deletion and / or addition (insertion) of one or several amino acid residues, so long as it retains its function.

[0030] In a construct for introducing a foreign substance in which a foreign substance is bound to the C-terminus of a carrier peptide fragment, the α-amino group of the amino acid residue on the N-terminus of the carrier peptide fragment is preferably acetylated. Specifically, since the amino acid residue on the N-terminus of the amino acid sequences shown in SEQ ID NOs: 1 to 4 is lysine, it is preferable that the α-amino group of such lysine is acetylated. Although the detailed mechanism is unknown, many proteins in eukaryotic cells are modified by acetylation at the α-amino group of the amino acid on the N-terminus, such a configuration increases the stability of the construct in the cell, and the foreign substance can be more stably retained in the cytoplasm and nucleolus.

[0031] It is preferable that the amino acid residue on the C-terminus side of the construct for introducing a foreign substance is amidated. Amidation of the carboxyl group of an amino acid residue (typically the C-terminal amino acid residue of a peptide chain) can improve the structural stability (e.g., protease resistance) of the construct in the cytoplasm and nucleolus. In addition, amidation of the carboxyl group improves the hydrophilicity of the construct, thereby improving the solubility of the construct in aqueous solvents. Examples of aqueous solvents include water, various buffer solutions, physiological saline (e.g., PBS), cell culture medium, and the like. For example, when a foreign substance is bound to the N-terminus of a carrier peptide fragment consisting of any of the amino acid sequences shown in SEQ ID NOs: 1 to 4, it is preferable to amidate the carboxyl group of arginine at the C-terminus of the carrier peptide fragment. In addition, when the foreign substance is a polypeptide and such a polypeptide is bound to the C-terminus of the carrier peptide fragment, it is preferable to amidate the carboxyl group of the C-terminal amino acid residue of the polypeptide.

[0032] Among the constructs for introducing foreign substances, those having relatively short peptide chains (including polypeptides, carrier peptide fragments, and peptidic linkers constituted as foreign substances) can be easily produced according to general chemical synthesis methods. For example, any of the conventionally known solid-phase synthesis methods or liquid-phase synthesis methods may be adopted. A solid-phase synthesis method using Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the amino group is preferable. That is, the above-mentioned peptide chains having the desired amino acid sequence and modified (N-terminal acetylation, C-terminal amidation, etc.) portions can be synthesized by solid-phase synthesis using a commercially available peptide synthesizer. Note that only a part of the peptide chain may be synthesized by the above method, for example, a peptide chain containing only a carrier peptide fragment or a carrier peptide fragment and a peptidic linker portion may be synthesized.

[0033] Alternatively, the peptide portion may be produced by biosynthesis based on genetic engineering techniques. That is, a polynucleotide (typically DNA) of a nucleotide sequence (including an ATG start codon) encoding a desired amino acid sequence is synthesized. Then, a recombinant vector having an expression gene construct consisting of the synthesized polynucleotide (DNA) and various regulatory elements (including promoters, ribosome binding sites, terminators, enhancers, and various cis elements that control the expression level) for expressing the amino acid sequence in a host cell is constructed according to the host cell. This recombinant vector is introduced into a given host cell (e.g., yeast, insect cell, plant cell) by a general technique, and the host cell or a tissue or an individual containing the cell is cultured under given conditions. This allows the target peptide to be produced within the cell. The peptide portion is then isolated from the host cell (or from the medium if secreted), and the target peptide portion can be obtained by refolding, purification, etc. as necessary. The method for constructing a recombinant vector and the method for introducing the constructed recombinant vector into a host cell may be any method conventionally used in the field, and such methods themselves do not characterize the technology disclosed herein, so detailed explanations will be omitted.

