WO2020054126A1 - Procédé d'introduction d'une substance dans une cellule cible - Google Patents

Procédé d'introduction d'une substance dans une cellule cible Download PDF

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WO2020054126A1
WO2020054126A1 PCT/JP2019/018861 JP2019018861W WO2020054126A1 WO 2020054126 A1 WO2020054126 A1 WO 2020054126A1 JP 2019018861 W JP2019018861 W JP 2019018861W WO 2020054126 A1 WO2020054126 A1 WO 2020054126A1
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peptide
cell
sequence
target
dtat
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PCT/JP2019/018861
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Japanese (ja)
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圭司 沼田
ジョアン チュア
崇 小山内
昂明 宮本
真樹 小田原
豊 児玉
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国立研究開発法人理化学研究所
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Priority to JP2020546684A priority Critical patent/JPWO2020054126A1/ja
Publication of WO2020054126A1 publication Critical patent/WO2020054126A1/fr
Priority to JP2024016042A priority patent/JP2024033010A/ja

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention provides a cell-permeable sequence, a linker containing a sarcosine residue, a peptide containing a domain capable of escape from intracellular vesicles, and an agent for introducing a target substance into a target plant cell or microalgae comprising the peptide. And a method for introducing a target substance into a target plant cell or microalgae using a peptide containing a cell-permeable sequence, a linker, and a domain capable of escaping from intracellular vesicles.
  • Non-Patent Documents 1-3 Non-Patent Documents 1-2.
  • the cell permeability of CPP is reported in Non-Patent Documents 3 and 4.
  • Non-Patent Document 5 uses animal cells as target cells for delivering macromolecules, and does not examine plant cells. Plant cells, unlike animal cells, have a thick and strong cell wall, and because plant cells have a decomposing activity on substances such as proteins, it is not possible to deliver substances into cells. It is considered difficult.
  • An object of the present invention is to provide a new method for delivering a substance to a plant cell or the like.
  • the present inventors have constructed a fusion peptide containing a cell-permeable sequence, a linker, and a domain capable of exiting from intracellular vesicles.
  • the present inventor surprisingly found that simply adding a target substance to be introduced in the presence of this peptide to a cell culture solution or intercellular space allows the target substance to be easily introduced into plant cells or the like.
  • the inventors have found that the present invention has been completed, and have completed the present invention.
  • the present invention includes the following aspects.
  • the peptide according to any one of (1) to (3), wherein the domain capable of exiting from intracellular vesicles comprises an amino acid sequence of GWWG (SEQ ID NO: 73) or GWFWG (SEQ ID NO: 74).
  • a method for producing a target plant cell or microalgae into which a target substance has been introduced comprising the step of contacting a target substance with a target plant cell or microalgae in the presence of a peptide containing a cell-permeable sequence, a linker, and a domain capable of exiting an intracellular vesicle.
  • a method for producing a transformed target plant cell or microalgae Contacting a nucleic acid molecule with a target plant cell or microalgae in the presence of a peptide comprising a cell-permeable sequence, a linker, and a domain capable of exiting an intracellular vesicle.
  • a method for producing a genome-modified target plant cell or microalgae Contacting a genome-editing protein with a target plant cell or microalgae in the presence of a peptide comprising a cell-permeable sequence, a linker, and a domain capable of escape from intracellular vesicles.
  • a peptide comprising a cell-permeable sequence, a linker, and a domain capable of escape from intracellular vesicles.
  • the method according to any one of (9) to (11) wherein the peptide is the peptide according to any one of (1) to (5) and (7).
  • a new method for delivering a substance to a plant cell or the like is provided.
  • the method of the present invention does not require a pretreatment such as mixing or conjugation of a peptide and a target substance, and introduces a substance into a target cell simply by mixing the target substance and the target cell in the presence of the peptide. Therefore, it can be simple and highly versatile.
  • FIG. 1 shows dTat (d (RRRQRRKKR)), dTat-EED4 (d (RRRQRRKKR)-(Sar) 6 -GWWG, dTat-EED5 (d (RRRQRRKKR)-(Sar) 6 -GFWFG), and Retro-Tat ( 57-49) shows the CD spectrum of (RRRQRRKKR) in water.
  • FIG. 1 shows dTat (d (RRRQRRKKR)), dTat-EED4 (d (RRRQRRKKR)-(Sar) 6 -GWWG, dTat-EED5 (d (RRRQRRKKR)-(Sar) 6 -GFWFG), and Retro-Tat ( 57-49) shows the CD spectrum of (RRRQRRKKR) in water.
  • FIG. 1 shows dTat (d (RRRQRRKKR)), dTat-EED4 (d (RRRQRRKKR)-(S
  • Figure 3 shows (a) dextran alone or dTat-EED4 at various concentrations ((b) 90 ⁇ M, (c, d) 225 ⁇ M, (e, f) 450 ⁇ M, (g, h) 675 ⁇ M, (i, j) 900 ⁇ M, (k, l) 2.3 mM, (m, n) 4.5 mM, (o, p) 6.8 mM, and (q, r) 9 mM) and dextran-treated tobacco BY- for 24 hours It is an image of a confocal microscope showing two cells.
  • Images are zoomed at 0.5 ⁇ (ac, e, g, i, k, m, o, q) or 2.0 ⁇ (d, f, h, j, l, n, p, r).
  • the scale bar indicates 50 ⁇ m.
  • Figure 4 shows (a, b) citrine alone or various concentrations ((c) 9 nM, (d) d90 nM, (e) 900 nM, (f) 9 ⁇ M, (g, h) 90 ⁇ M, (i) (225) ⁇ M, (j) (675) ⁇ M, (k) (900) ⁇ M, (l) (2.3) mM, (m) (4.5) mM, (n, o) (6.8) mM) dTat-EED4 and citrine treated tobacco BY for 1 hour.
  • 2 is an image of a confocal microscope showing cells.
  • FIG. 5 shows a time-course test of citrine internalization (a: immediately after treatment, b: 2 minutes and 11 seconds after treatment, c: 5 minutes and 12 seconds after treatment, d: 12 minutes and 30 seconds after treatment).
