Peptide fragments and uses thereof

Abstract

The present invention pertains to a peptide fragment containing the amino acid sequence: RRRRSNRRG (SEQ ID NO: 1), the total of the amino acid residues being 10 or less.

Description

Technical Field

[0001] This invention relates to peptide fragments and their uses. It should be noted that this application claims priority based on Japanese Patent Application No. 2023-081456, filed on May 17, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0002] For a long time, the introduction of exogenous substances such as polypeptides and nucleic acids into the cells (eukaryotic cells) of humans and non-human mammals has been used to induce biotransformation of the cells (and the tissues and organs composed of these cells) or to improve and enhance the function of the cells.

[0003] The aforementioned technologies employ, for example, cell penetrating peptides (CPPs) that allow exogenous substances to be introduced into the cytoplasm from outside the cell through the cell membrane. For instance, patent documents 1-3 disclose technologies for introducing nucleic acid drugs for disease treatment into cells using cell penetrating peptides.

[0004] Existing technical documents Patent documents Patent Document 1: Publication Specification of U.S. Patent Application No. 2022 / 0387601 Patent Document 2: Publication Specification of U.S. Patent Application No. 2022 / 0364085 Patent Document 3: Publication Specification of U.S. Patent Application No. 2022 / 0364084 Summary of the Invention

[0005] The problem that the invention aims to solve As mentioned above, cell membrane permeable peptides are used as tools to introduce exogenous substances such as nucleic acids into the cell, and there is a need to improve their introduction efficiency.

[0006] Technical solutions for solving the problem According to the present invention, a peptide fragment (synthetic peptide) with cell membrane permeability will be provided. One embodiment of the peptide fragment comprises the amino acid sequence: RRRRRSNRRG (Sequence No. 1), with a total of 10 or fewer amino acid residues. In a preferred embodiment, the peptide fragment consists of the amino acid sequence RRRRRSNRRG (Sequence No. 1).

[0007] The peptide fragment containing the amino acid sequence shown in Serial No. 1 exhibits excellent cell membrane permeability. Therefore, the construct containing this peptide fragment can be introduced from outside the cell into the cell (at least into the cytoplasm).

[0008] Furthermore, according to the present invention, a construct for introducing a target exogenous substance from outside a eukaryotic cell into the cell (hereinafter also referred to as the "construct") will be provided. The construct of the present invention comprises a peptide fragment and an exogenous substance bonded to the N-terminus and / or C-terminus of the peptide fragment, and consists of the amino acid sequence: RRRRSNRRG (Sequence No. 1).

[0009] With such a construct, the aforementioned exogenous substances can be introduced from the outside of a eukaryotic cell into the cell interior (e.g., at least into the cytoplasm).

[0010] In some embodiments, the exogenous substance is at least one organic compound selected from polypeptides, nucleic acids, pigments, and drugs. This allows for the introduction of multifunctional organic compounds into eukaryotic cells.

[0011] In some embodiments, the exogenous substance is disposed at the C-terminus of the peptide fragment. This enables the efficient introduction of the construct into eukaryotic cells.

[0012] Furthermore, according to the present invention, a method is also provided for introducing a target exogenous substance from outside a eukaryotic cell into at least the cytoplasm of the cell in an in vitro or in vivo environment. The method of the present invention includes (1) a step of preparing the construct described in the present invention; and (2) a step of supplying the construct to a sample containing the target eukaryotic cell. This enables the introduction of the target exogenous substance into the eukaryotic cell.

[0013] In some embodiments, the eukaryotic cell used as the object of the above-described construct is a mammalian cell. This allows the target exogenous substance to be introduced into the mammalian cell. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the average fluorescence intensity (MFI) of each NSC-34 cell obtained by adding the constructs or FAMs of Examples 1 to 8 to the culture medium and then analyzing the cells using flow cytometry. Detailed Implementation

[0015] The following will describe several specific embodiments of the technology described in this invention. Matters required for implementing the technology of this invention other than those specifically mentioned in this specification (such as chemical synthesis methods of peptides, cell culture techniques, and the preparation of constructs containing peptides and nucleic acids as components, etc.) can be considered as designable matters by those skilled in the art based on existing technologies in the fields of cell engineering, physiology, medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, and genetics.

[0016] Furthermore, the technology described in this invention can be implemented based on the content set forth in this specification and common technical knowledge in the field. It should be noted that in this specification, amino acids may be represented using single-letter symbols conforming to the relevant nomenclature of amino acids as shown in the IUPAC-IUB guidelines. It should also be noted that, unless otherwise stated, in this specification, "amino acid residue" refers to both the N-terminal and C-terminal amino acids of the peptide chain.

