Peptide fragments and their uses

Abstract

The present disclosure provides an artificially synthesized peptide fragment that makes it possible to introduce a target foreign substance from outside a eukaryotic cell into at least the cytoplasm of the cell with high efficiency. The peptide fragment disclosed herein is a peptide fragment that can be used for introducing a target foreign substance from outside a eukaryotic cell into at least the cytoplasm of the cell, the peptide fragment being configured such that at least two amino acid sequences VVKSLVK (SEQ ID NO:1) are arranged in series, and another amino acid residue is not included between the amino acid sequences or a peptide linker composed of 1 to 5 amino acid residues is interposed between the amino acid sequences.

Description

[Technical Field]

[0001] The present invention relates to a method for introducing (transporting) a foreign substance from the outside of a eukaryotic cell to the inside of the cell, a construct for introducing a foreign substance comprising a carrier peptide fragment used in the method, and a peptide fragment that constitutes the carrier peptide fragment. This application claims priority based on Japanese Patent Application No. 2021-75809, filed on April 28, 2021, the entire contents of which are incorporated herein by reference. [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 to transform the characteristics of the cells (and even the tissues and organs made up of those cells) or to improve or enhance the functions of those cells.

[0003] For example, Patent Document 1 discloses a construct for introducing a foreign substance, which contains the amino acid sequence set forth in SEQ ID NO: 2, which is known as the nucleolar localization signal of LIM kinase 2, a protein kinase involved in intracellular signal transduction present in human endothelial cells (see Non-Patent Document 1), and a foreign substance of interest. Because this amino acid sequence is an excellent cell membrane-permeable peptide, the construct can pass through the cell membrane of a eukaryotic cell with high efficiency. This allows the foreign substance of interest to be efficiently introduced from the outside of a eukaryotic cell into the cytoplasm of the cell. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2011 / 013700 [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-330206 [Non-patent literature]

[0005] [Non-Patent Document 1] JOUNAL OF BIOLOGICAL CHEMISTRY, Vol. 281, No. 35, 2006, pp. 25223-25230 Summary of the Invention

[0006] In recent years, interest in peptides with excellent cell membrane permeability has been growing, and from the perspective of medical treatment and other fields, there is a desire to develop technologies that can more efficiently introduce foreign substances into target cells than has been possible up to now.

[0007] The present invention was created to address these needs, and aims to provide a peptide fragment that can efficiently introduce a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell. Another aim is to provide a construct for introducing a foreign substance, which comprises such a peptide fragment and the foreign substance. A further aim is to provide a method for efficiently introducing such a construct into at least the cytoplasm of a eukaryotic cell from the outside of the cell.

[0008] To achieve the above-mentioned object, the present inventors searched for an amino acid sequence that has excellent cell membrane permeability and can introduce foreign substances from the outside of eukaryotic cells (i.e., outside the cell membrane) into at least the cytoplasm of the cells, and focused on an amino acid sequence (VHL-related peptide motif) consisting of 15 consecutive amino acid residues from positions 157 to 171 of the amino acid sequence of the von Hippel-Lindt (VHL) protein shown in SEQ ID NO: 3. This VHL-related peptide motif is an amino acid sequence that the present inventors discovered as a peptide motif involved in neuronal differentiation induction. After extensive investigation, the present inventors found that excellent cell membrane permeability can be achieved by tandemly repeating the seven amino acid residues at the C-terminus of the VHL-related peptide motif and further substituting lysine residues for the arginine residues.

[0009] That is, the peptide fragment disclosed herein is a peptide fragment for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell, The amino acid sequence: VVKSLVK (SEQ ID NO: 1) are arranged in tandem, with no other amino acid residues present between the amino acid sequences, or with a peptide linker consisting of 1 to 5 amino acid residues interposed therebetween. A peptide fragment having such a structure has excellent cell membrane permeability, and therefore can efficiently introduce a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell.

[0010] In a preferred embodiment of the peptide fragment disclosed herein, no other amino acid residues are present between the amino acid sequences. In a preferred embodiment, two amino acid sequences shown in SEQ ID NO: 1 are arranged in tandem. This configuration allows for excellent cell membrane permeability with a shorter sequence.

[0011] In order to achieve the above object, in another aspect, there is provided an artificially constructed construct for introducing (transporting) a target foreign substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell. That is, the construct for introducing foreign substances disclosed herein has a carrier peptide fragment consisting of the peptide fragment disclosed herein and the above-mentioned foreign substance of interest bound to the N-terminus and / or C-terminus of the carrier peptide fragment. According to this configuration, a foreign substance bound to a carrier peptide fragment can be efficiently introduced into eukaryotic cells. Here, the term "foreign substance" refers to inorganic and organic compounds that can be bound directly or indirectly via an appropriate linker to the N-terminus or C-terminus of the carrier peptide fragment, and that have a molecular size and chemical properties that allow them to be introduced into eukaryotic cells.

