WO2024128081A1 - 合成ペプチド及び構築物 - Google Patents

合成ペプチド及び構築物 Download PDF

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Publication number
WO2024128081A1
WO2024128081A1 PCT/JP2023/043553 JP2023043553W WO2024128081A1 WO 2024128081 A1 WO2024128081 A1 WO 2024128081A1 JP 2023043553 W JP2023043553 W JP 2023043553W WO 2024128081 A1 WO2024128081 A1 WO 2024128081A1
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Prior art keywords
amino acid
synthetic peptide
cell
construct
acid sequence
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English (en)
French (fr)
Japanese (ja)
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菜穂子 ベイリー小林
徹彦 吉田
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Toagosei Co Ltd
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Toagosei Co Ltd
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Priority to EP23903370.7A priority Critical patent/EP4635971A1/en
Priority to CN202380085554.3A priority patent/CN120344547A/zh
Priority to JP2024564312A priority patent/JPWO2024128081A1/ja
Publication of WO2024128081A1 publication Critical patent/WO2024128081A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention relates to a synthetic peptide having cell membrane permeability and a construct comprising the synthetic peptide.
  • CPPs Cell-penetrating peptides
  • Japanese Patent Publication No. 7041853 discloses a carrier peptide fragment that functions as a CPP, and a technology for introducing a foreign substance of interest into a eukaryotic cell by using the carrier peptide fragment.
  • the amino acid sequence constituting a CPP contains basic amino acids (e.g., arginine, lysine), and it is said that basic amino acids can contribute to the efficiency of cell membrane permeability.
  • basic amino acids e.g., arginine, lysine
  • cytotoxicity it is generally known that when the ratio of basic amino acids in a CPP is high, there is a tendency for cytotoxicity to be high.
  • the synthetic peptide disclosed herein is a CPP capable of introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell.
  • the synthetic peptide has the following amino acid sequence: (1) an amino acid sequence in which two or more minimum building blocks, each of which is a serine residue-alanine acid residue, are linked in series; and (2) an amino acid sequence in which one to three glycine residues are linked to the C-terminus of the amino acid sequence of (1) above; It is composed of either
  • the synthetic peptide of the above configuration does not contain arginine, lysine, or histidine residues, which are known as basic amino acids.
  • arginine, lysine, or histidine residues which are known as basic amino acids.
  • conventional amino acid residues can exhibit high cell membrane permeability because they contain basic amino acids, but the synthetic peptide disclosed here can exhibit high cell membrane permeability despite not containing basic amino acids.
  • the amino acid sequences of (1) and (2) above have 2 to 4 of the above minimum building blocks.
  • the synthetic peptide disclosed herein may have an amino acid sequence in which the above minimum building blocks are repeated 2 to 4 times.
  • the synthetic peptides disclosed herein include, for example, the following amino acid sequence: SASAG (SEQ ID NO: 1); SASASAG (SEQ ID NO: 2); and SASASAG (SEQ ID NO: 3); Any of the above amino acid sequences can exhibit high cell membrane permeability.
  • the present disclosure provides a construct for introducing a foreign substance (hereinafter, also simply referred to as a "construct") that is constructed for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell.
  • the construct disclosed herein has the synthetic peptide disclosed herein and the foreign substance of interest bound to the N-terminus and/or C-terminus of the synthetic peptide. Since such a construct has a synthetic peptide that functions as a CPP, it can be efficiently introduced into the interior of a eukaryotic cell, and the foreign substance of interest can be efficiently introduced into the interior of the cell.
  • the exogenous substance may be at least one organic compound selected from the group consisting of a polypeptide, a nucleic acid, a dye, and a drug.
  • polypeptide refers to a polymer having a structure in which multiple amino acids are bound by peptide bonds. Polypeptides are not limited by the number of peptide bonds (i.e., the number of amino acid residues). That is, polypeptides include those generally called peptides having 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, polypeptides and proteins are not strictly distinguished.
  • nucleic acid refers to a polymer of nucleotides, and includes DNA and RNA.
  • Nucleic acid is not limited by the number of bases.
  • the foreign substance may be located at the C-terminus of the synthetic peptide.