[0017] Furthermore, in this specification, "synthetic peptide" refers to a peptide (peptide fragment) that is not stably and independently present in nature, but is manufactured through artificial chemical synthesis or biosynthesis (i.e., genetic engineering-based production) and is capable of being stably present in a given composition. "Peptide" is a term for an amino acid polymer (including dimers, trimers, and oligomers) having one or more peptide bonds, and is not limited by the number of amino acid residues.

[0018] Furthermore, unless otherwise stated, the amino acid residues constituting peptides and proteins may be either L-isomers or D-isomers in this specification.

[0019] It should be noted that the amino acid sequences described in this specification always indicate the N-terminal side on the left and the C-terminal side on the right.

[0020] In some embodiments, the peptide fragment of the present invention comprises the amino acid sequence: RRRRRSNRRG (Sequence No. 1). Alternatively, the peptide fragment may consist of, for example, the amino acid sequence: RRRRRSNRRG (Sequence No. 1). The peptide fragment of the present invention may be, for example, a synthetic peptide.

[0021] Patent documents 1-3 disclosed a cell membrane permeable peptide composed of the amino acid sequence RRRRRRG (serial number 2). To improve the cell membrane permeability of this peptide, the inventors of this invention conducted in-depth research and developed the peptide fragment described in this invention. As clearly demonstrated by the test examples described later, the peptide fragment described in this invention exhibits superior cell membrane permeability compared to the cell membrane permeable peptide composed of serial number 2. Therefore, a construct containing the peptide fragment described in this invention can be efficiently introduced into eukaryotic cells from outside the cell.

[0022] The number of amino acid residues in the peptide fragments described in this invention is not limited, as long as it does not impair cell membrane permeability. The number of amino acid residues constituting the peptide fragments can be, for example, 12 or less, 11 or less, or 10 or less.

[0023] In addition, the peptide fragments described in this invention may also contain the modification sequence of sequence number 1, as long as it does not impair cell membrane permeability. Herein, "modification sequence" refers to an amino acid sequence (modified amino acid sequence) formed by replacing, deleting, and / or adding (inserting) one or more (usually two or three) amino acid residues.

[0024] Typical examples of modified sequences in this specification include sequences resulting from so-called conservative amino acid replacements, such as sequences involving the conserved substitution of one, two, or three amino acid residues, and sequences in which one, two, or three amino acid residues are added (inserted) or deleted within a specified amino acid sequence. Typical examples of conservative substitutions include sequences in which a basic amino acid residue is replaced by another basic amino acid residue (e.g., the substitution of lysine residues with arginine residues).

[0025] The construct described in this invention comprises the peptide fragment and the exogenous substance described in this invention. The exogenous substance binds to the N-terminal and / or C-terminal side of the peptide fragment.

[0026] Constructs can be designed and constructed, for example, by linking (binding) a foreign substance directly or indirectly to the N-terminal and / or C-terminal side of a peptide fragment. In the case of indirect bonding between the peptide fragment and the foreign substance, a linker is configured between, for example, the peptide fragment and the foreign substance. The linker is not particularly limited and can be either a peptide linker or a non-peptide linker. Furthermore, both peptide linkers and non-peptide linkers can be used in combination. It should be noted that the method of direct or indirect bonding between the peptide fragment and the foreign substance is not particularly limited and can be carried out according to various currently known scientific methods.

[0027] The peptide linker may consist of one or more amino acid residues. The number of amino acid residues constituting the peptide linker is not particularly limited. For example, the number of amino acid residues constituting the peptide linker may be one or more, or two or more. Alternatively, the number of amino acid residues constituting the peptide linker may be, for example, 10 or less, or 5 or less. In some embodiments, the amino acid sequence constituting the peptide linker is a steric sequence that does not cause steric hindrance and is compliant. Such a peptide linker may contain, for example, glycine, alanine, and / or serine. Furthermore, in some embodiments, the peptide linker may contain β-alanine.

[0028] Examples of non-peptide linkers include, but are not specifically limited to, alkyl linkers, PEG (polyethylene glycol) linkers, and aminocaproyl spacers. Additionally, crosslinking agents possessing homo- or hetero-bifunctional groups can be used as non-peptide linkers. Common functional groups found in crosslinking agents include N-hydroxysuccinimide activated esters (NHS esters), maleimides, azides, and iodoacetamide. NHS esters, in particular, can react efficiently with amines at pH values ​​above neutral. Furthermore, maleimides can selectively react with SH groups. For example, when using crosslinking agents containing maleimides, the bonding between the peptide fragment and the crosslinking agent can be easily achieved by adding cysteine ​​residues as linkers to the N- or C-terminus of the peptide fragment, or by preparing peptide fragments with cysteine ​​residues at the N- or C-terminus.