[0012] In a preferred embodiment of the construct for introducing an exogenous substance disclosed herein, the exogenous substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes, and drugs. Here, "polypeptide" refers to a polymer having a structure in which multiple amino acids are linked by peptide bonds. Polypeptides are not limited by the number of peptide bonds (i.e., the number of amino acid residues). That is, polypeptides include those generally called peptides, which have from 10 to less than 300 amino acid residues, and those generally called proteins (polymeric compounds typically consisting of 300 or more amino acid residues). In the art, 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. Furthermore, "nucleic acid" refers to a polymer of nucleotides, and includes DNA and RNA. "Nucleic acid" is not limited by the number of bases.

[0013] Furthermore, preferably, the foreign substance is a mature polypeptide or its precursor polypeptide derived from any biological species, and is a synthetic polypeptide having an amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance and the amino acid sequence of the carrier peptide fragment. More preferably, the amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance is located on the N-terminal side of the carrier peptide fragment.

[0014] Furthermore, in order to achieve the above object, the technology disclosed herein provides a method for efficiently introducing (transporting) a foreign substance of interest from the outside of a eukaryotic cell into at least the cytoplasm of the cell. That is, the method for introducing a foreign substance disclosed herein comprises the steps of: (1) providing a construct for introducing an exogenous substance as disclosed herein; (2) providing the construct for introducing a foreign substance into a sample containing a target eukaryotic cell; (3) incubating the sample to which the exogenous substance introduction construct has been supplied, thereby introducing the exogenous substance introduction construct into eukaryotic cells in the sample.

[0015] According to the method for introducing foreign substances having the above-described configuration, a foreign substance introduction construct constructed by binding the foreign substance of interest (typically an organic compound such as a polypeptide, nucleic acid, dye, drug, etc.) directly or indirectly via a suitable linker to the N-terminal and / or C-terminal side of the carrier peptide fragment is supplied to a sample containing the target eukaryotic cells (typically a culture containing the cells) (i.e., added to living eukaryotic cells), thereby enabling the foreign substance of interest to be introduced highly efficiently from the outside of the eukaryotic cells (outside the cell membrane) through the cell membrane into the cytoplasm.

[0016] 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 polypeptides, nucleic acids, dyes, and drugs. A construct containing this type of organic compound can be efficiently introduced into a target cell.

[0017] In another preferred embodiment of the method for introducing a foreign substance disclosed herein, the foreign substance is a mature polypeptide or its precursor polypeptide derived from any biological species, and the construct for introducing a foreign substance is a synthetic polypeptide having an amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance and the amino acid sequence of a carrier peptide fragment. According to this configuration, a synthetic peptide having the amino acid sequence of the mature polypeptide or its precursor polypeptide and the amino acid sequence of the carrier peptide fragment can be efficiently introduced into a target eukaryotic cell.

[0018] In another preferred embodiment of the method for introducing a foreign substance disclosed herein, the amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance is located on the N-terminal side of the carrier peptide fragment. According to this configuration, the amino acid sequence of the mature polypeptide or its precursor polypeptide can be introduced more efficiently into the target eukaryotic cells.

[0019] In another preferred embodiment of the method for introducing an exogenous substance disclosed herein, the eukaryotic cells into which the above-mentioned construct for introducing an exogenous substance is introduced are cells of a human or non-human mammal. This configuration allows for efficient introduction of foreign substances into the cytoplasm of human or non-human mammalian cells. [Brief explanation of the drawings]

[0020] [Figure 1] 1 is a histogram showing the relationship between fluorescence intensity and cell number obtained by flow cytometric analysis of cells after the addition of the constructs (Sample 1) having the amino acid sequence of SEQ ID NO: 4 and FAM to HeLa cell culture medium (Example 1), (Sample 2) having the amino acid sequence of SEQ ID NO: 5 and FAM (Example 2), (Sample 3) having the amino acid sequence of SEQ ID NO: 6 and FAM (Example 3), and (Sample 4) having FAM. The X-axis (horizontal axis) represents fluorescence intensity, and the Y-axis (vertical axis) represents cell number. DETAILED DESCRIPTION OF THE INVENTION

[0021] Preferred embodiments of the technology disclosed herein are described below. Matters necessary for carrying out the technology disclosed herein other than those specifically mentioned in the present specification (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 matters of design by 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. Furthermore, the technology disclosed herein can be implemented based on the contents disclosed in this specification and the common general technical knowledge in the relevant field. In the following explanation, amino acids are sometimes represented by one-letter symbols in accordance with the nomenclature for amino acids set forth in the IUPAC-IUB guidelines (however, in the sequence listing, they are represented by three-letter symbols). 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.