  • FIG. 7 shows cell growth and death over time, monitored by spectrophotometry.
  • FIG. 8 is a confocal microscope image showing Arabidopsis epidermal cells treated with dextran or citrin alone and dextran or citrin in combination with the 4.4 mM ⁇ dTat-EED4 peptide. Images were obtained at 63 ⁇ magnification. The scale bar indicates 10 ⁇ m.
  • FIG. 9 is a confocal microscopy image showing Euglena cells treated with dextran alone and dextran in combination with 9 nM to 900 ⁇ M dTat-EED4 peptide for 24, 48, or 72 hours. Images were obtained at 10 ⁇ magnification. The scale bar indicates 50 ⁇ m.
  • FIG. 8 is a confocal microscope image showing Arabidopsis epidermal cells treated with dextran or citrin alone and dextran or citrin in combination with the 4.4 mM ⁇ dTat-EED4 peptide. Images were obtained at 63 ⁇ magnification. The scale bar indicates 10 ⁇ m.
  • FIG. 10 shows that dextran was brought into contact with tobacco BY-2 cells in the absence of peptide (a), the presence of 900 ⁇ M dTatT-EED4 (b), and the presence of 90 ⁇ M dTat- EED5 (c).
  • Fig. 1 is a photograph observed after 1 hour incubation.
  • FIG. 11 shows that citrin was brought into contact with tobacco BY-2 cells under the peptide-free condition (a), the condition (b) in which 90 ⁇ M dTat-EED4 was present, and the condition (c) in which 90 ⁇ M dTat-EED5 was present.
  • Fig. 1 is a photograph observed after 1 hour incubation.
  • FIG. 12 shows the results of observation with a confocal laser scanning microscope after germinating spores of moss were mixed with dTat-EED4 and citrine and incubated. As a control, the results using germinated spores mixed with the same concentration of citrine alone are also shown (no peptide).
  • FIG. 13 shows the results of observation by confocal laser scanning microscopy after mixing Paramecium bursaria wild type cells (cc125 +) with dTat- ⁇ EED5 and citrine. As a control, the results using germinated spores mixed with the same concentration of citrine alone are also shown (no peptide).
  • FIG. 14 shows the results of evaluating the gene transfer efficiency based on the luciferase assay.
  • the present invention relates to a peptide comprising a cell-permeable sequence, a linker comprising a sarcosine residue, and a domain capable of exiting an intracellular vesicle.
  • a linker comprising a sarcosine residue
  • a domain capable of exiting an intracellular vesicle.
  • the peptide of the present invention may be a peptide that promotes the introduction of a substance into a plant cell.
  • the peptide of the present invention is characterized by including a cell-permeable sequence, a linker containing a sarcosine residue, and a domain capable of exiting from intracellular vesicles.
  • the cell-penetrating sequence, the linker containing a sarcosine residue, and the domain having the ability to escape from intracellular vesicles contained in the peptide of the present invention are described below.
  • Cell permeable sequence means a sequence of a cell permeable peptide (CPP: Cell Penetrating Peptide).
  • the cell-penetrating peptide means a peptide that can penetrate a cell membrane and enter a cell.
  • Examples of cell-penetrating peptides include BP100 (Appl Environ Microbiol 72 (5), 3302, 2006), HIV Tat (Journal Biological Chemistry, 272, pp. 16010-16017, 1997), Tat 2 (Biochim Biophys Acta 1768 ( 3), 419, 2007), Penetratin, pVEC, pAntp (Journal Biological Chemistry, 269, pp.
  • HSV-1 VP22 Cell, 88, pp. 223-233, 1997)
  • MAP Model amphiphilic peptide
  • Transportan FEBS Journal, 12, pp. 67-77, 1998)
  • R7 Nature Medicine, 6, pp. 1253-1257, 2000
  • MPG Nucleic Acid Research 25, pp. 2730-2736, 1997)
  • Pep-1 Pep-1 (Nature Biotechnology, 19, pp. 1173-1176, 2001), but are not limited thereto.
  • cell permeable sequence examples include, for example, the following sequences: KKLFKKILKYL (SEQ ID NO: 1), RKKRRQRRRRKKRRQRRR (SEQ ID NO: 2), RKKRRQRRR (SEQ ID NO: 3), PLSSIFSRIGDP (SEQ ID NO: 4), PISSIFSRTGDP (SEQ ID NO: 5), AISSILSKTGDP (SEQ ID NO: 6), PILSIFSKIGDL (SEQ ID NO: 7), PLSSIFSKIGDP (SEQ ID NO: 8), PLSSIFSHIGDP (SEQ ID NO: 9), PLSSIFSSIGDP (SEQ ID NO: 10), RQIKIWFQNRRMKWKK (SEQ ID NO: 11), DAATATRGRSAASRPTERPRAPARSASRPRRPVAP (VALID NO.
  • SEQ ID NO: 13 AAVLLPVLLAAP (SEQ ID NO: 14), VTVLALGALAGVGVG (SEQ ID NO: 15), GALFLGWLGAAGSTMGA (SEQ ID NO: 16), MGLGLHLLVLAAALQGA (SEQ ID NO: 17), LGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO: 18), GWTLNSAGYLLKINLALKLA, KLTLAAGLAKLA (SEQ ID NO: 20).
  • a peptide sequence in which one to several amino acid residues contained in these peptide sequences have substitution, insertion, and / or deletion and have cell permeability can be suitably used.
  • ⁇ several '' means, for example, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, preferably 5 or less or 4 or less, more preferably 3 or less or Means 2 or less.
  • examples of cell permeable sequences that can be used are shown in Table 1 below.
  • the cell permeable sequence is an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOS: 1 to 72, or one to several amino acid residues contained in these amino acid sequences are substituted. , Insertions, and / or deletions, and sequences having cell permeability.
  • the cell permeable sequence is an amino acid sequence of Tat (SEQ ID NO: 33), or one to several amino acid residues contained in the amino acid sequence are substituted, inserted, and / or deleted, and Contains cell permeable sequences.