[0029] Preferred crosslinking agents with homologous bifunctional groups include N-hydroxysuccinimide (NHS), bis(succinimide) octanoate (DSS), and bis(sulfosuccinimide) octanoate (BS). 3 Dithiobis(succinimide propionate) (DSP), dithiobis(sulfosuccinimide propionate) (DTSSP), ethylene glycol bis(succinimide succinate) (EGS), ethylene glycol bis(sulfosuccinimide succinate) (sulfon-EGS), tartaric acid disuccinimide (DST), tartaric acid disulfosuccinimide (sulfon-DST), etc. Particularly preferred are bis(sulfosuccinimide) octanoate (BS). 3 ).

[0030] Preferred crosslinking agents with heterodifunctional groups include N-(6-maleimide hexanoyloxy)succinimide (EMCS), m-maleimide benzoyl-N-hydroxysuccinimide ester (MBS), succinimide-4-[maleimide phenyl]butyrate (SMPB), succinimide-4-(maleimide methyl)-1-cyclohexanecarboxylate (SMCC), N-(γ-maleimide butoxy)succinimide ester (GMBS), m-maleimide propionate-N-hydroxysuccinimide ester (MPS), and N-succinimide-4-iodoacetyl)aminobenzoate (SIAB). EMCS and MBS, which possess both NHS ester and maleimide reactive functional groups, are particularly preferred.

[0031] There are no particular limitations on the exogenous substance; any compound with the molecular size and chemical properties suitable for introduction into eukaryotic cells is acceptable. It can be an organic or inorganic compound. Organic compounds include, for example, amino acids, polypeptides, nucleic acids, pigments, and drugs. The exogenous substance can be a single substance or two or more. When two or more exogenous substances are included, they can bind to either the N-terminal or C-terminal side of the peptide fragment. Alternatively, one or more exogenous substances can bind to both the N-terminal and C-terminal sides of the peptide fragment, respectively.

[0032] When the exogenous material is a polypeptide, the polypeptide (amino acid sequence) used is not particularly limited. In this specification, "polypeptide" refers to a polymer (including dimers, trimers, and oligomers) having a structure in which multiple amino acids are linked by peptide bonds. The number of peptide bonds (i.e., the number of amino residues) is not limited to a polypeptide. Compounds with a relatively large number of amino acid residues, such as peptides with 2 to 100 amino acid residues, polypeptides with 100 to 300 amino acid residues, or proteins (typically high molecular weight compounds containing more than 300 amino acid residues), can also be used as exogenous materials. In this technical field, polypeptides and proteins are not strictly distinguished.

[0033] There is no particular upper limit to the number of amino acid residues constituting a polypeptide, but from the perspective of ease of synthesis (biosynthesis, chemical synthesis), it is preferred to be, for example, less than 1000, less than 600, less than 500, less than 400, or less than 300.

[0034] Polypeptides can be mature polypeptides or polypeptide precursors (including pro- and pre-pro-) that are related to various functions such as development, differentiation, reproduction, carcinogenesis, homeostasis, and metabolic regulation of various cells and tissues (organs). In addition, polypeptides can also be polypeptides with unknown functions. According to the technology described in this invention, the functions of polypeptides with unknown functions within cells (living tissues) can be clearly elucidated.

[0035] For example, when the eukaryotic cell to which the exogenous substance is introduced is a mammalian stem cell (human or other mammalian), it is preferable to use a mature polypeptide or polypeptide precursor having various physiological activities related to the induced differentiation of the stem cell. It should be noted that "stem cell" includes somatic stem cells, embryonic stem cells, and induced pluripotent stem cells (iPS cells). Furthermore, when the eukaryotic cell to which the exogenous substance is introduced is a cancer cell (tumor cell), it is preferable to use various polypeptides related to the induction of apoptosis in the cancer cell (tumor cell). Alternatively, in this case, it is preferable to use polypeptides that can inhibit the function of cancer cells (tumor cells) in suppressing immune surveillance mechanisms. Furthermore, when the eukaryotic cell to which the exogenous substance is introduced is a bacterial-infected cell or a virus-infected cell, it is preferable to use various polypeptides related to the induction of apoptosis in the infected cell, polypeptides that can inhibit the proliferation of bacteria or viruses in the infected cell, and polypeptides that can inhibit the spread of bacterial or viral infection from the infected cell.