  • FIG. 1 is a graph showing MFI values obtained by adding the constructs (additives) shown in Examples 1 to 3 and Reference Example 1 to a culture medium of NSC-34 cells, culturing the cells, and then analyzing the cells using a flow cytometer.
  • Matters other than those specifically mentioned in this specification that are necessary for implementing this technology (for example, general matters related to chemical synthesis of peptides, cell culture techniques, and preparation of constructs containing peptides or nucleic acids as components) can be understood as design matters of a person skilled in the art based on conventional techniques in the fields of cell engineering, physiology, medicine, pharmacology, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc.
  • the technology disclosed here can be implemented based on the contents disclosed in this specification and the technical common sense in the relevant field.
  • amino acids are sometimes represented by one-letter notation in accordance with the nomenclature for amino acids set forth in the IUPAC-IUB guidelines.
  • amino acid residue includes the N-terminal amino acid and the C-terminal amino acid of a peptide chain, unless otherwise specified.
  • synthetic peptide refers to a peptide fragment whose peptide chain does not exist stably in nature independently, but is produced by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and can exist stably in a given composition.
  • peptide is a term that refers to an amino acid polymer (including dimers, trimers, oligomers, etc.) having peptide bonds, and is not limited by the number of amino acid residues.
  • amino acid residues constituting a peptide or protein may be in the L- or D-form.
  • left side of the amino acid sequences described in this specification always represents the N-terminus and the right side represents the C-terminus.
  • the synthetic peptide has the amino acid sequence shown in (1) or (2) below: (1) an amino acid sequence in which two or more SA (serine residue-alanine acid residue) minimum building blocks are continuously linked in tandem; (2) an amino acid sequence in which 1 to 3 glycine residues are bound to the C-terminus of the amino acid sequence of (1) above; It consists of:
  • linked consecutively in series means that one minimum structural unit is linked to the N-terminus and/or C-terminus of another minimum structural unit via a peptide bond.
  • the minimum building blocks may be, for example, 2 to 10 consecutively linked in series.
  • the minimum building blocks may be 2 to 6, 2 to 4, or 3 to 4 consecutively linked in series.
  • 4 of the minimum building blocks are consecutively linked in series.
  • An amino acid sequence in which 4 of the minimum building blocks are consecutively linked in series may exhibit particularly excellent cell membrane permeability.
  • the synthetic peptide disclosed herein may have 1 to 3 glycine residues bound to the C-terminus of an amino acid sequence in which the minimum building blocks are bound in series, but the number of glycine residues is preferably 1 to 2, and more preferably 1.
  • Glycine residues are neutral amino acids with the smallest side chain among amino acids. Therefore, it can be understood from the common general knowledge in this technical field that if an amino acid sequence in which the minimum building blocks are bound in series has cell membrane permeability, even if 1 to 3 glycine residues are bound to the C-terminus of the amino acid sequence, the cell membrane permeability will not be significantly impaired.
  • the number of amino acid residues in the synthetic peptide disclosed herein is 4 or more, and is 5 or more, 6 or more, 7 or more, or 8 or more, since the synthetic peptide contains at least two of the smallest building blocks.
  • the upper limit of the number of amino acid residues in the synthetic peptide is not particularly limited, and may be, for example, 21 or less, 19 or less, 17 or less, 15 or less, 13 or less, 12 or less, 11 or less, 10 or less, or 9 or less. If the synthetic peptide has too many amino acid residues, the synthetic peptide may become too bulky, which may reduce cell membrane permeability.
  • synthetic peptides disclosed herein include the amino acid sequences shown in (2) above, such as SASAG (SEQ ID NO: 1), SASAG (SEQ ID NO: 3), and SASASASAG (SEQ ID NO: 4).
  • amino acid sequences shown in (1) above include SASA (SEQ ID NO: 4), SASAS (SEQ ID NO: 5), and SASASASA (SEQ ID NO: 6). All of the synthetic peptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 6 can exhibit cell membrane permeability.
  • the synthetic peptide disclosed herein may be a modified sequence of the amino acid sequence shown in (1) or (2) above, so long as the modified sequence does not significantly impair cell membrane permeability.