[0034] For example, a fusion protein expression system can be used to efficiently produce large amounts of the polypeptide in a host cell. That is, a gene (DNA) encoding the amino acid sequence of the polypeptide of interest is chemically synthesized, and the synthetic gene is introduced into a suitable site of a suitable fusion protein expression vector (for example, a GST (Glutathione S-transferase) fusion protein expression vector such as the pET series provided by Novagen and the pGEX series provided by Amersham Biosciences). Then, a host cell (typically Escherichia coli) is transformed with the vector. The resulting transformant is cultured to prepare the fusion protein of interest. The protein is then extracted and purified. The purified fusion protein obtained is then cleaved with a specific enzyme (protease) to release the peptide fragment of interest (i.e., The resulting construct (designed artificial polypeptide) is then recovered by a method such as affinity chromatography. By using such a conventionally known fusion protein expression system (for example, the GST / His system provided by Amersham Biosciences) it is possible to produce the desired construct for introducing a foreign substance (artificial polypeptide). Alternatively, a template DNA for a cell-free protein synthesis system (i.e., a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of the peptide portion of the construct for introducing a foreign substance) is constructed, and various compounds (ATP, RNA polymerase, amino acids, etc.) necessary for the synthesis of the peptide portion are used to adopt a so-called cell-free protein synthesis system to synthesize the target polypeptide in vitro. For cell-free protein synthesis systems, for example, Shimizu et al.'s paper (Shimizu et al., Nature Biotechnology, 19, 751-755(2001)) and Madin et al.'s paper (Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564(2000)) are useful references. Based on the techniques described in these papers, many companies have already contracted to produce polypeptides at the time of filing the present application, and cell-free protein synthesis kits (available, for example, from Cell Free Science Co., Ltd. in Japan) are commercially available.

[0035] A single-stranded or double-stranded polynucleotide containing a nucleotide sequence encoding the peptide portion of a construct for introducing a foreign substance and / or a nucleotide sequence complementary to said sequence can be easily produced (synthesized) by a conventional method. That is, by selecting a codon corresponding to each amino acid residue constituting a designed amino acid sequence, a nucleotide sequence corresponding to said amino acid sequence can be easily determined and provided. Once the nucleotide sequence is determined, a polynucleotide (single-stranded) corresponding to a desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. Furthermore, the obtained single-stranded DNA can be used as a template to obtain a desired double-stranded DNA by employing various enzymatic synthesis means (typically PCR). The polynucleotide may be in the form of DNA or RNA (mRNA, etc.). The DNA may be provided in a double-stranded or single-stranded form. When provided in a single-stranded form, it may be a coding strand (sense strand) or a non-coding strand (antisense strand) of a sequence complementary thereto. The polynucleotides thus obtained can be used as materials for constructing recombinant genes (expression cassettes) for peptide production in various host cells or in cell-free protein synthesis systems, as described above.

[0036] The construct for introducing an exogenous substance may be suitably used as an active ingredient of a composition for use based on the function of the exogenous substance. The construct for introducing an exogenous substance may be in the form of a salt as long as the function of the exogenous substance is not lost. For example, an acid addition salt that can be obtained by addition reaction of a commonly used inorganic acid or organic acid according to a conventional method can be used. Therefore, the "construct for introducing an exogenous substance" described in this specification and claims includes such salt forms.

[0037] The construct for introducing an exogenous substance can be provided as a composition which can contain, in addition to the construct for introducing an exogenous substance as an active ingredient, various medicamentally (pharmacologically) acceptable carriers depending on the form of use. The above-mentioned carrier is preferably a carrier generally used in peptide medicines as a diluent, excipient, etc. The carrier may vary depending on the purpose and form of the construct for introducing a foreign substance, but typically includes water, physiological buffer solutions, and various organic solvents. In addition, the carrier may be an aqueous solution of alcohol (such as ethanol) at an appropriate concentration, glycerol, a non-drying oil such as olive oil, or a liposome. In addition, examples of secondary components that may be contained in the pharmaceutical composition include various fillers, bulking agents, binders, wetting agents, surfactants, dyes, fragrances, etc.

[0038] The form of the composition is not particularly limited. For example, typical forms include liquids, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, and ointments. In addition, for use in injections, etc., the composition can be made into a freeze-dried product or granulated product that is dissolved in physiological saline or a suitable buffer solution (e.g., PBS) just before use to prepare a drug solution. The process of preparing various forms of drugs (compositions) using a construct for introducing a foreign substance (main component) and various carriers (secondary components) may be in accordance with a conventionally known method, and detailed explanations of such formulation methods are omitted since they do not characterize the present invention. For example, Comprehensive Medicinal Chemistry, edited by Corwin Hansch, published by Pergamon Press (1990) is an example of a detailed information source regarding prescriptions.