[0036] It should be noted that polypeptides and peptide fragments, as exogenous substances, can also contain modified amino acid sequences formed by the substitution, deletion, and / or addition (insertion) of one or more amino acid residues, as long as their function is maintained.

[0037] When the exogenous substance is a polypeptide, a construct in which the polypeptide and the peptide fragment are directly bonded can be made by designing the peptide chain, for example, in a manner that includes the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the peptide fragment, and by biosynthesizing or chemically synthesizing the peptide chain.

[0038] The types of nucleic acids that can be used as exogenous substances may include, but are not specifically limited to, so-called nucleic acid drugs. In this specification, "nucleic acid" may refer to polymers of nucleotides, including DNA, RNA, and DNA / RNA hybrid fragments containing both DNA and RNA (also known as DNA-RNA chimeras). Furthermore, "nucleic acid" is not limited by the number of bases. Nucleic acids can be single-stranded (including hairpin single-stranded) or double-stranded. Additionally, the phosphate, sugar, and base portions of the nucleotides constituting nucleic acids may also contain modifying groups. Examples of nucleic acid drugs include, but are not specifically limited to, siRNA, miRNA, antisense strands, (nucleic acid) aptamers, inducers, ribozymes, CpG oligonucleotides, etc.

[0039] There are no particular limitations on the types of pigments used as exogenous substances. Pigments can include various fluorescent pigment compounds such as FAM and FITC. Because the construct contains pigments, the introduction of the construct is easier to evaluate. The efficiency of introduction into eukaryotic cells can also be evaluated using methods such as introduction microscopy (e.g., fluorescence microscopy), flow cytometry, and immunochemical methods (e.g., Western blotting, immunocellular staining, etc.).

[0040] There are no particular restrictions on the types of drugs used as exogenous substances. Drugs can include various organic compounds such as nucleic acid-based antitumor drugs like 5-fluorouracil (5FU), antiviral drugs like azidothymidine (AZT), and other nucleic acid-based antitumor drugs.

[0041] In the construct, when the exogenous substance binds to the C-terminus of the peptide fragment, it is preferable to convert the α-amino group of the amino acid residues on the N-terminus of the peptide fragment to an acetyl group. Although the detailed mechanism has not been elucidated, since most proteins in eukaryotic cells have their α-amino groups of the N-terminal amino acids modified to acetyl groups, this structure can improve the intracellular stability of the construct.

[0042] Furthermore, the construct preferably comprises amino acid residues on the C-terminus. Amidation of the carboxyl groups of amino acid residues (typically C-terminal amino acid residues of a peptide chain) improves the structural stability (e.g., protease resistance) of the construct in the cytoplasm and nucleus. In addition, amidation of the carboxyl groups improves the hydrophilicity of the construct, thus enhancing its solubility in aqueous solvents. Examples of such aqueous solvents include water, various buffer solutions, physiological saline (e.g., PBS), and cell culture media.

[0043] In the case of a construct in which, for example, a foreign substance binds to the N-terminal side of a peptide fragment, it is preferable to amidate the carboxyl groups of the amino acid residues on the C-terminal side of the peptide fragment. Furthermore, in the case where, for example, the foreign substance is a polypeptide, and the polypeptide binds to the C-terminal side of the peptide fragment, it is preferable to amidate the carboxyl groups of the C-terminal amino acid residues of the polypeptide.

[0044] Peptide moieties (including polypeptides, peptide fragments, and peptide linkers as exogenous substances) in peptide fragments or constructs can be readily synthesized using, for example, conventional chemical synthesis methods. For instance, either solid-phase synthesis or liquid-phase synthesis, currently known methods, can be employed. Solid-phase synthesis methods using Boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as protecting groups for the amino group are suitable. That is, solid-phase synthesis using commercially available peptide synthesis equipment can also synthesize the aforementioned peptide moieties having the desired amino acid sequence and modified (N-terminal acetylation, C-terminal amidation, etc.) moieties. It should be noted that it is also possible to synthesize only a portion of the peptide chain using the above methods; for example, only peptide fragments can be synthesized, or peptide chains containing both peptide fragments and peptide linker moieties can be synthesized.

[0045] Alternatively, peptide fragments and peptide portions of the construct can also be biosynthesized using genetic engineering methods. That is, a polynucleotide (usually DNA) encoding a nucleotide sequence (including the ATG start codon) that encodes the desired amino acid sequence is synthesized. Then, a recombinant vector containing an expression gene construct, comprising the synthesized polynucleotide (DNA) and various active agents (including promoters, ribosome binding sites, terminators, enhancers, and various cis-acting agents controlling expression levels) for expressing that amino acid sequence within the host cell, is constructed based on the host cell.