  • the "modified sequence” refers to an amino acid sequence (modified amino acid sequence) formed by substituting, deleting and/or adding (inserting) one or several (typically two or three) amino acid residues.
  • modified sequences in this specification include sequences resulting from conservative substitution of one, two, or three amino acid residues, so-called conservative amino acid replacement, and sequences in which one, two, or three amino acid residues are added (inserted) or deleted from a given amino acid sequence, etc.
  • conservative substitutions include sequences in which alanine residues, which are nonpolar amino acids, are replaced with other nonpolar amino acid residues such as glycine residues.
  • the synthetic peptides disclosed herein as described above can have cell membrane permeability, and therefore can be used to introduce a foreign substance of interest from outside a eukaryotic cell into at least the cytoplasm (and even into the nucleus) of the cell. Therefore, the present disclosure provides a construct for introducing a foreign substance that includes the synthetic peptides disclosed herein.
  • the construct disclosed herein has the synthetic peptide disclosed herein and a foreign substance of interest bound to the N-terminus and/or C-terminus of the synthetic peptide.
  • the constructs disclosed herein can be designed and constructed by binding (linking) a desired foreign substance directly or indirectly via a suitable linker to the N-terminus and/or C-terminus of the above-mentioned synthetic peptide.
  • the linker is not particularly limited, but may be a peptidic linker or a non-peptidic linker.
  • the amino acid sequence constituting the peptidic linker is preferably an amino acid sequence that does not cause steric hindrance and is flexible.
  • the peptidic linker may be, for example, a linker consisting of 10 or less amino acid residues (more preferably 1 to 5, for example, 1, 2, 3, 4, or 5 amino acid residues) containing one or more amino acid residues selected from glycine, alanine, serine, etc.
  • ⁇ -alanine may be used as such a linker.
  • the non-peptidic linker although not particularly limited, for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminohexanoyl spacer, etc. may be used.
  • the exogenous substance can be, for example, an organic compound such as a polypeptide, a nucleic acid, a dye, or a drug.
  • a peptide chain is designed to include the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the synthetic peptide, and the peptide chain is biosynthesized or chemically synthesized to prepare a construct for introducing a foreign substance of interest.
  • a construct can be prepared by directly or indirectly binding 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 synthetic peptide described above by various scientific techniques known in the art.
  • the function of the foreign substance may be, for example, promotion of stem cell differentiation induction (stem cell differentiation induction activity), inhibition of tumor cell proliferation (antitumor activity), inhibition of virus-infected cell proliferation (antiviral activity), etc.
  • the number of foreign substances bound to the synthetic peptide is not particularly limited.
  • one or more foreign substances may be bound to one synthetic peptide.
  • a polypeptide, nucleic acid, drug, etc. may be bound to the C-terminus of one synthetic peptide, and a dye may be bound to the N-terminus. Binding a dye to a synthetic peptide is preferable because it makes it easier to evaluate the efficiency of introduction of the construct into eukaryotic cells and its localization within the cells.
  • the polypeptide (amino acid sequence) to be adopted is not particularly limited.
  • a foreign substance having a relatively large number of amino acid residues such as a polypeptide or protein having about 100 to 1000 amino acid residues, may be adopted.
  • 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 or more (e.g., 10 or more), and is suitably 1000 or less, preferably 600 or less, more preferably 500 or less, and particularly preferably 300 or less (e.g., 10 to 300).
  • Polypeptides of such a length are easy to synthesize (biosynthesis or chemical synthesis) and are easy to use.
  • Preferred foreign substances are mature or precursor (including pro and prepro forms) of polypeptides involved in functions such as the development, differentiation, proliferation, canceration, homeostasis, and metabolic regulation of various cells and tissues (organs).
  • the foreign substance introduction method disclosed herein can also be carried out to introduce a polypeptide whose function has not been previously known into a cell and elucidate the function of the polypeptide within the cell (within a living tissue).
  • the eukaryotic cells to which a foreign substance is introduced are human or other mammalian stem cells, it is preferable to use mature polypeptides or their precursors having various physiological activities involved in the differentiation induction of the stem cells.