[0022] Furthermore, as used herein, the term "synthetic peptide" refers to a peptide fragment whose peptide chain does not exist independently and stably in nature, 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, the term "peptide" refers to an amino acid polymer having multiple peptide bonds and is not limited by the number of amino acid residues. In the amino acid sequences described herein, the left side always represents the N-terminus and the right side represents the C-terminus. Furthermore, when a numerical range is described herein as A to B (where A and B are arbitrary numerical values), this is interpreted as generally meaning A or more and B or less, and includes a range exceeding A and below B.

[0023] The peptide fragment disclosed herein contains multiple (two or more) amino acid sequences: VVKSLVK shown in SEQ ID NO: 1. The amino acid sequences shown in SEQ ID NO: 1 are arranged in tandem.

[0024] The amino acid sequence shown in SEQ ID NO: 1 is a sequence in which the third arginine residue from the N-terminus of the seven amino acid residues (positions 165 to 171) constituting the von Hippel-Lindt (VHL) protein, which is known to be expressed in neurons of the central nervous system, is replaced with a lysine residue. VHL protein is known to have a VHL-related peptide motif (an amino acid sequence consisting of a total of 15 amino acid residues (positions 157 to 171) from the N-terminus of the VHL protein (SEQ ID NO: 3)), which exhibits neuronal differentiation-inducing properties (see Patent Document 2). Although the VHL-related peptide motif possesses cell membrane permeability, its permeation efficiency is not high. However, the present inventors' studies have revealed that a peptide fragment containing a repeat sequence of seven amino acid residues from the C-terminus of the VHL-related motif possesses excellent cell membrane permeability. Furthermore, the replacement of the arginine residue in this repeat sequence with a lysine residue significantly improved cell membrane permeability beyond the range predicted by conservative amino acid substitutions (so-called conservative substitutions).

[0025] In the peptide fragment disclosed herein, two or more amino acid sequences shown in SEQ ID NO: 1 are arranged in tandem. The number of sequences arranged may be, for example, three or more, or four or more. Furthermore, although not particularly limited, the number of sequences arranged in tandem may be, for example, ten or less, or five or less. In this specification, "n (n is a natural number of two or more) sequences of the amino acid sequence shown in SEQ ID NO: 1 are arranged" means that n units are arranged, with the amino acid sequence consisting of seven amino acid residues shown in SEQ ID NO: 1 being one unit.

[0026] The amino acid sequences of SEQ ID NO: 1 contained in the peptide fragment may be directly (contiguously) linked to each other without the inclusion of any other amino acid residues, or may be indirectly linked via a peptide linker consisting of other amino acid residues. The number of amino acid residues constituting this peptide linker is not particularly limited as long as it does not significantly impair cell membrane permeability, but is, for example, 1 to 5, and preferably 1 to 3. The type of amino acid residues constituting the peptide linker may include any amino acid residues as long as it does not significantly impair cell membrane permeability. Among these, preferred examples include amino acids whose side chains are uncharged and which are unlikely to cause steric hindrance. Examples of such amino acids include glycine, alanine, serine, etc., and it is preferable to select one or more of these to constitute the peptide linker.

[0027] Furthermore, the peptide fragment may contain other amino acid residues at the N-terminal and / or C-terminal ends, to the extent that cell membrane permeability is not significantly impaired. The number of other amino acid residues is not particularly limited, but may be, for example, 5 or less at each end, and preferably 3 or less. The type of other amino acid is not particularly limited, but may be, for example, an amino acid residue whose side chain is uncharged and which is unlikely to cause steric hindrance, such as glycine, alanine, or serine.

[0028] When the total number of amino acid residues constituting the peptide fragment is taken as 100%, the proportion of the amino acid sequence shown in SEQ ID NO: 1 is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more, and is preferably 100% (i.e., composed only of repeats of the amino acid sequence shown in SEQ ID NO: 1). The higher the proportion of the amino acid sequence shown in SEQ ID NO: 1, the more effectively the cell membrane permeability can be exerted.

[0029] The total number of amino acid residues constituting the peptide fragment is not particularly limited, but may be, for example, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, or 15 or less.