[0046] Using conventional techniques, the recombinant vector is introduced into a designated host cell (e.g., yeast, insect cells, plant cells), and the host cell or tissue or individual containing that cell is cultured under specified conditions. This enables the intracellular production of the target peptide. The target peptide can then be obtained by isolating the peptide from the host cell (or, in the case of secretion, in a culture medium) and performing refolding, purification, etc., as needed.

[0047] It should be noted that the methods for constructing the recombinant vector and the methods for introducing the constructed recombinant vector into host cells can directly adopt the methods that have been used in the art. Since these methods themselves do not bring specific features to the technology described in this invention, detailed descriptions are omitted.

[0048] Furthermore, in the manufacturing process of the peptide portion of the construct, fusion protein expression systems can be utilized to achieve, for example, efficient and large-scale production within host cells. For instance, a gene (DNA) encoding the amino acid sequence of the target polypeptide is chemically synthesized and inserted into a suitable site on an appropriate fusion protein expression vector (e.g., the pET series from Novogene and the pGEX series from Amersham Biosciences, both GST (glutathione S-transferase) fusion protein expression vectors). Then, the vector is used to biotransform host cells (usually *E. coli*). The resulting biotransformants are cultured to prepare the target fusion protein. This protein is then extracted and purified. The purified fusion protein is subsequently cleaved with a specific enzyme (protease), and the released target peptide fragment (i.e., the designed artificial polypeptide) is recovered using methods such as affinity chromatography. By employing this existing known fusion protein expression system (e.g., the GST / His system from Amersham Biosciences), the target polypeptide (artificial polypeptide) can be manufactured.

[0049] Alternatively, a cell-free protein synthesis system can be constructed using template DNA (i.e., a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of the peptide moiety of the construct), and various compounds required for peptide synthesis (ATP, RNA polymerase, amino acids, etc.) can be used to synthesize the target peptide moiety in vitro using this so-called cell-free protein synthesis system. For more information on cell-free protein synthesis systems, please refer to, for example, the paper by Shimizu et al. Nature Biotechnology , 19, 751-755 (2001)) and the paper by Madin et al. (Madin et al., Proc.Natl.Acad.Sci. USA, 97(2), 559-564 (2000). Based on the techniques described in these papers, at the time of this application, many companies were already accepting contract manufacturing of peptides and they were already being marketed as kits for cell-free protein synthesis (e.g., available from Cellfree Science Co., Ltd., Japan).

[0050] Single-stranded or double-stranded polynucleotides, including nucleotide sequences encoding the peptide moiety of the construct and / or nucleotide sequences complementary to that sequence, can be readily produced (synthesized) using existing known methods. For example, by selecting codons corresponding to the amino acid residues constituting the peptide moiety, the nucleotide sequence corresponding to that amino acid sequence can be readily determined and provided. Moreover, once the nucleotide sequence is determined, the polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be readily obtained using DNA synthesis equipment, etc. The obtained single-stranded DNA can then be used as a template to obtain the target double-stranded DNA using various enzymatic synthesis methods (usually PCR). Furthermore, the polynucleotide can be in the form of DNA or RNA (mRNA, etc.). DNA can be provided in double-stranded or single-stranded form. When provided in single-stranded form, it can be either the coding strand (sense strand) or the non-coding strand (antisense strand) with a complementary sequence. The polynucleotides obtained in this way, as described above, can be used as materials for constructing recombinant genes (expression cassettes) for peptide production in various host cells or cell-free protein synthesis systems.

[0051] It should be noted that nucleic acids, as exogenous substances, can also be prepared using the same manufacturing method described above.

[0052] The constructs described in this invention are suitable for use as active components in compositions for applications based on the function of exogenous substances. It should be noted that the constructs can also be in the form of salts, as long as the function of the exogenous substance is not lost. For example, acid adduct salts obtained by conventional methods through the addition reaction of commonly used inorganic or organic acids can be used. Therefore, the term "construct" as used in this specification and claims also includes compounds in this salt form.

[0053] The construct can also be used as an active component of a composition that may contain a variety of pharmaceutically acceptable carriers, depending on the form of use.

[0054] As the aforementioned carrier, a preferred carrier is one commonly used as a diluent or excipient in peptide drugs. Such a carrier may vary depending on the intended use and morphology of the exogenous substance delivery construct, but water, physiological buffers, and various organic solvents are commonly cited. Alternatively, the carrier may be an aqueous solution of alcohol (ethanol, etc.) of appropriate concentration, a non-drying oil such as glycerol or olive oil, or liposomes. Furthermore, as secondary components that may be included in the pharmaceutical composition, various fillers, fillers, binders, wetting agents, surfactants, pigments, fragrances, etc., are cited.