  • cell-permeable peptide two or more kinds of cell-permeable peptides may be used in combination. It is also possible to select a cell-penetrating peptide specific to a particular cell of interest.
  • Linker containing sarcosine residue Means a linker comprising or consisting of a sarcosine residue represented by
  • the number of sarcosine residues contained in the linker is not limited, but is, for example, 2 to 20, 3 to 12, 4 to 8, 5 to 7, or 6, or 6 or more. Good.
  • Sarcosine residues also show higher hydrophilicity compared to PEG, and linkers containing sarcosine residues contribute to the stability of peptides in aqueous solution, both in vivo and in vitro. Further, since the excluded volume of the linker containing the sarcosine residue is small, efficient uptake into cells is expected.
  • domain capable of escape from intracellular vesicles means that a substance escapes from intracellular vesicles (eg, endosomes) after translocating a substance across a cell membrane and into cells via macropinocytosis or the like. Refers to the domain that prompts.
  • domains capable of exiting from intracellular vesicles those described in Lonn, P. et al., Scientific Reports, 2016, 6, 32301 can be used.
  • examples of domains capable of exiting from intracellular vesicles include GWWG (SEQ ID NO: 73), GWFWG (SEQ ID NO: 74), GFWG (SEQ ID NO: 75), GFWFG (SEQ ID NO: 76), GWGGWG (SEQ ID NO: 77) ) Or GWWWG (SEQ ID NO: 78), preferably an amino acid sequence comprising or consisting of GWWG (SEQ ID NO: 73).
  • the peptide of the present invention comprises, in order from the N-terminus, a cell-permeable sequence, a linker containing a sarcosine residue, and a domain capable of exiting an intracellular vesicle.
  • the peptide of the present invention can be synthesized, for example, according to a general peptide synthesis method such as a solid phase method, or can be bioengineered using genetic recombination.
  • a separately prepared cell-permeable sequence and a domain capable of exiting from intracellular vesicles can be chemically linked via a linker, for example, by a crosslinking reaction.
  • a DNA fragment encoding a cell permeable sequence may be added to at least one end of a DNA fragment encoding a domain capable of exiting from intracellular vesicles by using an appropriate DNA It can be bound by a ligation reaction with an adapter.
  • Such methods of genetic manipulation are well known to those skilled in the art of molecular biology.
  • the peptide of the present invention may further contain an arbitrary sequence, for example, an organelle translocation sequence, in addition to a cell-permeable sequence, a linker containing a sarcosine residue, and a domain capable of exiting from an intracellular vesicle.
  • Organelle translocation sequence refers to the sequence of a peptide that has affinity or permeability for a particular intracellular organelle. By adding an organelle translocating sequence, it becomes possible to deliver a target substance to any organelle in a plant cell.
  • the organelle translocation sequence include, for example, a nuclear translocation signal (nuclear localization signal, NLS) targeting the nucleus and a peroxisomal targeting signal (PTS).
  • NLS nuclear translocation signal
  • PTS peroxisomal targeting signal
  • a peptide sequence having affinity or permeability for mitochondria or chloroplasts can also be used. More specifically, a chloroplast transit peptide derived from Chlamydomonas ferredoxin (Cf) and Chlamydomonas Rubisco activase (CRa), a mitochondrial matrix target signal peptide (Biochemical and Biophysical Research and Communications, 1996, 226, pp. 561-565), mitochondria The inner membrane target signal peptides SS01, SS02, SS31, and SS20 (The AAAPS Journal, 2006, 8, pp.
  • E277-E283 50S ribosomal protein L28, 50S ribosomal protein L24, 50S ribosomal protein L27, RuBisCo small chain
  • Examples include, but are not limited to, LHCII type 1 and the like.
  • organelle translocation sequence for example the following sequence: PKKKRKV (SEQ ID NO: 79), SKL (SEQ ID NO: 80), MAMAMRSTFAARVGAKPAVRGARPASRMSCMA (SEQ ID NO: 81), MQVTMKSSAVSGQRVGGARVATRSVRRAQLQV (SEQ ID NO: 82), MATMVAGISLRGPVMSSHRTFSVTKRASLPQSKLSSELSFVTSQLSGLKISSTHFISSSAPLSVPFKPSLQPVA (SEQ ID NO: 83), MAALQSSFAGLSTSFFGQRFSPPLSLPPLVKSTEGPCLIQA (SEQ ID NO: 84), MAVSFSLVGAFKGLSLASSSSFLKGDFGAAFPVAPKFSVSFPLKSPLTIES (SEQ ID NO: 85), MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC (SEQ ID NO: 86), MAASTMALS
  • the peptide of the present invention has a function of introducing a target substance into plant cells or microalgae (in this specification, “plant cells” and “microalgae” are also simply referred to as “target cells”). I can do it. Therefore, in one aspect, the present invention relates to an agent for introducing a substance of interest into target cells, comprising the peptide of the present invention. In addition, the present invention relates to a composition for introducing a target substance into target cells, which comprises an agent for introducing the substance.
  • the present composition may contain, for example, a medium such as water or oil, a buffer, and / or a salt in addition to the above-mentioned introducing agent.
  • the peptide or substance introduction agent of the present invention is used in combination with a carrier peptide containing a polycation sequence or a cell-permeable sequence and a polycation sequence.
  • the present invention relates to a combination of the peptide or substance introducing agent of the present invention and the above-mentioned carrier peptide, or a peptide or substance introducing agent of the present invention for use in combination with the above-mentioned carrier peptide.
  • the carrier peptide contains a polycation sequence or a cell permeable sequence and a polycation sequence.
  • a cell-permeable sequence When a cell-permeable sequence is included, the details of the sequence, for example, specific sequences are as described herein.
  • the polycation sequence is a peptide sequence containing at least three amino acid residues selected from lysine (K), arginine (R) and histidine (H), and forming a stable bond with a nucleic acid under physiological conditions. .
  • the polycationic component is sufficiently cationic in its overall properties to interact with nucleic acids under physiological conditions.
  • Neutral amino acids can also be included, provided that they form stable bonds. This can be tested with a simple experiment of adding nucleic acids.