[0030] The peptide fragments disclosed herein may contain modified sequences of the amino acid sequence shown in SEQ ID NO: 1 to the extent that cell membrane permeability is not impaired. Here, a "modified sequence" refers to an amino acid sequence (modified amino acid sequence) formed by the substitution, deletion, and / or addition (insertion) of one or several (e.g., two or three) amino acid residues. Examples of modified sequences in the technology disclosed herein include sequences resulting from so-called conservative amino acid replacement, in which one or two amino acid residues are conservatively substituted for the total amino acid residues constituting the amino acid sequence shown in SEQ ID NO: 1 contained in the peptide fragment (e.g., a total of 14 amino acid residues when two copies of the amino acid sequence shown in SEQ ID NO: 1 are contained), and sequences in which one, two, or three amino acid residues are added (inserted) or deleted. Such slightly modified sequences can be easily utilized by those skilled in the art based on the information disclosed herein, and are therefore encompassed by the "peptide fragment (carrier peptide fragment)" as a technical concept disclosed herein. Specific examples of conservative substitutions include sequences in which a hydrophobic amino acid residue is substituted for another hydrophobic amino acid residue (for example, mutual substitution of leucine, isoleucine, and valine residues).

[0031] The construct for introducing foreign substances disclosed herein has a carrier peptide fragment consisting of the peptide fragment disclosed herein and the above-mentioned foreign substance of interest bound to the N-terminus and / or C-terminus of the carrier peptide fragment. The "carrier peptide fragment" disclosed herein is a sequence defined (understood) by the amino acid sequence of the peptide fragment disclosed herein, and is an amino acid sequence that exerts cell membrane permeability in eukaryotic cells. Therefore, a construct for introducing an exogenous substance, which has such a carrier peptide fragment and a target exogenous substance bound to the N-terminus and / or C-terminus of the carrier peptide fragment, can be introduced with high efficiency into at least the cytoplasm of a eukaryotic cell from outside the cell.

[0032] A construct for introducing a foreign substance can be designed and constructed by binding (linking) the desired foreign substance directly or indirectly via a suitable linker to the N-terminus and / or C-terminus of the above-mentioned carrier peptide fragment. The linker is not particularly limited, but may be a peptide linker or a non-peptide linker. It is preferable, but not particularly limited, that the amino acid sequence constituting the peptide linker is one that does not cause steric hindrance and is flexible. The peptide linker may be, for example, a linker consisting of 10 or less amino acid residues (more preferably 1 to 5, e.g., 1, 2, 3, 4, or 5) containing one or more amino acid residues selected from glycine, alanine, serine, etc. Furthermore, β-alanine may be used as a component of such a linker. The non-peptide linker is not particularly limited, but may be, for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminohexanoyl spacer, or the like.

[0033] The foreign substance is typically an organic compound such as a polypeptide, nucleic acid, dye, or drug. The foreign substance may be, for example, a polypeptide. When the foreign substance is a polypeptide, a peptide chain containing the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the carrier peptide fragment can be designed and biosynthesized or chemically synthesized to produce a desired construct for introducing a foreign substance. Alternatively, a construct for introducing a foreign substance can be constructed by directly or indirectly linking 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)) to the N-terminus and / or C-terminus of the carrier peptide fragment described above using various scientific techniques known in the art. Although not particularly limited, the function of the foreign substance may be, for example, promoting stem cell differentiation induction (stem cell differentiation induction activity), inhibiting tumor cell proliferation (antitumor activity), inhibiting the proliferation of virus-infected cells (antiviral activity), etc.

[0034] In a construct for introducing an exogenous substance, the number of exogenous substances bound to a carrier peptide fragment is not particularly limited. That is, one or more exogenous substances may be bound to one carrier peptide fragment. Although not particularly limited, for example, a polypeptide, nucleic acid, drug, etc. may be bound to the N-terminus of one carrier peptide fragment, and a dye may be bound to the C-terminus. Binding a dye to a carrier peptide fragment is preferred because it facilitates evaluation of the efficiency of introduction of an exogenous substance into eukaryotic cells and its localization within the cells.

[0035] When the foreign substance is a polypeptide, the polypeptide (amino acid sequence) to be used is not particularly limited. For example, a foreign substance may be a polypeptide or protein having a relatively large number of amino acid residues, such as about 100 to 1000 amino acid residues. 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 lengths are easy to synthesize (biosynthesize or chemically synthesize) and are easy to use.