[0055] The form of the composition is not particularly limited. Examples include liquid formulations, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, etc. In addition, for use in injections, lyophilized or granulated products prepared by dissolving the drug solution in physiological saline or a suitable buffer solution (such as PBS) just before use may also be used.

[0056] The processes for preparing various forms of drugs (compositions) using the construct (major component) and various carriers (minor components) as materials can be carried out according to currently known methods. These manufacturing methods are not inherently characteristic of the present invention, and therefore detailed descriptions are omitted. For example, sources of information regarding formulation details can be found in, for instance, *Comprehensive Medicinal Chemistry*, edited by Corwin Hansch and published by Pergamon Press. Comprehensive Medicinal Chemistry (1990).

[0057] This invention provides a method for introducing a target exogenous substance into the interior of a eukaryotic cell from the outside of the cell using the construct described herein in an in vivo or in vitro environment. The "interior of the eukaryotic cell" refers to the inner side of the eukaryotic cell covered by the cell membrane, including, for example, the cytoplasm, cytosol, and organelles (e.g., the nucleus, mitochondria, endoplasmic reticulum, microtubules, lysosomes, Golgi apparatus, etc.). In the method described in this invention, the construct can be introduced, for example, at least into the cytoplasm (and even organelles) of the eukaryotic cell.

[0058] The method described in this invention mainly includes the following steps (1) to (2): (1) The preparation process of the construct described in this invention; and (2) The process of supplying the above-mentioned construct to a sample containing target eukaryotic cells.

[0059] In addition, the method described in this invention may also include, as step (3), a step after step (2) above, a step of incubating a sample to which the above-mentioned construct is supplied and introducing the relevant construct into eukaryotic cells in the sample.

[0060] In vivo, the aforementioned "eukaryotic cells" include, for example, various tissues, viscera, organs, blood, and lymph. In vitro, the aforementioned "eukaryotic cells" include, for example, various cell clusters, tissues, viscera, organs, blood, and lymph, as well as cell lines, removed from living organisms. Examples of eukaryotic cells include, for example, cells derived from the animal kingdom such as mammals, birds, fish, amphibians, reptiles, and insects; cells derived from the fungi kingdom; and cells derived from the plant kingdom. Preferably, they are cells from humans or non-human mammals (mammalian cells).

[0061] The method described in this invention, for example, involves providing a sample containing eukaryotic cells after preparing a composition comprising the construct described in this invention. In vivo, the composition can be used according to the method and dosage corresponding to its form and purpose. For example, as a liquid formulation, it can be administered to the affected area (e.g., malignant tumor tissue, virus-infected tissue, inflamed tissue, etc.) of a patient (i.e., a living organism) in an appropriate amount by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. Alternatively, solid-form formulations such as tablets, gel-like or aqueous gel-like formulations such as ointments can be applied directly to the targeted tissue (i.e., the affected area including tissues or organs such as tumor cells, virus-infected cells, inflammatory cells, etc.). Alternatively, solid-form formulations such as tablets can also be administered orally. In the case of oral administration, capsules or protective (coating) materials capable of inhibiting the breakdown by digestive enzymes in the digestive tract are preferred.

[0062] For eukaryotic cells cultured in vitro, an appropriate amount of the construct or composition may be supplied to the target eukaryotic cell culture medium at least once. The amount and frequency of each supply vary depending on the type of eukaryotic cells being cultured, cell density (cell density at the start of culture), passage number, culture conditions, and type of culture medium, and therefore there are no particular limitations. For example, it is preferable to perform one, two, or more additions so that the peptide fragment concentration in the culture medium is typically in the range of 0.05 μM to 100 μM, for example, 0.5 μM to 50 μM, or, for example, 1 μM to 30 μM. Furthermore, the incubation time after adding the construct also varies depending on the type of eukaryotic cells and various conditions, and therefore there are no particular limitations. 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. It should be noted that the incubation conditions also vary depending on the type of eukaryotic cells, and therefore there are no particular limitations; for example, incubation can be carried out in a 5% CO2 environment at a temperature of 37°C.

[0063] It should be noted that an example of an in vitro importation method will be given in the test cases described later.

[0064] As described above, the following solutions can be cited as specific embodiments of the technology described in this specification.

[0065] Item 1: A peptide fragment, wherein the peptide fragment contains the amino acid sequence: RRRRSNRRG (sequence number 1), and the total number of amino acid residues is less than 10.

[0066] Item 2: The peptide fragment according to Item 1, wherein the peptide fragment consists of the amino acid sequence: RRRRRSNRRG (Sequence No. 1).