  • a peptide that forms a peptide-nucleic acid complex that is sufficiently stable to cause nucleic acid band delay in agarose gel electrophoresis is suitable. This delay in the nucleic acid band is an indication that the peptide-nucleic acid complex is retained during agarose gel electrophoresis.
  • the polycation sequence contains at least three lysines, arginines or histidines, with no upper limit. Polycation sequences can contain up to 450 amino acid residues and are still known to function (Proc Natl Acad Sci USA 87, 3410-3414, 1990). However, the length of the polycation sequence is preferably from 5 to 100 amino acid residues, more preferably from 5 to 50, even more preferably from 7 to 20 amino acid residues.
  • the proportion of the cationic amino acid residues in the polycation sequence is preferably at least 40 mol%, more preferably at least 60 mol%, further preferably at least 80 mol%, most preferably at least 90 mol%. A polycation sequence consisting of only polycationic amino acid residues is most preferably used.
  • Polycation sequence is preferably 4 or more, more preferably 5 or more, more preferably 7 or more, preferably 30 or less, more preferably 25 or less, more preferably 20 or less lysine, arginine and And / or contains histidine residues. Further, the polycation sequence preferably has a series of three or more consecutive lysine, arginine and / or histidine residues, and preferably has a series of five or more consecutive lysine, arginine and / or histidine residues. More preferably, it is particularly preferred to have a series of 7 or more consecutive lysine, arginine and / or histidine residues.
  • the rate of introduction into cells can be controlled by appropriately selecting the polycation sequence.
  • Preferred examples of the polycation sequence include KH or RH repeating sequences, for example, 3 to 20 repeating sequences of KH or RH, more preferably 5 to 15 repeating sequences of KH or RH, and still more preferably 7 to 12 repeating sequences. Sequences.
  • R arginine
  • K contiguous sequence of arginine
  • H histidine
  • a contiguous sequence, preferably a 5-15 contiguous sequence of H, more preferably a 7-12 contiguous sequence of H is also mentioned by way of example.
  • Specific examples of the polycation sequence include, for example, the following sequences. RRRRRR (SEQ ID NO: 89), KHKHKHKHKHKHKHKHKHKH (SEQ ID NO: 90).
  • the carrier peptide contains a component corresponding to a linear fusion of a cell permeable sequence and a polycation sequence. In this fusion, it is preferred that the polycation sequence is attached to the N-terminal and / or C-terminal of the cell-permeable sequence.
  • One or more polycation sequences described above can be bound to the cell-permeable sequence, preferably one to several, more preferably one to three or so, particularly preferably a cell-permeable sequence To one polycation sequence.
  • the conjugation may be performed chemically according to a conventional peptide conjugation reaction, or may be performed biologically using an enzyme such as ligase. For example, it can be carried out according to a general peptide synthesis method such as a solid phase method.
  • a suitable oligopeptide linker or the like may be interposed between the two.
  • a linker consisting of one to several amino acids can be interposed, and the amino acid residues constituting the linker can be appropriately selected. Since cell-penetrating peptides exhibit their properties at the N-terminus, it is preferred that the cell-penetrating sequence be attached to the N-terminus of the polycation sequence.
  • Carrier peptides can also be obtained by recombinant DNA technology.
  • a DNA fragment encoding a polycation sequence is linked to one or both ends of a DNA fragment encoding a cell-permeable sequence by ligation with a suitable DNA adapter or by in vitro mutagenesis.
  • ligation with a suitable DNA adapter or by in vitro mutagenesis.
  • the carrier peptide forms a complex with the nucleic acid.
  • the complex can be formed, for example, by mixing the carrier peptide and the nucleic acid in a solution.
  • the concentration of the carrier peptide is usually 10 ⁇ g / mL to 10 mg / mL, preferably 100 ⁇ g / mL to 1 mg / mL. mL
  • the concentration of the nucleic acid solution can be generally 1 ⁇ g / mL to 10 mg / mL, preferably 10 ⁇ g / mL to 1 mg / mL.
  • the present inventors have proposed a macropinocytosis inhibitor (specifically, amiloride (amiloride; Commisso, C. et al. Nature 497, 633-637 (2013)).
  • amiloride amiloride
  • Commisso C. et al. Nature 497, 633-637 (2013)
  • cytochalasin D Cytochalasin D; Nakase, I. et al. Mol. Ther. 10, 1011-1022 (2004)
  • the introduction of a substance into target cells by the peptide of the present invention is clearly inhibited. Therefore, without being limited by theory, it is confirmed that the peptide of the present invention promotes the incorporation of a target substance by promoting the uptake of an external solution into cells by macropinocytosis. It is thought to get.
  • target substance to be introduced into plant cells or microalgae
  • the substance of interest may be, for example, a protein, a polysaccharide, a nucleic acid, a compound, or a mixture thereof.
  • proteins include any of structural proteins, secreted proteins, enzymes, antibodies, labeled proteins, regulatory proteins, and selectable marker proteins (eg, NPT (neomycin phosphotransferase) II that produces kanamycin resistance, ⁇ -lactamase that produces ampicillin resistance, etc.), and the like. It may be. Specifically, for example, BSA (Bovine serum albumin), ADH (alcohol dehydrogenase), modified YFP Citrine, NPT II and the like can be mentioned.
  • Preferred examples of the target protein include a genome editing protein.
  • genome editing or “genome modification” means specifically cutting and editing a target site on a genome, for example, knocking in or knocking out a specific gene with respect to a wild-type genomic gene. Point to.
  • genome editing proteins include Transcription activator-like effector nuclease (TALEN), Cas9 (CRISPR associated protein 9), and ZFN (zinc finger nuclease), preferably TALEN and Cas9.
  • TALEN Transcription activator-like effector nuclease
  • Cas9 Cas9
  • ZFN zinc finger nuclease
  • Two or more target proteins can be simultaneously introduced into target plant cells.For example, by simultaneously introducing a selectable marker protein and another target protein, cells into which the protein has been introduced based on the selectable marker can be selected. It may be possible.
