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Method of introducing nucleic acid into plant cells UPDATE_EN

Foreign code F190009894
File No. 07999-EP
Posted date Aug 26, 2019
Country EPO
Application number 13755612
Gazette No. 2821486
Gazette No. 2821486
Date of filing Feb 27, 2013
Gazette Date Jan 7, 2015
Gazette Date Dec 26, 2018
International application number JP2013056062
International publication number WO2013129698
Date of international filing Feb 27, 2013
Date of international publication Sep 6, 2013
Priority data
  • P2012-040622 (Feb 27, 2012) JP
  • 201261691833 (Aug 22, 2012) US
  • 2013JP56062 (Feb 27, 2013) WO
Title Method of introducing nucleic acid into plant cells UPDATE_EN
Abstract The object of the present invention is to provide a method of introducing a nucleic acid into plant cells, which is simple and widely applicable to various types of plant cells and nucleic acids. The present invention relates to a method of introducing a nucleic acid into a target plant cell, comprising a step of forming a complex by bringing a carrier peptide, wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic sequence, into contact with a nucleic acid and a step of bringing the obtained complex into contact with a target plant cell.
Outline of related art and contending technology Background Art
Providing useful traits that cannot be provided by cross breeding to a plant by introducing foreign genes to plant cells is extremely meaningful in future improvement of crops and development of agriculture. Furthermore, in order to establish a substance production method by plants using carbon dioxide as a raw material, as a novel substance production method alternating a petroleum-dependent substance production method, it is indispensable to use a plant genetic recombination technique.
As a method of introducing a gene into a plant cell, an Agrobacterium method using infection with a soil bacterium, i.e., Agrobacterium, a particle-gun method and a viral vector method are known. The Agrobacterium method is the most frequently used in introducing a gene into a plant cell. However, there are many economically important plants to which a gene cannot be introduced by the Agrobacterium method, at present. The particle-gun method has been much more generally used compared to the Agrobacterium method; however, device cost is high. In addition to this problem, there is a risk of damaging a gene and transformation efficiency is low. The viral vector method is advantageous compared to the Agrobacterium method since transformation efficiency and gene expression efficiency are high; however, the method has a problem in that the size of the gene to be introduced is limited and viral infectious ability is low. In the circumstances, it has been desired to develop a novel method of introducing a gene, which is simple and widely applicable to all types of plants and genes.
It is known that a cell-penetrating peptide (CPP) has a function of transporting a complex comprising the peptide and another substance (for example, a protein and a nucleic acid) through biomembrane of mammalian and human cell strains. However, use of CPP in plant cells is limited. Unlike animal cells, plant cells have double barriers, i.e., cell wall and cell membrane, for preventing internalization of a complex comprising CPP. In the meantime, it is disclosed that a polycationic peptide concentrates negatively charged DNA by ionic interaction to form a complex that can be used for gene delivery, and that such a complex is useful for introducing a gene into animal cells (Patent Document 1: WO2011/006133). It has been also reported that a polycationic peptide was used for introducing a gene into a plant protoplast (Patent Document 2: JP Patent Publication (Kokai) No. 7-213285). However, the method employs a protoplast without a cell wall. In addition, it cannot be said that gene introduction efficiency into a plant cell is sufficient, in this method.
Prior Art Documents
Patent Documents:
Patent Document 1: WO2011/006133
Patent Document 2: JP Patent Publication (Kokai) No. 7-213285
Scope of claims [claim1]
1. A method of introducing a nucleic acid into a target plant cell, comprising a step of forming a complex by bringing a carrier peptide, wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic sequence comprising:
(a) a continuous series of 5 or more residues of lysine (K), arginine (R) or histidine (H);
(b) a continuous series of 7 or more residues of lysine (K), arginine (R) and/or histidine (H); or
(c) 3 to 20 repeat sequences of KH;
into contact with a nucleic acid such that the ratio of the number of amine groups from the carrier peptide and the number of phosphate groups from the nucleic acid (N/P ratio) is 0.2 or more; and
a step of bringing the obtained complex into contact with a target plant cell.