[0067] Item 3: A construct comprising a peptide fragment and an exogenous substance bound to the N-terminal side and / or C-terminal side of the peptide fragment, wherein the peptide fragment consists of the amino acid sequence: RRRRRSNRRG (Sequence No. 1).

[0068] Item 4: The construct according to Item 3, wherein the exogenous substance is selected from at least one organic compound of polypeptides, nucleic acids, pigments and drugs.

[0069] Item 5: The construct according to Item 3 or Item 4, wherein the above-mentioned exogenous substance binds to the C-terminal side of the above-mentioned peptide fragment.

[0070] Item 6: A method for introducing a target exogenous substance from the outside of a eukaryotic cell into the interior of the cell in vitro, wherein the method includes... (1) The process of preparing the construct as described in any one of items 3 to 5; and (2) The process of supplying the above-mentioned construct to a sample containing target eukaryotic cells.

[0071] Item 7: According to the method described in Item 6, wherein the eukaryotic cell of the object into which the above-described construct is imported is a mammalian cell.

[0072] The following describes several relevant test examples of the technology described in this specification, but it is not intended to limit the technology described in this specification to the content shown in these test examples.

[0073] <Construction Preparation> In Examples 1-8, the constructs shown in Table 1 were prepared. As shown in Table 1, in each example, a peptide fragment (synthetic peptide) comprising the components of Examples 1-8 was prepared, and FAM (C-terminus of the luciferase) as a fluorescent dye was attached to the C-terminus of the peptide fragment. 21 H 12 The construct of O7: 5(6)-carboxyfluorescein (molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) was prepared. It should be noted that in each example, compounds were prepared in which the amino groups of the N-terminal amino acid residues of the peptide fragments were acetylated. Furthermore, the constructs of each example were dissolved in dimethyl sulfoxide (DMSO) to prepare sample solutions with a construct concentration of 2 mM. Additionally, a 2 mM FAM solution was prepared by dissolving the fluorescent dye FAM in DMSO. For ease of explanation, the construct used in Example X will be referred to as Construct X, and the sample solution will be referred to as Sample Solution X.

[0074] <Evaluation of cell membrane permeability> The cell membrane permeability of the construct prepared as described above was evaluated using NSC-34 cells (mouse motor neuron-like hybrid cell line) as eukaryotic cells.

[0075] (Example 1) NSC-34 cells were cultured in DMEM (Dulbecco's modified Eagle's medium, manufactured by Fujifilm and Kohden Corporation, catalog number 044-29765) containing 10% FBS (fetal bovine serum).

[0076] After washing the NSC-34 cells adhered to the culture plate with PBS, 0.25% trypsin / EDTA solution was added, and the cells were incubated at 37°C for 3 minutes. Following this incubation, DMEM containing 10% FBS was added to inactivate the trypsin, and the cells were then centrifuged at 150×g for 5 minutes to pellet the cells. After removing the supernatant from the centrifugation, DMEM containing 10% FBS was added to the pellet (cell pellets), preparing approximately 2×10⁻⁶ cells. 5 A cell suspension of 1 cell / mL was prepared. 1 mL of this cell suspension was added to the culture wells of a commercially available 6-well plate (manufactured by AGC Technical Glass Co., Ltd.) for cell seeding (approximately 2 × 10⁶ cells / mL). 5 Cells were incubated at 100 cells / well for 2–3 hours until they adhered to the bottom of the well. Then, the 2 mM sample solution 1 prepared above was diluted with DMEM containing 10% FBS to prepare a 20 μM sample solution 1. Next, 1 mL of the 20 μM sample solution 1 was added to the well (i.e., the concentration of construct 1 in the culture medium in the well was 10 μM, and the concentration of DMSO was 0.5%). The cells were then incubated at 37°C for 20 hours under 5% CO2 conditions.

[0077] After 20 hours of incubation, the culture supernatant was removed from the wells, and the cells were washed twice with 1 mL of PBS. Then, 100 μL of 0.25% trypsin / EDTA solution was added to the wells, and incubation was performed at 37°C for 3 minutes. Following this incubation, 900 μL of the aforementioned DMEM containing 10% FBS was added to the wells to inactivate the trypsin, and the cell suspension was then transferred to test tubes for cell recovery. Subsequently, the cells remaining in the wells were further recovered by rinsing and transferred to the aforementioned test tubes. These test tubes were centrifuged at 4°C and 210 × g for 5 minutes. After centrifugation, the supernatant was removed, and the precipitate (cell pellet) was resuspended in 1 mL of PBS (washed) and centrifuged under the same conditions. The supernatant was then removed, yielding cells (cell pellet) cultured in medium containing the construct.