  • the guide RNA may be delivered into the cell together with Cas9, or may be introduced into the cell by other techniques. Examples of such a technique include transfection of a guide RNA or a vector such as a plasmid containing the guide RNA.
  • polysaccharides examples include water-soluble polysaccharides such as starch, glycogen, agarose, pectin, and dextran, and water-insoluble polysaccharides such as cellulose and chitin, and are preferably water-soluble polysaccharides.
  • nucleic acid examples include DNA or RNA (for example, the above guide RNA in the case of genome editing), and the nucleic acid may be linear or circular. It may be single-stranded or double-stranded.
  • Nucleic acids are intended to include nucleic acids of all types and sizes, including cDNA, plasmids, genomic DNA and derivatives thereof.
  • the nucleic acid can be chemically modified. Examples of suitable modified nucleic acids include, for example, thioate and dithioate. In this regard, other suitable nucleic acid derivatives are mentioned, for example, in Uhlmann & Peymann, Chemical Reviews, 90 (4), 543-584, 1990. Furthermore, nucleic acids in which nucleotide bases have been chemically modified can also be used. By introducing the nucleic acid into the cells, the cells can be transformed.
  • Target plant cells or microalgae the type of target plant cells is not particularly limited, and includes any angiosperms including monocotyledons and dicotyledons, gymnosperms, bryophytes, ferns, herbaceous plants, woody plants, etc.
  • the present invention can be applied to plant cells.
  • plants include, for example, Solanaceae (eggplant (Solanum melongena L.), tomato (Solanum lycopersicum), peppers (Capsicum annuum L. var. Angulosum Mill.), Pepper (Capsicum annuum L.), and tobacco (Nicotiana).
  • a plant cell derived from any tissue can be used and is not particularly limited. Examples thereof include an embryo, callus, pollen, leaf, anther, root, root tip, flower, seed, pod, stem, tissue culture, and the like. Can be used.
  • the target cells may be microalgae.
  • the microalgae include cyanobacteria, photosynthetic bacteria, diatoms, yellow-green algae, dinoflagellates, euglena, and the like, and algae of the Chlamydomonas family.
  • the microalgae are euglena, such as Euglena, such as E. glacilis.
  • the Chlamydomonas family algae is a Chlamydomonas algae, such as Chlamydomonas reinhardtii.
  • the present invention provides a target substance to be introduced into a target plant cell or microalgae, and introduction of a peptide or a substance described herein.
  • the present invention relates to a kit for introducing a target substance into a target plant cell or microalgae, comprising an agent.
  • the peptide, the target substance to be introduced into the target cell, and the configuration of the target cell are as described above, and thus description thereof is omitted here.
  • the kit of the present invention may include an instruction manual, a carrier peptide described in the present specification, a reagent for cell introduction, an instrument, and the like, in addition to the target substance and the peptide or substance introduction agent.
  • the invention provides cell permeable sequences, linkers, and escape from intracellular vesicles
  • the present invention relates to a method for introducing a target substance into cells or microalgae.
  • the peptide may be added to the target plant cell or microalgae simultaneously with the target substance, or may be added at a different timing (for example, before adding the target substance).
  • the cell-permeable sequence contained in the peptide and the domain capable of exiting from intracellular vesicles are as described in the above “1. Peptide”.
  • the linkers included in the peptide include, but are not limited to, chemical linkers such as disulfide linkers, maleimide linkers, and PEG linkers (linkers containing 2-20, 3-12, or 4-8 PEGs), sarcosine Examples include a linker containing a residue, and a peptide linker, for example, a linker composed of glycine and serine (GGS linker and GS linker).
  • the linker may be a linker containing a nitrogen atom, such as a linker containing a sarcosine residue.
  • the peptide in this embodiment may be, for example, the peptide described in “1. Peptide” above.
  • the step of bringing the target substance into contact with the target cell can be performed by a method known in the art, and is not limited as long as the target substance can be introduced into the target cell.
  • a solution in which one or more target substances are dissolved in a predetermined solvent for example, water, a mixed solvent of water and a hydrophilic organic solvent, etc.
  • a predetermined solvent for example, water, a mixed solvent of water and a hydrophilic organic solvent, etc.
  • the conditions of this step can be determined in consideration of the type of target substance, peptide, and target cell, toxicity to cells, and the like.
  • the conditions include temperature, pressure, time, temperature of peptide and target substance, and the like.
  • a solution of a substance of interest is brought into contact with target cells in the presence of the peptide described herein, and then, for example, at room temperature (20 ° C. to 35 ° C., 22 ° C. to 30 ° C., 24 ° C. to 28 ° C. or about 26 ° C.) At a temperature of 2).
  • Incubation time may be, for example, 1 minute or more, 2 minutes or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more, 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, Or it may be up to 2 hours, for example 1 minute to 48 hours, 5 minutes to 12 hours or 10 minutes to 2 hours.
  • the concentration of the peptide or substance introducing agent in this step may be, for example, 0.1 ⁇ M or more, 1 ⁇ M or more, or 10 ⁇ M or more, and may be 100 mM or less, 10 mM or less, or 1 mM or less, for example, 0.1 ⁇ M to 100 mM, 1 ⁇ M It may be 1010 mM, or 10 ⁇ M-1 mM.
  • the concentration of the target substance in this step may be, for example, 1 ⁇ g / ml or more, 10 ⁇ g / ml or more, 50 ⁇ g / ml or more, 75 ⁇ g / ml or more, 10 mg / ml or less, 1 mg / ml or less, 200 ⁇ g / ml or less, Alternatively, it may be less than or equal to 125 ⁇ g / ml, for example 1 ⁇ g / ml to 10 mg / ml, 50 ⁇ g / ml to 200 ⁇ g / ml, or about 100 ⁇ g / ml.
  • the contact may be performed under atmospheric pressure, under reduced pressure, under increased pressure, or under a combination of these pressure conditions.
  • a pretreatment step of bringing the peptide of the present invention into contact with target cells or the like may be performed in advance.
  • the target substance complexed with the carrier peptide is introduced into a target cell or the like, it is preferable to perform the pretreatment step before performing the contacting step.