[0039] There is provided a method for introducing a construct for introducing a foreign substance in vivo or outside the body (in vitro) using the construct (composition) for introducing a foreign substance disclosed herein. The method includes the following steps (1) to (3): (1) preparing a construct for introducing a foreign substance, the construct having a carrier peptide fragment consisting of any one of the amino acid sequences shown in SEQ ID NOs: 1 to 4 and a foreign substance of interest bound to the N-terminus and / or C-terminus of the carrier peptide fragment; (2) providing a construct for introducing an exogenous substance into a sample containing a target eukaryotic cell; (3) incubating the sample supplied with the construct for introducing an exogenous substance to introduce the construct into eukaryotic cells in the sample.

[0040] The above-mentioned "eukaryotic cells" include, in vivo, various tissues, organs, blood, lymph, etc., and in vitro, various cell masses, tissues, organs, blood, lymph, cell lines, etc., extracted from a living body.

[0041] The composition containing the construct disclosed herein can be used in vivo in a manner and dosage appropriate for its form and purpose. For example, as a liquid agent, it can be administered in a desired amount to the affected area (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient (i.e., living body) by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. Alternatively, a solid form such as a tablet or a gel or aqueous jelly such as an ointment can be administered directly to a specific tissue (i.e., an affected area such as a tissue or organ containing, for example, tumor cells, virus-infected cells, inflammatory cells, etc.). Alternatively, a solid form such as a tablet can be administered orally. In the case of oral administration, it is preferable to encapsulate or apply a protective (coating) material to suppress digestive enzyme decomposition in the digestive tract.

[0042] Alternatively, an appropriate amount of the composition disclosed herein (i.e., an appropriate amount of the construct for introducing a foreign substance) may be supplied at least once to the culture medium of the eukaryotic cells being cultured outside the body (in vitro). The amount and number of times of supply per time are not particularly limited, since they may vary depending on the type of eukaryotic cells to be cultured, cell density (cell density at the start of culture), number of passages, culture conditions, type of medium, and other conditions. For example, it is preferable to add once, twice, or more times so that the carrier peptide fragment concentration in the culture medium is within the range of approximately 0.05 μM to 100 μM, for example, 0.5 μM to 50 μM, for example, 1 μM to 20 μM, and for example, 1 μM to 10 μM. In addition, the incubation time after addition of the construct is also not particularly limited, since it may vary depending on the type of eukaryotic cells and various conditions. For example, it may be 0.5 hours or more, 1 hour or more, 4 hours or more, 8 hours or more, or 20 hours or more. Incubation conditions are not particularly limited as they may vary depending on the type of eukaryotic cell, but for example, incubation can be performed in a 5% CO2 atmosphere at 37°C. An example of an in vitro introduction method is shown in the following Examples.

[0043] The method for evaluating the introduction efficiency of a construct for introducing a foreign substance is not particularly limited. For example, when a dye (typically a fluorescent dye compound) is bound to the construct, the introduction efficiency into eukaryotic cells can be evaluated by microscopic observation (e.g., confocal laser microscope observation) or flow cytometry. The introduction efficiency of the construct can also be evaluated by immunochemical techniques (e.g., Western blot, immunocytostaining, etc.) using an antibody that specifically recognizes the peptide portion of the construct.

[0044] Several examples of the technology disclosed herein will be described below, but it is not intended that the technology disclosed herein be limited to those shown in these examples.