[0036] 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). Furthermore, the method for introducing foreign substances disclosed herein can also be carried out to introduce into cells a polypeptide whose function has not previously been known, in order to elucidate the function of the polypeptide within the cell (in a living tissue). For example, when the eukaryotic cells to be introduced with a foreign substance are human or other mammalian stem cells, it is preferable to use mature forms or precursors of polypeptides with various physiological activities involved in inducing the differentiation of the stem cells. Note that "stem cells" encompass somatic stem cells, embryonic stem cells, and induced pluripotent stem cells (hereinafter referred to as iPS cells). Furthermore, when the eukaryotic cells to be introduced with a foreign substance are cancer cells (tumor cells), it is preferable to use various polypeptides involved in inducing apoptosis of the cancer cells (tumor cells). Alternatively, in this case, it is preferable to use polypeptides that can inhibit the suppression of the immune surveillance mechanism of cancer cells (tumor cells). Furthermore, when the eukaryotic cells to be introduced with a foreign substance are bacterially or virally infected cells, it is preferable to use various polypeptides involved in inducing apoptosis of the infected cells, polypeptides that can inhibit bacterial or viral proliferation in the infected cells, or polypeptides that can inhibit the spread of bacterial or viral infection from the infected cells. As with peptide fragments, a polypeptide as a foreign substance may contain a modified amino acid sequence formed by substitution, deletion, and / or addition (insertion) of one or several amino acid residues, as long as it retains its function.

[0037] The construct for introducing an exogenous substance preferably has at least one amino acid residue 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 for introducing an exogenous substance in the cytoplasm and nucleus. For example, when a foreign substance is bound to the N-terminus of a carrier peptide fragment, it is preferable to amidate the C-terminal amino acid residue of the carrier peptide fragment.Also, when the foreign substance is a polypeptide and the polypeptide is bound to the C-terminus of a carrier peptide fragment, it is preferable to amidate the C-terminal amino acid residue of the polypeptide.

[0038] Among the peptide fragments and constructs for foreign substance introduction disclosed herein, those with relatively short peptide chains (including the polypeptide constituting the foreign substance, the carrier peptide fragment, and the peptide linker) can be easily produced using standard chemical synthesis methods. For example, either conventional solid-phase or liquid-phase synthesis methods may be employed. Solid-phase synthesis using Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as the amino group protecting group is preferred. That is, the above-mentioned peptide chains having the desired amino acid sequence and modifications (e.g., C-terminal amidation) can be synthesized by solid-phase synthesis using a commercially available peptide synthesizer. Note that only a portion of the peptide chain may be synthesized using the above method; for example, a peptide chain containing only the carrier peptide fragment, or a peptide chain containing the carrier peptide fragment and a peptide linker portion, may be synthesized.

[0039] Alternatively, the peptide portion may be biosynthesized using genetic engineering techniques. That is, a polynucleotide (typically DNA) with a nucleotide sequence (including an ATG initiation codon) encoding the desired amino acid sequence is synthesized. Then, a recombinant vector containing 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 expression levels) for expressing the amino acid sequence in host cells is constructed according to the host cell. This recombinant vector is introduced into a specific host cell (e.g., yeast, insect cell, or plant cell) using standard techniques, and the host cell or a tissue or individual containing the cell is cultured under specific conditions. This allows the target peptide to be produced intracellularly. The target peptide portion can then be isolated from the host cell (or from the culture medium if secreted), and, if necessary, refolded, purified, or the like, to obtain the target peptide portion. 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 relevant field, and such methods themselves do not particularly characterize the present technology, so detailed explanations thereof will be omitted.

[0040] For example, a fusion protein expression system can be used to efficiently mass-produce a target polypeptide in a host cell. Specifically, a gene (DNA) encoding the amino acid sequence of the target polypeptide is chemically synthesized, and the synthetic gene is introduced into a suitable site of a suitable fusion protein expression vector (e.g., a GST (Glutathione S-transferase) fusion protein expression vector such as the pET series from Novagen and the pGEX series from Amersham Biosciences). Host cells (typically Escherichia coli) are then transformed with the vector. The resulting transformant is cultured to prepare the target fusion protein. The protein is then extracted and purified. The purified fusion protein is then cleaved with a specific enzyme (protease), and the released target peptide fragment (i.e., the designed artificial polypeptide) is recovered by affinity chromatography or other methods. A target construct for introducing a foreign substance (artificial polypeptide) can be produced using such a conventionally known fusion protein expression system (e.g., the GST / His system from Amersham Biosciences). 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) can be constructed, and various compounds (ATP, RNA polymerase, amino acids, etc.) necessary for synthesizing the peptide portion can be used to employ a so-called cell-free protein synthesis system to synthesize the desired polypeptide in vitro. Regarding cell-free protein synthesis systems, for example, Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)) and Madin et al. (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 were already contracted to produce polypeptides at the time of filing the present application, and cell-free protein synthesis kits (available, for example, from CellFree Science Co., Ltd. in Japan) were commercially available.