  • stem cells include somatic stem cells, embryonic stem cells, and induced pluripotent stem cells (iPS cells).
  • iPS cells induced pluripotent stem cells
  • 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, so long as it retains its function.
  • the ⁇ -amino group of the amino acid residue on the N-terminus of the synthetic peptide is acetylated.
  • the ⁇ -amino group of the amino acid on the N-terminus of many proteins in eukaryotic cells is acetylated, so this type of configuration can improve the stability of the construct within the cell.
  • the construct is preferably amidated at the C-terminal amino acid residue.
  • Amidation of the carboxyl group of an amino acid residue can improve the structural stability (e.g., protease resistance) of the construct in the cytoplasm and nucleolus.
  • amidation of the carboxyl group improves the hydrophilicity of the construct, thereby improving the solubility of the construct in aqueous solvents.
  • aqueous solvents include water, various buffer solutions, physiological saline (e.g., PBS), cell culture medium, and the like.
  • the carboxyl group of the amino acid residue at the C-terminus of the synthetic peptide is amidated.
  • the carboxyl group of the C-terminal amino acid residue of the polypeptide is amidated.
  • relatively short peptide chains can be easily produced according to general chemical synthesis methods.
  • any of the conventionally known solid-phase synthesis methods or liquid-phase synthesis methods may be adopted.
  • Solid-phase synthesis methods using Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as the protecting group for the amino group are preferable.
  • the above-mentioned peptide chains having the desired amino acid sequence and modified portions 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 by the above-mentioned method, for example, a peptide chain containing only a synthetic peptide or a synthetic peptide and a peptidic linker portion can be synthesized.
  • the peptide portion may be produced by biosynthesis based on genetic engineering techniques. That is, a polynucleotide (typically DNA) of a nucleotide sequence (including an ATG start codon) encoding a desired amino acid sequence is synthesized. Then, a recombinant vector having an expression gene construct consisting of the synthesized polynucleotide (DNA) and various regulatory elements (including promoters, ribosome binding sites, terminators, enhancers, and various cis elements that control the expression level) for expressing the amino acid sequence in a host cell is constructed according to the host cell.
  • DNA synthesized polynucleotide
  • various regulatory elements including promoters, ribosome binding sites, terminators, enhancers, and various cis elements that control the expression level
  • This recombinant vector is introduced into a given host cell (e.g., yeast, insect cell, or plant cell) using a common technique, and the host cell or a tissue or individual containing the cell is cultured under given conditions. This allows the target peptide to be produced within the cell. The peptide portion is then isolated from the host cell (or from the medium if secreted), and the target peptide portion can be obtained by, if necessary, refolding, purification, or the like.
  • a given host cell e.g., yeast, insect cell, or plant cell
  • the method of constructing a recombinant vector and the method of introducing the constructed recombinant vector into a host cell may be any method conventionally used in the field, and since the method itself does not particularly characterize the present technology, a detailed description thereof will be omitted.
  • a fusion protein expression system can be used to efficiently produce a large amount of the polypeptide in a host cell. That is, a gene (DNA) encoding the amino acid sequence of the polypeptide of interest is chemically synthesized, and the synthetic gene is introduced into a suitable site of a suitable fusion protein expression vector (for example, a GST (Glutathione S-transferase) fusion protein expression vector such as the pET series provided by Novagen and the pGEX series provided by Amersham Biosciences). Then, a host cell (typically Escherichia coli) is transformed with the vector. The obtained transformant is cultured to prepare the desired fusion protein. The protein is then extracted and purified.
  • a suitable fusion protein expression vector for example, a GST (Glutathione S-transferase) fusion protein expression vector such as the pET series provided by Novagen and the pGEX series provided by Amersham Biosciences.
  • a host cell typically Escherichia
  • the obtained purified fusion protein is then cleaved with a specific enzyme (protease), and the released peptide fragment of interest (i.e., the designed artificial polypeptide) is recovered by a method such as affinity chromatography.
  • a specific enzyme protease
  • the desired construct artificial polypeptide
  • 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
  • various compounds ATP, RNA polymerase, amino acids, etc.