[0045] <Preparation of constructs for introducing foreign substances> Five synthetic peptides (peptides 1 to 5) shown in Table 1 were prepared. Peptides 1 to 5 are peptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 5, respectively. In the amino acid sequences shown in SEQ ID NOs: 1 to 4, the 8th and 9th amino acid residues from the N-terminus of the amino acid sequence known as NoLS of LIM kinase 2 shown in SEQ ID NO: 5 are all substituted with basic amino acids (arginine or lysine). Peptides 1 to 5 were all synthesized by solid-phase synthesis (Fmoc method) using a commercially available peptide synthesizer according to the manual. In addition, the α-amino group of the N-terminal lysine of peptides 1 to 5 was all acetylated. It should be noted that the mode of use of the peptide synthesizer itself does not characterize the technology disclosed herein, and therefore a detailed description thereof will be omitted.

[0046] [Table 1]

[0047] Next, the C-terminal amino acids of peptides 1 to 5 were treated with a fluorescent dye, FAM (C 21 H 12O7:5(6)-carboxyfluorescein (molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) was directly bound according to a conventional method. As a result, a foreign substance introduction construct comprising peptide 1 (also referred to as "sample 1"), a foreign substance introduction construct comprising peptide 2 (also referred to as "sample 2"), a foreign substance introduction construct comprising peptide 3 (also referred to as "sample 3"), a foreign substance introduction construct comprising peptide 4 (also referred to as "sample 4"), and a foreign substance introduction construct comprising peptide 5 (also referred to as "sample 5") were obtained. Samples 1 to 5 were each diluted with DMSO to prepare sample solutions 1 to 5 with a sample concentration of 2 mM.

[0048] <Test 1> <Evaluation of cell membrane permeability by flow cytometry> HeLa cells (an established cell line derived from human cervical cancer cells) were used as eukaryotic cells to analyze the cell membrane permeability of peptides 1 to 5. In this test, as shown in Table 2, samples 1 to 5 prepared above were used in Examples 1 to 5, respectively, and an FAM solution was used in Example 6.

[0049] [Table 2]

[0050] (Example 1) HeLa cells were cultured in a common culture medium, Dulbecco's modified Eagle's medium (DMEM, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 043-30085), containing 10% FBS (fetal bovine serum). After washing the HeLa cells attached to the culture plate with PBS, a 0.25% trypsin / EDTA solution was added and incubated at 37°C for 3 minutes. After the incubation, DMEM containing 10% FBS was added to inactivate the trypsin, and the cells were precipitated by centrifugation at 150 × g for 5 minutes. After removing the supernatant generated by centrifugation, DMEM containing 10% FBS was added to the precipitate (cell pellet), and approximately 1 × 10 5A cell suspension of 10 cells / mL was prepared. 2 mL of the cell suspension was added to the wells of a commercially available 6-well plate (AGC Technoglass Co., Ltd.), and the cells were seeded (approximately 2 × 10 5 The cells were then cultured at 37°C under 5% CO2 for 2 hours to allow the cells to adhere to the bottom of the wells.

[0051] Next, the 2 mM sample solution 1 was diluted with the 10% FBS-containing DMEM to prepare a sample solution 1 with a concentration of 20 μM. After removing 1 mL of the culture supernatant from the well after the 2-hour culture, 1 mL of the 20 μM sample solution 1 was added to the well (i.e., the concentration of sample 1 in the culture solution in the well was 10 μM and the DMSO concentration was 0.5%). Then, the cells were incubated at 37° C. for 20 hours under 5% CO2 conditions. After the 20-hour incubation, the culture supernatant was removed from the well, and the cells in the well were washed twice with 1 mL of PBS. Next, 200 μL of 0.25% trypsin / EDTA solution was added to the well, and the well was incubated at 37° C. for 3 minutes. After the incubation, 400 μL of 10% FBS-containing DMEM was added to the well to inactivate trypsin, and the cell suspension in the well was transferred to a tube and the cells were collected. Then, 600 μL of PBS was further added to the well to wash the well. Then, the PBS in the well was transferred to the tube, and the cells remaining in the well were collected in the tube. The tube was centrifuged at 4° C. and 210×g for 5 minutes. After centrifugation, the supernatant was removed, and the precipitate (cell pellet) was suspended (washed) in 1 mL of PBS, and then centrifuged under the same conditions as above. After repeating this operation twice, the supernatant was removed to obtain cells (cell pellet) cultured in the medium containing Sample 1.