[0041] Single-stranded or double-stranded polynucleotides containing a nucleotide sequence encoding the peptide portion of a construct for introducing an exogenous substance and / or a nucleotide sequence complementary to said sequence can be easily produced (synthesized) by conventional methods. Specifically, by selecting codons corresponding to each amino acid residue constituting a designed amino acid sequence, the 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 the 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 the desired double-stranded DNA by various enzymatic synthesis methods (typically PCR). Furthermore, the polynucleotide may be in the form of DNA or RNA (e.g., mRNA). DNA can be provided as either double-stranded or single-stranded. When provided as a single-stranded DNA, it may be the coding strand (sense strand) or the non-coding strand (antisense strand) of a complementary sequence. 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.

[0042] The construct for introducing an exogenous substance can be suitably used as an active ingredient in 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 obtainable by addition reaction of a commonly used inorganic or organic acid according to standard methods can be used. Therefore, the "construct for introducing an exogenous substance" described in this specification and claims encompasses such salt forms.

[0043] The construct for introducing an exogenous substance can be provided as a composition that 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 carrier is preferably one that is commonly used in peptide drugs as a diluent, excipient, or the like. While such a carrier may vary depending on the application and form of the exogenous substance introduction construct, typical examples include water, physiological buffer solutions, and various organic solvents. Furthermore, such a carrier may be an aqueous solution of an appropriate concentration of alcohol (e.g., ethanol), glycerol, a non-drying oil such as olive oil, or a liposome. Secondary components that may be contained in the pharmaceutical composition include various fillers, extenders, binders, humectants, surfactants, dyes, fragrances, and the like.

[0044] The form of the composition is not particularly limited. For example, typical forms include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, and ointments. Furthermore, for use in injections, etc., the composition may be in the form of a freeze-dried product or granulated product that can be dissolved in physiological saline or an appropriate buffer solution (e.g., PBS) immediately before use to prepare a medicinal 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 conventionally known methods, and since such formulation methods do not characterize the present technology, detailed explanations will be omitted. For example, Comprehensive Medicinal Chemistry, edited by Corwin Hansch, published by Pergamon Press (1990), is an example of a source of detailed information on formulations.

[0045] There is provided a method for introducing a construct for introducing a foreign substance in vivo or in vitro using the construct (composition) for introducing a foreign substance disclosed herein. The method roughly comprises the following steps (1) to (3): (1) providing a construct for introducing an exogenous substance as disclosed herein; (2) providing the construct for introducing a foreign substance into a sample containing a target eukaryotic cell; (3) incubating the sample to which the exogenous substance introduction construct has been supplied, thereby introducing the exogenous substance introduction construct into eukaryotic cells in the sample.

[0046] The "eukaryotic cells" mentioned above include, for example, various tissues, organs, blood, lymph, etc. in vivo. The "eukaryotic cells" mentioned above include, for example, various cell masses, tissues, organs, blood, lymph, and cell lines extracted from a living body in vitro.

[0047] The compositions containing the constructs disclosed herein can be used in vivo in a manner and dosage appropriate for their form and purpose. For example, as a liquid formulation, they 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 organism) by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. Alternatively, solid forms such as tablets, or gels or aqueous jelly forms such as ointments, can be administered directly to the affected area (e.g., affected areas such as tissues or organs containing tumor cells, virus-infected cells, inflammatory cells, etc.). Alternatively, solid forms such as tablets can be administered orally. For oral administration, encapsulation or application of a protective (coating) material is preferred to prevent degradation by digestive enzymes in the digestive tract.

[0048] Alternatively, an appropriate amount of the composition disclosed herein (i.e., an appropriate amount of a construct for introducing an exogenous substance) may be added at least once to the culture medium of eukaryotic cells being cultured in vitro. The amount and frequency of addition per addition are not particularly limited, as they may vary depending on the type of eukaryotic cells being 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 the carrier peptide fragment once, twice, or more times so that the concentration of the carrier peptide fragment in the culture medium is approximately in the range of 0.05 μM to 100 μM, e.g., 0.5 μM to 50 μM, or e.g., 1 μM to 20 μM. An example of an in vitro introduction method is shown in the Examples below.

[0049] The method for evaluating the efficiency of introduction 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 efficiency of introduction into eukaryotic cells can be evaluated using microscopic observation (e.g., fluorescence microscopic observation) or flow cytometry. The efficiency of introduction of the construct can also be evaluated by immunochemical techniques (Western blotting, immunocytostaining, etc.) using an antibody that specifically recognizes the peptide portion of the construct.

[0050] Hereinafter, several examples of the technology disclosed herein will be described, but it is not intended that the technology disclosed herein be limited to those shown in these examples.