  • ATP ATP, RNA polymerase, amino acids, etc.
  • cell-free protein synthesis systems for example, Shimizu et al.'s paper (Shimizu et al., Nature Biotechnology, 19, 751-755(2001)) and Madin et al.'s paper (Madin et al., Proc. Natl.
  • a single-stranded or double-stranded polynucleotide containing a nucleotide sequence encoding the peptide portion of the construct and/or a nucleotide sequence complementary to said sequence can be easily produced (synthesized) by a conventional method. That is, by selecting a codon corresponding to each amino acid residue constituting a designed amino acid sequence, a nucleotide sequence corresponding to said amino acid sequence can be easily determined and provided. Once the nucleotide sequence is determined, a polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like.
  • the obtained single-stranded DNA can be used as a template to obtain the desired double-stranded DNA by employing various enzymatic synthesis means (typically PCR).
  • the polynucleotide may be in the form of DNA or in the form of RNA (mRNA, etc.).
  • the DNA can be provided in a double-stranded or single-stranded form. When provided in a single-stranded form, it may be a coding strand (sense strand) or a non-coding strand (antisense strand) of a sequence complementary thereto.
  • the polynucleotides thus obtained can be used as materials for constructing recombinant genes (expression cassettes) for peptide production in various host cells or in cell-free protein synthesis systems, as described above.
  • constructs disclosed herein may be suitably used as active ingredients in compositions for applications based on the function of the foreign substance.
  • the constructs may be in the form of a salt, so long as the function of the foreign substance is not lost.
  • acid addition salts that can be obtained by addition reaction of commonly used inorganic or organic acids according to standard methods can be used. Therefore, the "constructs" described in this specification and claims may include those in such salt form.
  • the construct can be used as an active ingredient of a composition which can contain various medicamentally (pharmacologically) acceptable carriers depending on the form of use.
  • the above-mentioned carrier is preferably a carrier generally used in peptide medicines as a diluent, excipient, etc.
  • the carrier may vary depending on the purpose and form of the construct for introducing a foreign substance, but typically includes water, physiological buffer solutions, and various organic solvents.
  • the carrier may be an aqueous solution of alcohol (such as ethanol) at an appropriate concentration, glycerol, a non-drying oil such as olive oil, or a liposome.
  • secondary components that may be contained in the pharmaceutical composition include various fillers, bulking agents, binders, wetting agents, surfactants, dyes, fragrances, etc.
  • compositions may be in the form of a liquid, suspension, emulsion, aerosol, foam, granule, powder, tablet, capsule, ointment, etc.
  • the composition may be in the form of a freeze-dried product or granules that are dissolved in physiological saline or a suitable buffer solution (e.g., PBS) immediately before use to prepare a drug solution for injection or the like.
  • physiological saline e.g., PBS
  • PBS physiological saline
  • the process of preparing various forms of drugs (compositions) using constructs (main components) and various carriers (secondary components) may be in accordance with conventionally known methods, and detailed explanations of such formulation methods are omitted since they do not characterize the present technology.
  • Comprehensive Medicinal Chemistry edited by Corwin Hansch, published by Pergamon Press (1990) is an example of a detailed information source regarding formulations.
  • synthetic peptides disclosed herein do not contain basic or acidic amino acids, it may be possible to create constructs equipped with foreign substances that were difficult to combine with conventional CPPs due to issues such as charge.
  • the construct disclosed herein may be used to introduce a foreign substance in vivo or outside the body (in vitro).
  • the method of introduction may broadly include a step of preparing the construct disclosed herein (preparation step) and a step of supplying the construct into a sample containing the target eukaryotic cells (supply step).
  • the method may further include a step of incubating the sample to which the construct has been supplied after the supply step, and introducing the construct into the eukaryotic cells in the sample (introduction step).
  • eukaryotic cells include, for example, various tissues, organs, blood, and lymph fluids in vivo.
  • the above-mentioned “eukaryotic cells” include, for example, various cell masses, tissues, organs, blood, and lymph fluids extracted from living organisms, as well as cell lines, etc. in vitro.
  • composition containing the construct disclosed herein can be used in vivo in a manner and dosage appropriate for its form and purpose.