[0052] The obtained cells (cell pellet) were analyzed for cell permeability in Sample 1 using a flow cytometer. The flow cytometer used was an On-Chip Flowcytometer (manufactured by On-Chip Biotechnologies Co., Ltd.). For this analysis, the obtained cell pellet was suspended in 50 μL of PBS, and 50 μL of the 2× sample buffer for the flow cytometer was further added to this suspension to prepare a cell suspension for analysis.

[0053] Using the above flow cytometer, gating based on forward scatter (FSC) and side scatter (SSC) was performed to set a gate for the cell population to be analyzed, and the fluorescence intensity of the cell population within the gate was measured. The analysis was performed so that the cell population had at least 10,000 cells. The fluorescence intensity was measured using the fluorescence detector FL2 (optimum detection wavelength: around 543 nm) of the above flow cytometer, which can detect the fluorescence wavelength of FAM. The measurement results were analyzed using commercially available analysis software "FlowJo (registered trademark)" (manufactured by TreeStar), and the mean fluorescent intensity (MFI) of the cell population to be measured was obtained.

[0054] (Example 2) The same procedure as in Example 1 was repeated, except that sample solution 1 was replaced with sample solution 2 prepared above. (Example 3) The same procedure as in Example 1 was repeated, except that sample solution 1 was replaced with sample solution 3 prepared above. (Example 4) The same procedure as in Example 1 was repeated, except that sample solution 1 was replaced with sample solution 4 prepared above. (Example 5) The same procedure as in Example 1 was repeated, except that sample solution 5 prepared above was used instead of sample solution 1. (Example 6) The same procedure as in Example 1 was carried out, except that sample solution 1 was a FAM solution diluted with DMSO. The concentration of the FAM solution was the same as that of sample 1 solution (i.e., the FAM concentration of the culture solution in the well was 10 μM, and the DMSO concentration was 0.5%).

[0055] The results obtained for Examples 1 to 6 are shown in Figure 1. Figure 1 shows the relative MFI values ​​for each Example when the MFI value for Example 6 is taken as 1.

[0056] As shown in FIG. 1, Examples 1 to 5 all had higher MFI values ​​than Example 6. This shows that Peptides 1 to 5 all have cell membrane permeability. Furthermore, Examples 1 to 4 all had MFI values ​​more than twice as high as the MFI value of Example 5, and among these, the MFI value of Example 1 was more than three times higher than the MFI value of Example 5. This shows that Peptides 1 to 4 have better cell membrane permeability than Peptide 5 (NoLS of LIM kinase 2). In addition, the inventors' investigations have revealed that foreign substances include not only fluorescent dyes but also polypeptides, nuclei, etc. It has been confirmed that such foreign substances, whether acids or drugs, can be efficiently introduced from the outside of the cell into the cytoplasm and even into the nucleolus.

[0057] <Test 2> In this study, the localization of samples 1 to 5 in HeLa cells was analyzed. HeLa cells were cultured on collagen-coated 8-well slides at approximately 2 × 10 4 The cells were seeded at 100 cells / well and cultured overnight at 37℃ in the presence of 5% CO2. DMEM containing 10% FBS was used as the cell culture medium. After overnight culture, sample solutions 1 to 5 were added to separate wells so that the sample concentration in the culture medium in the well was 5μM and the DMSO concentration was 0.5%, and the cells were cultured for an additional 20 hours.

[0058] Then, the supernatant (culture solution) was removed, and ice-cold PBS was added and washed three times on ice. Then, ice-cold methanol was added and the cells were left to stand at -20°C for 10 minutes to fix the cells. Then, methanol was removed and the cells were washed three times with ice-cold PBS. Next, mounting was performed using a DAPI-containing mounting solution (ThemoFisher Scientific) and a cover glass, and fluorescence observation was performed using a confocal laser microscope. , Figure 2A shows a fluorescence observation image of an example where sample 1 was added, Figure 2B shows a fluorescence observation image of an example where sample 2 was added, Figure 2C shows a fluorescence observation image of an example where sample 3 was added, Figure 2D shows a fluorescence observation image of an example where sample 4 was added, and Figure 2E shows a fluorescence observation image of an example where sample 5 was added. Note that Figures 2A to 2E all show images obtained by merging the fluorescence images of DAPI and FAM.