[0051] <Preparation of constructs for introducing foreign substances> Three synthetic peptides (Peptides 1 to 3) shown in Table 1 were prepared. Peptide 1 is a peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 4, and is composed of two amino acid sequences shown in SEQ ID NO: 1 linked directly in tandem. Peptide 2 is a peptide fragment consisting of the amino acid sequence (SEQ ID NO: 5) in which two seven amino acid residues from the C-terminus of a VHL-related motif are linked directly in tandem. Peptide 3 is a peptide fragment consisting of the amino acid sequence (SEQ ID NO: 6) in which the two lysine residues in the amino acid sequence shown in SEQ ID NO: 5 are each replaced with arginine residues. Peptides 1 to 3 were all synthesized by solid-phase synthesis (Fmoc method) using a commercially available peptide synthesizer according to the manual. In addition, peptides 1 to 3 were all synthesized in such a way that the carboxyl group (-COOH) of the C-terminal amino acid residue was amidated (-CONH2). It should be noted that the manner of use of the peptide synthesizer itself does not characterize the technology disclosed herein, and therefore a detailed description thereof will be omitted.

[0052] [Table 1]

[0053] Next, the fluorescent dye FAM (C 21 H 12 O7:5(6)-Carboxyfluorescein (molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) was directly coupled using standard methods to prepare constructs for introducing foreign substances (Samples 1 to 3). Samples 1 to 3 were each diluted with DMSO to prepare sample solutions 1 to 3 with a concentration of 2 mM.

[0054] <Evaluation of cell membrane permeability for samples 1 to 3> HeLa cells (an established cell line derived from human cervical cancer cells) were used as eukaryotic cells to evaluate the cell membrane permeability of Samples 1 to 3. As shown in Table 2, the test in which Sample Solution 1 was added to the HeLa cell culture medium was Example 1, the test in which Sample Solution 2 was added was Example 2, the test in which Sample Solution 3 was added was Example 3, and the test in which FAM solution diluted with DMSO was added was Example 4. In Table 2, "FAM" refers to the above-mentioned fluorescent dye.

[0055] [Table 2]

[0056] (Example 1) HeLa cells were cultured in a common culture medium, Dulbecco's modified Eagle's medium (DMEM) (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 this incubation, the above-mentioned 10% FBS-containing DMEM was added to inactivate the trypsin, and the cells were precipitated by centrifugation at 150 × g for 5 minutes. After removing the supernatant resulting from centrifugation, the above-mentioned 10% FBS-containing DMEM was added to the precipitate (cell pellet), and approximately 1 × 10 5 A cell suspension of 200 cells / mL was prepared. 2 mL of the cell suspension was added to each well 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 3 hours to allow them to adhere to the bottom of the wells.

[0057] Next, the 2 mM sample solution 1 was diluted with 10% FBS-containing DMEM to prepare a sample solution 1 with a sample 1 concentration of 20 μM. After the 3-hour culture, 1 mL of the culture supernatant was removed from the well, and 1 mL of the 20 μM sample solution 1 was added to the well (i.e., the sample 1 concentration in the culture solution in the well was 10 μM and the DMSO concentration was 0.5%). The cells were then 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 the 10% FBS-containing DMEM was added to the well to inactivate the trypsin, and the cell suspension in the well was transferred to a tube and the cells were collected. After that, 600 μL of PBS was added to the wells to wash them. The PBS in the wells was then transferred to the tube, and the cells remaining in the wells were collected in the tube. The tubes were 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 centrifuged under the same conditions as above. This procedure was repeated twice, and then the supernatant was removed to obtain cells (cell pellet) cultured in Sample 1-containing medium.

[0058] The obtained cells (cell pellet) were analyzed for cell membrane 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 added to this suspension to prepare a cell suspension for analysis.

[0059] 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 this gate was measured. The analysis was performed so that the cell population consisted of approximately 10,000 cells. The fluorescence intensity was measured using the FL2 fluorescence detector of the above flow cytometer, which is capable of detecting the fluorescence wavelength of FAM (optimal detection wavelength: approximately 543 nm). The measurement results were analyzed using commercially available analysis software "FlowJo (registered trademark)" (TreeStar), and the mean fluorescence intensity (MFI) of the cell population to be measured was obtained.

[0060] (Examples 2-3) The same procedure as in Example 1 was carried out except that the above sample solution 1 was used as the sample solution for each example. (Example 4) The same procedure as in Example 1 was carried out, except that Sample Solution 1 was an FAM solution diluted with DMSO. The concentration of the FAM solution was the same as that of Sample 1 solution (i.e., the culture solution in the wells had an FAM concentration of 10 μM and a DMSO concentration of 0.5%).