[0078] The cell membrane permeability of the cells (cytogranules) obtained above was analyzed using flow cytometry. An On-Chip flow cytometer (manufactured by On-Chip Biotechnologies Co., LTD.) was used as the flow cytometer.

[0079] To perform this analysis, the obtained cell pellets were suspended in 50 μL of On-Chip T buffer to prepare the analytical cell suspension.

[0080] The aforementioned flow cytometer was used to perform gating based on forward scatter (FSC) and side scatter (SSC) to select cell populations for analysis, and the fluorescence intensity of the cells within each gate was measured. It should be noted that the analysis was performed with approximately 10,000 cells in the cell population. Fluorescence intensity measurements were performed using the FL2 fluorescence detector (optimal detection wavelength around 543 nm) of the aforementioned flow cytometer, which is capable of detecting FAM fluorescence wavelengths. The measurement results were analyzed using the commercially available analysis software "FlowJo" (manufactured by TreeStar) to obtain the mean fluorescence intensity (MFI) of the target cell population.

[0081] (Examples 2~8) In Examples 2-8, the same procedures as in Example 1 were followed, except that Sample Solution 1 was replaced with Sample Solution 2-8.

[0082] (Example 9) In Example 9, the procedure was the same as in Example 1, except that FAM solution was used instead of sample solution 1. It should be noted that the concentration of the FAM solution was the same as that of sample solution 1 (i.e., the FAM concentration in the culture medium in the culture well was 10 μM and the DMSO concentration was 0.5%).

[0083] The MFI results obtained from Examples 1-9 are shown in Table 1 and Figure 1 .

[0084] [Table 1] As described above, the peptide fragment contained in the construct used in Example 8 is a cell membrane permeability peptide disclosed in Patent Documents 1-3, etc. In this test, cell membrane permeability was evaluated by adding or inserting SN (serine residue-asparagine residue) between the chain ends or amino acid residues of this peptide fragment. The results are shown in Table 1 and... Figure 1 As shown, surprisingly, the construct containing the peptide fragment consisting of the amino acid sequence RRRRRSNRRG used in Example 5 exhibited particularly excellent cell membrane permeability.

[0085] It should be noted that, although detailed data is not shown, the inventors’ research according to the present invention confirms that peptide fragments containing an amino acid sequence consisting of RRRSNRRG and constructs containing polypeptides, nucleic acids or drugs as exogenous substances can be efficiently introduced from outside the eukaryotic cell into the cell.

[0086] The specific examples of the technology described in this invention have been described in detail above, but these are merely illustrative examples and are not intended to limit the scope of protection claimed. The technology described in the scope of protection claims includes solutions with various modifications and alterations to the specific examples described above.

[0087] According to the present invention, a peptide fragment capable of introducing a target exogenous substance from the outside of a eukaryotic cell (particularly human cells without cell walls and various animal cells, including non-human mammals) into the cell interior, and a construct containing the peptide fragment, will be provided. Using this construct, the target exogenous substance can be efficiently introduced into target cells, resulting in living tissues such as cells and organs infused with the exogenous substance. Furthermore, by using the peptide fragment or construct described in this invention in drug delivery technology, targeted therapeutic drugs for various diseases can be provided.

Claims

1. A peptide fragment, characterized by comprising an amino acid sequence: RRRRSNRRG (SEQ ID NO: 1), the total number of amino acid residues being 10 or less.

2. The peptide fragment according to claim 1, characterized by consisting of an amino acid sequence: RRRRSNRRG (SEQ ID NO: 1).

3. A construct, characterized by comprising a peptide fragment, and an exogenous substance bound to the N-terminal side and / or the C-terminal side of the peptide fragment, the peptide fragment consisting of an amino acid sequence: RRRRSNRRG (SEQ ID NO: 1).

4. The construct according to claim 3, characterized in that the exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments, and drugs.

5. The construct according to claim 4, characterized in that the exogenous substance is bound to the C-terminal side of the peptide fragment.

6. A method for introducing a construct into a target eukaryotic cell, comprising: (1) a step of preparing the construct according to any one of claims 3 to 5; and (2) a step of supplying the construct into a sample containing the target eukaryotic cell.

7. The method according to claim 6, characterized in that the eukaryotic cell into which the construct is introduced is a mammalian cell. ​ ​ ​ ​ 6. A method for introducing a target exogenous substance from the outside of a eukaryotic cell to the inside of the cell in vitro, characterized by ​ ​ ​ ​ ​ ​

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