[0059] As is clear from Figures 2A to 2D, the fluorescence of FAM in Samples 1 to 4 was observed not only in the cytoplasm but also in the nucleoli present in the cell nuclei. In particular, the fluorescence intensity of FAM in the nucleoli was significantly stronger than that in the cytoplasm, and the outline of the nucleoli was clearly observed. On the other hand, as shown in Figure 2E, the fluorescence of FAM in Sample 5 was slightly observed in the nucleoli, but the fluorescence intensity was weaker than that in Figures 2A to 2D, and the outline of the nucleoli was unclear. These results show that peptides 1 to 4 have significantly superior nucleolar transportability than peptide 5 (NoLS of LIM kinase 2), and can introduce the desired foreign substance (here, FAM) into the nucleolus.

[0060] Although specific examples of the technology disclosed herein have been described in detail above, these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and alterations of the specific examples exemplified above. [Industrial Applicability]

[0061] According to the technology disclosed herein, an artificially produced construct is provided for introducing a foreign substance of interest from the outside of a eukaryotic cell (particularly various animal cells such as humans and other mammals that do not have a cell wall) into at least the cytoplasm, and further into the nucleolus. By using such a construct, it is possible to effectively introduce the foreign substance of interest into a target cell, and obtain living tissues such as cells into which the foreign substance has been introduced and organs containing cells containing the foreign substance. In addition, by using such a construct, it is possible to provide a therapeutic drug for a disease. In addition, since the construct disclosed herein has high nucleolar migration property, for example, a construct equipped with a dye can be used as a nucleolus marker. In addition, since the construct disclosed herein has significantly low cytotoxicity and can introduce a foreign substance of interest into the nucleolus, it can be used as a therapeutic drug for cancer and various diseases that target the nucleolus. [Sequence List Free Text]

[0062] SEQ ID NOs: 1 to 6 Synthetic peptides

Claims

1. A method for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell in vitro, comprising the steps of: (1) the following amino acid sequence: KKRTLRKKRKKR (SEQ ID NO: 1); KKRTLRKRRRKKR (SEQ ID NO:2); KKRTLRKRKRKRKKR (SEQ ID NO:3); KKRTLRKKRRKKR (SEQ ID NO: 4); A carrier peptide fragment consisting of any one of The foreign substance bound to the N-terminus and / or C-terminus of the carrier peptide fragment; Providing a construct for introducing an exogenous substance, comprising: (2) providing the construct for introducing an exogenous substance into a sample containing a target eukaryotic cell; (3) incubating the sample to which the construct for introducing an exogenous substance has been supplied, thereby introducing the construct into eukaryotic cells in the sample; The method includes:

2. The method of claim 1 , wherein the exogenous substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes and drugs.

3. the foreign substance is bound to the C-terminus of the carrier peptide fragment, The method according to claim 1 or 2, wherein the α-amino group of the lysine on the N-terminal side of the carrier peptide fragment is acetylated.

4. The method according to any one of claims 1 to 3, wherein the eukaryotic cell into which the construct for introducing an exogenous substance is introduced is a human or non-human mammalian cell.

5. A construct for introducing a foreign substance, which is prepared for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell, comprising: The amino acid sequence: KKRTLRKKRKKR (SEQ ID NO: 1); KKRTLRKRRRKKR (SEQ ID NO:2); KKRTLRKRKRKRKKR (SEQ ID NO:3); KKRTLRKKRRKKR (SEQ ID NO: 4); A carrier peptide fragment consisting of any one of the foreign substance of interest bound to the N-terminus and / or C-terminus of the carrier peptide fragment; A construct for introducing an exogenous substance comprising the above structure.

6. The construct according to claim 5 , wherein the foreign substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes and drugs.

7. the foreign substance is bound to the C-terminus of the carrier peptide fragment, The construct according to claim 5 or 6, wherein the α-amino group of the lysine on the N-terminal side of the carrier peptide fragment is acetylated.

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