[0061] The results obtained for Examples 1 to 4 are shown in Figure 1 and Table 3. Figure 1 shows a histogram of fluorescence intensity (horizontal axis) and cell number (vertical axis) measured by the fluorescence detector of the flow cytometer. Table 3 shows the MFI values ​​for each example obtained by such measurements.

[0062] [Table 3]

[0063] As shown in FIG. 1 , the histograms for Examples 1 to 3 were shifted to the right on the horizontal axis compared to Example 4, in which only FAM was added. This indicates that all of the sequences of Peptides 1 to 3 have cell membrane permeability and can at least introduce foreign substances into the cytoplasm. Furthermore, comparing Examples 1 to 3, Example 1, which used Peptide 1, in which the arginine residue of Peptide 2 was substituted with a lysine residue, had an MFI value more than twice that of Example 2, indicating that it exhibits particularly excellent cell membrane permeability. On the other hand, Example 3, which used Peptide 3, in which the lysine residue of Peptide 2 was substituted with an arginine residue, showed a significantly lower MFI value compared to Example 2. These results indicate that conservative substitution of basic amino acids (lysine, arginine) contained in the amino acid sequence of Peptide 2 significantly affects cell membrane permeability.

[0064] Furthermore, although detailed data are not shown, the inventors' investigations have confirmed that foreign substances, whether they are fluorescent dyes, polypeptides, nucleic acids, or drugs, are efficiently introduced from outside the cell at least into the cytoplasm.

[0065] As is clear from the above, according to the technology disclosed herein, by using a peptide fragment consisting of two or more amino acid sequences shown in SEQ ID NO: 1 arranged in series as a carrier peptide fragment, it is possible to efficiently introduce a target foreign substance from outside a eukaryotic cell into at least the cytoplasm of the cell.

[0066] While 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]

[0067] The technology disclosed herein provides artificially engineered peptide fragments and constructs comprising the peptide fragments for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell (particularly various animal cells not having a cell wall, such as humans and other mammalian cells) from the outside of the cell. By utilizing such constructs, it is possible to effectively introduce the foreign substance of interest into the target cell, thereby obtaining cells into which the foreign substance has been introduced, as well as biological tissues such as organs containing cells containing the foreign substance. Furthermore, by utilizing such constructs, it is possible to provide therapeutic drugs for diseases. [Sequence List Free Text]

[0068] SEQ ID NOs: 1 to 6 Synthetic peptides

Claims

1. A peptide fragment for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell, comprising: The following amino acid sequence: VVKSLVK (SEQ ID NO: 1) A peptide fragment in which two or more of the following are arranged in tandem, with no other amino acid residues present between the amino acid sequences or with a peptide linker consisting of 1 to 5 amino acid residues interposed therebetween.

2. The peptide fragment of claim 1 , which does not contain any other amino acid residues between the amino acid sequences.

3. The peptide fragment according to claim 1 or 2, wherein two amino acid sequences shown in SEQ ID NO: 1 are arranged in tandem.

4. 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, A carrier peptide fragment consisting of the peptide fragment according to any one of claims 1 to 3; the foreign substance of interest bound to the N-terminal side and / or C-terminal side of the carrier peptide fragment; A construct for introducing an exogenous substance, comprising:

5. The construct according to claim 4 , wherein the exogenous substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes and drugs.

6. The construct described in claim 5, wherein the foreign substance is a mature polypeptide or its precursor polypeptide derived from any biological species, and is a synthetic polypeptide having an amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance and the amino acid sequence of the carrier peptide fragment.

7. The construct of claim 6 , wherein the amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance is located on the N-terminal side of the carrier peptide fragment.

8. A method for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell in vitro from the outside of the cell, comprising: (1) preparing a construct for introducing an exogenous substance according to any one of claims 4 to 7; (2) providing the construct for introducing a foreign substance into a sample containing a target eukaryotic cell; (3) incubating the sample to which the exogenous substance introduction construct has been supplied, thereby introducing the exogenous substance introduction construct into eukaryotic cells in the sample; A method that encompasses

9. The method of claim 8, wherein the foreign substance is any organic compound selected from the group consisting of polypeptides, nucleic acids, dyes, and drugs.

10. The method of claim 9, wherein the foreign substance is a mature polypeptide or its precursor polypeptide derived from any biological species, and the construct for introducing a foreign substance is a synthetic polypeptide having an amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance and the amino acid sequence of the carrier peptide fragment.

11. The method according to claim 10, wherein the amino acid sequence corresponding to the mature polypeptide or its precursor polypeptide as the foreign substance is located on the N-terminal side of the carrier peptide fragment.

12. The method according to any one of claims 8 to 11, wherein the eukaryotic cells into which the construct for introducing an exogenous substance is introduced are cells of a human or non-human mammal.

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