  • a liquid formulation it can be administered in a desired amount to the affected area (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient (i.e., a living body) by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection.
  • a solid form such as a tablet or a gel or aqueous jelly such as an ointment can be administered directly to a specific tissue (i.e., an affected area such as a tissue or organ containing, for example, tumor cells, virus-infected cells, inflammatory cells, etc.).
  • a solid form such as a tablet can be administered orally.
  • an appropriate amount of the construct may be supplied at least once to the culture medium of the eukaryotic cells being cultured outside the body (in vitro).
  • the amount and number of times of supply per time are not particularly limited because they may vary depending on the type of eukaryotic cells to be cultured, cell density (cell density at the start of culture), number of passages, culture conditions, type of medium, and other conditions.
  • it is preferable to add the synthetic peptide once, twice, or more times so that the concentration of the synthetic peptide in the culture medium is generally within the range of 0.05 ⁇ M to 100 ⁇ M, for example, within the range of 0.5 ⁇ M to 50 ⁇ M, and for example, within the range of 1 ⁇ M to 30 ⁇ M.
  • the incubation time after addition of the construct is also not particularly limited because it may vary depending on the type of eukaryotic cells and various conditions. For example, it may be 0.5 hours or more, 1 hour or more, 4 hours or more, 8 hours or more, or 20 hours or more.
  • the incubation conditions are not particularly limited because they may vary depending on the type of eukaryotic cells, but for example, it can be incubated in a 5% CO 2 atmosphere at 37°C. An example of an in vitro introduction method is shown in the test example described below.
  • the method for evaluating the efficiency of introduction of the construct is not particularly limited.
  • a dye typically a fluorescent dye compound
  • the efficiency of introduction into eukaryotic cells can be evaluated using microscopic observation (e.g., fluorescent microscopic observation) or flow cytometry.
  • the efficiency of introduction of the construct can also be evaluated by immunochemical techniques (e.g., Western blotting, immunocytostaining, etc.) using an antibody that specifically recognizes the peptide portion of the construct.
  • Item 1 A synthetic peptide capable of 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 (1) an amino acid sequence in which two or more minimum building blocks, each of which is a serine residue-alanine acid residue, are linked in series; and (2) an amino acid sequence in which one to three glycine residues are linked to the C-terminus of the amino acid sequence of (1) above;
  • a synthetic peptide consisting of either Item 2 The synthetic peptide according to Item 1, wherein the amino acid sequences of (1) and (2) above each have 2 to 4 of the minimum structural units.
  • Item 3 A synthetic peptide capable of 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: SASAG (SEQ ID NO: 1); SASASAG (SEQ ID NO: 2); and SASASAG (SEQ ID NO: 3); A synthetic peptide consisting of either Item 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 synthetic peptide according to any one of items 1 to 3, the foreign substance of interest bound to the N-terminus and/or C-terminus of the synthetic peptide; A construct having the following structure: Item 5: The construct according to Item 4, wherein the foreign substance is at least one organic compound selected from the group consisting of a polypeptide, a nucleic acid, a dye and a drug. Item 6: The construct according to Item 4 or 5, wherein the foreign substance is located
  • item 3 shows a specific example that has the items specified in items 1 or 2, item 3 can be subordinate to item 1 or 2.
  • Test 1 A construct having a synthetic peptide composed of the amino acid sequence shown in Table 1 was prepared.
  • Sample n was a construct having peptide n (n is a natural number from 1 to 3) shown in Table 1, and samples 1 to 3 were obtained from Eurofins Genomics, Inc. Note that in samples 1 to 3, the ⁇ -amino groups of the amino acid residues on the N-terminus of peptides 1 to 3 were all acetylated.
  • peptides 1 to 3 were prepared in which the fluorescent dye FAM (C 21 H 12 O 7 :5(6)-Carboxyfluorescein, molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) was bound to the amino acid residues on the C-terminus of peptides 1 to 3 as a foreign substance.
  • FAM fluorescent dye
  • NSC-34 cells (mouse motor neuron-like hybrid cell line) were used as eukaryotic cells, and the cell membrane permeability of peptides 1 to 3 was analyzed.