  • the conditions of the pretreatment step can be adjusted from the same viewpoint as the contact step.
  • the subject is a callus
  • the contacting step can be performed on cells such as cultured cells, for example, plant tissues such as plant embryos, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods, stems, and the like. It can also be performed directly on the culture.
  • plant tissues such as plant embryos, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods, stems, and the like. It can also be performed directly on the culture.
  • the substance introduction method of the present invention is particularly excellent for introducing a substance in a relatively short time.
  • the method of the present invention does not require a pretreatment such as mixing or conjugation of the peptide and the target substance, and simply mixes the target substance and the target cell in the presence of the peptide to transfer the substance to the target cell. Since it can be introduced, it can be simple and highly versatile.
  • the invention provides a method for producing a transformed target plant cell or microalgae comprising contacting a nucleic acid molecule of interest with the target plant cell or microalgae in the presence of a peptide described herein. Or a method for transforming a target plant cell or microalgae.
  • the configuration other than using a nucleic acid molecule as the target substance is the same as the above-described method for introducing the target substance.
  • the transformation method of the present invention may further include a step of obtaining a transformant from the transformed plant cell obtained in the above step.
  • a transformant can be obtained by a conventionally known method.
  • a transformed tissue or cell can be dedifferentiated to obtain a callus, if necessary, and then re-differentiated to obtain a plant.
  • the method for introducing a substance of the present invention can be used as a genome editing method. That is, in one embodiment, the present invention relates to a method of producing a genome-modified cell, or a method of editing or modifying the genome of a target cell, wherein the method comprises editing the genome in the presence of the peptide described herein. Contacting the protein with a target plant cell or microalgae. The configuration of the peptide, the genome editing protein, the target cell, and the contacting step are as described above.
  • the contacting in the method of the present invention is performed in the presence of a carrier peptide or a complex of a carrier peptide and a nucleic acid described herein.
  • the details of the carrier peptide are as described in “1. Peptide”.
  • the concentration is not limited, but may be, for example, 0.1 ⁇ M or more, 1 ⁇ M or more, or 10 ⁇ M or more, and may be 100 mM or less, 10 mM or less, or 1 mM or less, for example, 0.1 mM or less. It may be from ⁇ M to 100 mM, from 1 ⁇ M to 10 mM, or from 10 ⁇ M to 1 mM.
  • the present invention relates to a method for producing a genome-modified plant, comprising a step of producing a genome-modified plant from the genome-modified plant cell obtained by the above method.
  • the step of producing a genome-modified plant from a genome-modified plant cell is known to those skilled in the art.For example, a tissue or cell subjected to genome modification is dedifferentiated as necessary to obtain a callus, and then re-differentiated to obtain a plant. You can get the body. Since a plant in which an arbitrary gene is modified can be obtained by the present method, the present method can be used for breeding and the like.
  • Dextran-Texas Red 70 kDa was purchased from Invitrogen (Carlsbad, CA) and Evans Blue was purchased from Sigma-Aldrich (St. Louis, MO).
  • BY-2 cell suspension culture was obtained from RIKEN BioResource Research Center. Cells were maintained at 130 rpm in a modified Linsmaier and Skoog medium in the dark at 26 ° C. and according to previous reports (Nagata, T. et al., International Review of Cytology, 1992, 132, pp. 1-30). Subculture was performed at weekly intervals.
  • AArabidopsis thaliana used as a model plant system in this example was grown under the same conditions as those used previously (Lakshmanan M. et al., Biomacromolecules, 2012, 14, 14, pp. 10-16). Euglena cells were maintained in CM medium (pH 3.5) at 26 ° C. and 100 rpm and subcultured at weekly intervals as previously described (Cramer M. et al., Arch. Mikrobiol. 1952, 17). , Pp. 384-402).
  • Circular dichroism (CD) spectroscopy The CD spectrum of the peptide (10 ⁇ M) in water was measured using a Jasco J-820 CD spectropolarimeter. Background scans were obtained with water. The measurement was performed using a quartz cuvette with a path length of 0.1 cm. Each spectrum was the average of 10 scans from 190 nm to 240 nm at 1 nm resolution and was obtained at 200 nm min -1 with a 1 nm bandwidth.
  • Peptide introduction method 1 Protein internalization into tobacco BY-2 cells was performed using a 96-well microplate. Exponentially growing cells (3 days after subculturing) were diluted with medium to an OD 600 of 0.5 and 80 ⁇ l was added to each well. The cells were then treated with 9 nM to 9 mM dTat-EED4 peptide in the presence of 100 ⁇ g / ml dextran or citrine. Medium was added to each well to a final volume of 100 ⁇ l and then incubated at 26 ° C. for 1 hour before analysis.
  • Cells were treated with 9 nM to 900 ⁇ M dTat-EED4 peptide in the presence of 100 ⁇ g / ml dextran. Medium was added to each well to a final volume of 100 ⁇ l, followed by incubation at 26 ° C. at 100 rpm for 24, 48, or 72 hours before analysis.
  • the peptides (dTat-EED4 and dTat-EED5) of the present invention were analyzed for secondary structure by measuring circular dichroism (CD) spectra.
  • CD circular dichroism
  • Retor-Tat (57-49) and dTat were similarly measured.
  • the Retro-Tat CD spectrum is that of a typical unstructured peptide with a minimum at 195 nm. The results are shown in Figure 1. From the results shown in FIG. 1, it can be understood that the CD spectra of dTat-EED4 and dTat-EED5 are all mirror images of the CD spectrum of Retro-Tat, and no clear secondary structure is observed.
  • the CD spectrum of dTat is less structurized compared to dTat-EED4 and dTat-EED5.
  • dTat-EED4 and dTat-EED5 both show excellent substance introduction properties that cannot be achieved with a dTat-only domain, but the results of this CD spectrum analysis show that the peptide of the present invention It can be understood that the material introduction property indicated by does not require secondary structuring.
  • BY-2 Cell Viability in the Presence of dTat-EED4 Peptide According to the cell viability analysis method 1 described above, the BY-2 cell viability at an effective peptide concentration and the incubation time (allowing for internalization of dextran and citrine) were determined. evaluated. Untreated or ineffective treatment (no dextran / citrin internalization; 9 ⁇ M, 1 hour) were included during comparative analysis. Cell viability was tested by exposing the peptide to effective treatment conditions. As a result, about 33 to 68% of the cells died after the treatment, whereas the survival rates of the untreated and the ineffectively treated cells were equivalent (about 25% of the dead cells) (FIG. 6).
  • dTat-EED4 Introduction of a substance into Arabidopsis thaliana cells using the dTat-EED4 peptide
  • the function of dTat-EED4 was tested using leaves of Arabidopsis thaliana, which is a model organism of a higher plant.
  • dTat-EED4 allowed cellular internalization of dextran to uniformly fill the cytosol and vacuolar compartments.
  • citrine was not internalized without the peptide, and clear internalization into cytosol was observed in the presence of dTat-EED4 (FIG. 8).
  • dTat-EED4 peptide was also used for intracellular delivery of dextran to E. glacilis cells.
  • internalization of dextran was observed in cells treated with 90 ⁇ M and 900 ⁇ M of dTat-EED4 after incubation for 24 h.
  • dextran internalized cells were also observed at lower concentrations of 900 nM and 9 ⁇ M (FIG. 9).
  • FIG. 10 (a) shows the condition without peptide, (b) shows the condition where 900 ⁇ M dTat -EED4 is present, and (c) shows the condition where 90 ⁇ M dTat- EED5 is present.
  • FIGS. 11A and 11B are photographs observed after contact with the cells and incubation for 1 hour.
  • FIG. 11A shows the condition where no peptide is present
  • FIG. 11B shows the condition where 90 ⁇ M dTat-EED4 is present
  • FIG. It is a photograph observed after contacting citrine with tobacco BY-2 cells under the condition where dTat-EED5 is present and incubating for 1 hour. Note that in FIGS. 10 and 11, (b) and (c) show two different scales.
  • BY-2 cell viability in the presence of dTat-EED4 peptide and dTat-EED5 peptide In the same manner as in cell viability analysis method 1, under conditions where various concentrations of dTat-EED4 and dTat-EED5 are present. The survival rate of BY-2 cells was determined. As described above, the effective concentration of dTat-EED4 introduced into BY-2 cells in dextran and citrine in 1 hour incubation was 900 ⁇ M and 90 ⁇ M, respectively, and the effective concentration of dTat-EED5 introduced under the same conditions was respectively 90 ⁇ M and 90 ⁇ M.
  • dTat-EED4 is more suitable for introducing substances into BY-2 cells.
  • the effective concentration and incubation conditions of each peptide are determined according to the substance to be introduced and the cell type, the cell viability at the effective concentration and incubation conditions is determined, and the determined cell viability is determined.
  • substance introduction can be achieved without significantly reducing cell viability.
  • dTat-EED4 or dTat-EED5 peptide Spores from spores of moss were incubated in 150 ⁇ L of Milli-Q water for 4 days.
  • the germinated spores were mixed with dTat-EED4 (4 mg / mL) and citrine (0.2 mg / mL) and incubated at 22 ° C. for 1 hour. Germinated spores mixed with only the same concentration of citrine were used as a control.
  • Citrin fluorescence was observed by a time-gating method (0.5 to 12.0 ns) using a confocal laser scanning microscope SP8X system (manufactured by Leica Microsystems). A 510 nm laser and a 546-566 nm wavelength were used for excitation and emission, respectively.
  • the results are shown in FIG.
  • the scale bar indicates 10 ⁇ m.
  • the observation results of a sample similarly prepared in the absence of the dTat-EED4 peptide are also shown. From the results shown in FIG. 12, it can be understood that citrine was introduced into the spores of the moss.
  • Chlamydomonas reinhardtii wild-type cells (cc125 +) were cultured in a TAP liquid medium at 23 ° C. under constant light irradiation conditions.
  • cultured cells of Euglena were mixed with 900 ⁇ M dTat-EED5 and 100 ⁇ g / mL citrine and incubated for 3 hours.
  • As a control cultured cells of E. coli were mixed with only the same concentration of citrine. In the same manner as above, the fluorescence of citrine was observed with a confocal laser scanning microscope.
  • Peptide dTat-EED4 was obtained in the same manner as described above.
  • BP100- (KH) 9 (sequence: KKLFKKILKYL-KHKHKHKHKHKHKHKHKHKH, SEQ ID NO: 91) used in the following examples, and (KH) 9 (sequence: KHKHKHKHKHKHKH, SEQ ID NO: 90) are each provided by RIKEN Brain Science Institute. The synthesized one was obtained.
  • the plasmid used in the following examples coded for oproforsul luciferase (Nluc) or green fluorescent protein (GFP) gene, together with the cauliflower mosaic virus 35S promoter and DNA derived from Agrobacterium tumefaciens. (Hereinafter referred to as “p35S-Nluc-tNOS” and “p35S-GFP-tNOS”, respectively).
  • p35S-Nluc-tNOS oproforsul luciferase
  • GFP-tNOS green fluorescent protein
  • Callus to be introduced was prepared as follows. Rice (Oryza sativa; Nipponbare) seeds are immersed in ethanol / water (70% v / v) for 1 minute while rotating at 20 rpm, then in bleach / water (50% v / v) for 30 minutes And sterilized. The seeds were washed 10 times with sterile water and, on a Petri dish, callus induction medium (N6D; lactose (30 g / L), casamino acid (0.3 g / L), l-proline (2.8 g / L), 2, Incubated at 30 ° C.
  • N6D callus induction medium
  • lactose (30 g / L)
  • casamino acid 0.3 g / L
  • l-proline 2.8 g / L
  • Arabidopsis thaliana (Cal-0) seeds were rotated in ethanol / water (70% v / v) for 1 minute while rotating at 20 rpm, then bleach / water (20% v / v). It was immersed for 30 minutes and sterilized. Thereafter, the seeds were washed 10 times with sterile water. After cooling the seeds at 4 ° C. for at least 3 days, half strength MurashigM and Skoog medium (MS salt (2.2 ⁇ g / L), MES (0.5 g / L, adjusted to pH ⁇ 5.7 with KOH), sucrose (10 g / L L) and phytagel (3 ⁇ g / L).) At 22 ° C. under dark conditions to obtain seedlings.