  • As the culture medium for NSC-34 cells 10% FBS (fetal bovine serum)-containing DMEM (Dulbecco's modified Eagle's medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 044-29765)) was used.
  • Samples 1 to 3 were each dissolved in dimethyl sulfoxide (DMSO) to prepare sample solutions 1 to 3 with a sample concentration of 4 mM.
  • the sample solutions were further diluted with the above culture medium to prepare sample solutions 1 to 3 with a concentration of 40 ⁇ M.
  • DMSO dimethyl sulfoxide
  • NSC-34 cells were suspended in the above culture medium to prepare a cell suspension of 2 ⁇ 10 5 cells/mL. 1 mL of the cell suspension was added to the wells of a commercially available 6-well plate (manufactured by AGC Technoglass Co., Ltd.) to seed NSC-34 cells at 2 ⁇ 10 5 cells/well. Next, 1 mL of 40 ⁇ M sample solution 1 was added to the wells so that the sample concentration in the culture medium in the wells was 20 ⁇ M. Thereafter, the 6-well plate was placed in a cell culture device and incubated at 37° C. under 5% CO 2 conditions for 20 hours.
  • the culture supernatant was removed from the wells, and the cells in the wells were washed twice with 1 mL of PBS.
  • 100 ⁇ L of 0.25% trypsin/EDTA solution was added to the wells, and the wells were incubated at 37°C for 3 minutes.
  • 900 ⁇ L of the above culture medium was added to the wells to inactivate the trypsin, and the cell suspension in the wells was transferred to a tube and the cells were collected. This tube was centrifuged for 5 minutes at 4°C and 210 ⁇ g.
  • the obtained cells were analyzed for cell membrane permeability of Sample 1 using a flow cytometer.
  • the cell pellet obtained above was suspended in 100 ⁇ L of On-Chip T buffer to prepare a cell suspension for analysis.
  • the above flow cytometer was used to perform gating based on forward scatter (FSC) and side scatter (SSC), set a gate for the cell population to be analyzed, and measured the fluorescence intensity of the cell population within the gate.
  • the analysis was performed so that the cell population had at least 10,000 cells.
  • the fluorescence intensity was measured using the fluorescence detector FL2 (optimum detection wavelength: around 543 nm) of the above flow cytometer, which is capable of detecting the fluorescence wavelength of FAM.
  • the measurement results were analyzed using the commercially available analysis software "FlowJo" (manufactured by TreeStar), and the fluorescence intensity value (mean fluorescence intensity: MFI) of the cell population to be measured was obtained.
  • Example 2 (Examples 2 to 3) The same procedure as in Example 1 was repeated, except that sample solution 1 was replaced with one of the above-prepared sample solutions 2 and 3.
  • the samples (constructs) used in each example are as shown in Table 2.
  • Example 1 The same procedure as in Example 1 was repeated, except that the fluorescent dye FAM was used instead of Sample 1.
  • the FAM concentration in the FAM-containing culture medium was the same as that of Sample 1 in Example 1 (i.e., the FAM concentration in the culture medium in the well was 20 ⁇ M).
  • Examples 1 to 3 had higher MFI values than Reference Example 1.
  • samples 1 to 3 in which either peptide 1 to 3 was bound to FAM were introduced into the cells in greater amounts than when FAM was added alone (Reference Example 1). This shows that peptides 1 to 3 have cell membrane permeability.
  • the technology disclosed herein provides a carrier peptide fragment capable of introducing a foreign substance of interest from the outside into the cytoplasm of a eukaryotic cell (particularly various animal cells such as humans and other mammals that do not have a cell wall) and a construct having the carrier peptide fragment.
  • a construct having the carrier peptide fragment By using such a construct, it is possible to effectively introduce a foreign substance of interest into a target cell, and obtain cells and biological tissues such as organs into which the foreign substance has been introduced.
  • the carrier peptide fragment disclosed herein can be used in drug delivery technology to provide therapeutic drugs for various diseases.

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  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/JP2023/043553 2022-12-16 2023-12-06 合成ペプチド及び構築物 Ceased WO2024128081A1 (ja)

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