  • the seedlings 10 days after germination are dissected and the hypocotyl is taken out.
  • the hypocotyl is called a callus induction medium (CIM; ⁇ Gamborg's B5 ⁇ salt, glucose (20 g / L), myo-inositol ⁇ (10 mg / L), thiamine (2 mg / L), nicotinic acid (0.1 mg / L), pyridoxine (0.1 mg / L), MES (0.5 g / L, adjusted to pH 5.7 with KOH), 2,4-D (0.5 ⁇ g / mL), kinetin ( 0.05 ⁇ g / L), biotin (1 ⁇ g / L), and phytagel (6 ⁇ g / L).
  • CIM callus induction medium
  • ⁇ Gamborg's B5 ⁇ salt glucose (20 g / L)
  • myo-inositol ⁇ 10 mg / L
  • thiamine 2 mg / L
  • nicotinic acid 0.1 mg
  • BP100- (KH) 9 (1 mg / mL, 1 ⁇ L) was prepared using Cy3-labeled pDNA (1 mg / mL), GFP-encoded pDNA (p35S-GFP-tNOS, 1 mg / mL) and luciferase.
  • a complex having an N / P ratio of 0.5 was prepared by mixing with Milli-Q water (2.5 ⁇ L) containing any one of -code pDNA (p35S-Nluc-tNOS, 1 mg / mL).
  • the N / P ratio is defined as the molar ratio of the nitrogen (N) of the cationic peptide to the phosphorus (P) of the anionic pDNA.
  • (KH) 9 / pDNA complex N / P ratio 0.5, (KH) 9 aqueous solution (1 mg / mL, 1 ⁇ L) and pDNA (p35S-Nluc-tNOS) aqueous solution (1.0 mg / mL, 2.5 ⁇ L) was prepared by mixing After incubating the sample solution containing each complex at 25 ° C. for 30 minutes, it was used in the following Examples.
  • CLSM Confocal Laser Scanning Microscope
  • the callus soaked was cut into small pieces with a spatula, degassed for 1 minute under the condition of -0.08 MPa, and then pressurized at 0.08 MPa for 1 minute. After incubation at 25 ° C. for 10 minutes, a sample solution of BP100- (KH) 9 / Cy3-pDNA complex (N / P 0.5, 3.5 ⁇ L) was added to the callus immersion liquid. The final concentration of Cy3-pDNA was 25 ⁇ g / mL. The complex was allowed to penetrate the callus by degassing at -0.08 MPa for 1 minute, followed by pressurization at 0.08 MPa for 1 minute followed by incubation at 25 ° C. for 3 hours.
  • the infiltrated callus was immersed in an aqueous solution (20 ⁇ g / mL) of Hoechst 33258 (Thermo Fisher Scientific. Waltham, Mass., USA) for 15 minutes, and then washed three times with Milli-Q water. Calli after these treatments were used for observation of fluorescence images using a confocal laser scanning microscope ("CLSM, Leica Microsystems" Wetzlar, Germany) (Hoechst, excitation (405 nm), emission (430-515 nm). Cy3, excitation (514 nm), emission (545-615 nm)).
  • CLSM confocal laser scanning microscope
  • Luciferase-encoding DNA (p35S-Nluc-tNOS) was introduced into calli, luciferase expression in the calli was quantified by a luciferase assay, and the quantitative value was evaluated as the transfer efficiency. Specifically, it is as follows.
  • pDNA p35S-Nluc-tNOS, 25 ⁇ g / mL
  • pDNA p35S-Nluc-tNOS, 25 ⁇ g / mL
  • the rice calli were cultured on a 1/2 MS medium plate in a bioincubator at 30 ° C. under continuous light irradiation.
  • Arabidopsis callus was cultured on a 1/2 MS medium plate at 25 ° C. in a dark room.
  • the supernatant obtained from the lysate was diluted with Milli-Q water, mixed with Bradford reagent (APRO SCIENCE, Tokushima, Japan), and the protein was quantified from the absorbance at 595 nm.
  • the RLU / mg value was obtained by dividing by the quantitative value. Background correction was performed by subtracting the average value of the RLU / mg of the callus without the introduction treatment from each RLU / mg of the callus subjected to the introduction treatment. An average value was calculated from the corrected RLU / mg values obtained from the four samples, and this value was used for quantitative evaluation of the introduction efficiency.
  • dTat-EED4 also improved the transfection efficiency of uncomplexed pDNA. This result is particularly unexpected, since it is believed that fluidically large molecules, such as pDNA, cannot cross the cell wall and reach the cell membrane. Although it is not clear what effect this result was obtained for, it is thought that dTat-EED4 condensed anionic pDNA by electrostatic interaction. This was confirmed by the dynamic scattered light method (DLS) that the hydrodynamic diameter of pDNA was comparable to that of the BP100- (KH) 9 / pDNA complex in the presence of dTat-EED4. Is consistent with being positive in the presence of dTat-EED4, although the zeta potential is negative (data not shown).
  • DLS dynamic scattered light method

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Abstract

Le problème à résoudre par la présente invention concerne la fourniture d'un nouveau procédé d'administration d'une substance à une cellule végétale ou similaire. La présente invention concerne : un peptide contenant une séquence de pénétration cellulaire, un lieur contenant un résidu de sarcosine, et un domaine capable de sortir d'une vésicule intercellulaire ; un agent permettant d'introduire une substance d'intérêt dans une cellule végétale cible ou une micro-algue cible, l'agent comprenant le peptide susmentionné ; un procédé d'introduction d'une substance d'intérêt dans une cellule végétale cible ou une micro-algue cible à l'aide d'un peptide contenant une séquence de pénétration cellulaire, un lieur, et un domaine capable de sortir d'une vésicule intercellulaire ; et autres.
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