[claim2]
2. The method according to Claim 1, wherein the N/P ratio is 2 or less.

[claim3]
3. The method according to Claim 1 or Claim 2, wherein the step of forming a complex is performed in the presence of the carrier peptide comprising an organelle transit sequence and a polycationic sequence.

[claim4]
4. The method according to any one of Claims 1 to 3, wherein incubation time for bringing the complex into contact with a target plant cell is 5 to 150 hours.

[claim5]
5. The method according to any one of Claims 1 to 4, wherein the cell-penetrating sequence is BP100 or Tat2.

[claim6]
6. The method according to any one of Claims 1 to 5, wherein the polycationic sequence comprises a sequence of 5 to 20 consecutive Rs.

[claim7]
7. The method according to any one of Claims 1 to 6, wherein the carrier peptide further comprises an organelle transit sequence.

[claim8]
8. Use of a carrier peptide for introducing a nucleic acid into a target plant cell, wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic sequence comprising:
(a) a continuous series of 5 or more residues of lysine (K), arginine (R) or histidine (H);
(b) a continuous series of 7 or more residues of lysine (K), arginine (R) and/or histidine (H); or
(c) 3 to 20 repeat sequences of KH, and
forms a complex with the nucleic acid, in which the ratio of the number of amine groups from the carrier peptide and the number of phosphate groups from the nucleic acid (N/P ratio) is 0.2 or more.

[claim9]
9. The use according to Claim 8, wherein the carrier peptide comprises:
(i) the cell-penetrating sequence BP100 or Tat2; and/or
(ii) a polycationic sequence comprising a sequence of 5 to 20 consecutive Rs.

[claim10]
10. The use according to Claims 8 or 9, wherein the carrier peptide further comprises an organelle transit sequence.

[claim11]
11. A complex for introducing a nucleic acid into a target plant cell, comprising a carrier peptide and a nucleic acid, wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic sequence comprising:
(a) a continuous series of 5 or more residues of lysine (K), arginine (R) or histidine (H);
(b) a continuous series of 7 or more residues of lysine (K), arginine (R) and/or histidine (H); or
(c) 3 to 20 repeat sequences of KH; and
forms a complex with the nucleic acid, in which the ratio of the number of amine groups from the carrier peptide and the number of phosphate groups from the nucleic acid (N/P ratio) is 0.5 to 2.

[claim12]
12. The complex according to Claim 11, wherein the carrier peptide comprises:
(i) the cell-penetrating sequence BP100 or Tat2; and/or
(ii) a polycationic sequence comprising a sequence of 5 to 20 consecutive Rs.

[claim13]
13. The complex according to claim 11 or 12, wherein the carrier peptide further comprises an organelle transit sequence.

[claim14]
14. A complex for introducing a nucleic acid into a target plant cell, comprising a first carrier peptide, a second carrier peptide comprising an organelle transit sequence and a polycationic sequence, and a nucleic acid,
wherein the first carrier peptide comprises a cell-penetrating sequence and a polycationic sequence comprising:
(a) a continuous series of 5 or more residues of lysine (K), arginine (R) or histidine (H);
(b) a continuous series of 7 or more residues of lysine (K), arginine (R) and/or histidine (H); or
(c) 3 to 20 repeat sequences of KH, and
forms a complex with the nucleic acid, in which the ratio of the number of amine groups from the carrier peptide and the number of phosphate groups from the nucleic acid, N/P ratio, is 0.2 or more.

[claim15]
15. The complex according to Claim 14, wherein the first carrier peptide comprises:
(i) the cell-penetrating sequence BP100 or Tat2; and/or
(ii) a polycationic sequence comprising a sequence of 5 to 20 consecutive Rs.

[claim16]
16. The complex according to any one of Claims 11 to 15, wherein the N/P ratio is 2 or less.

[claim17]
17. A kit for introducing a nucleic acid into a target plant cell, comprising a carrier peptide and the nucleic acid, wherein the carrier peptide comprises a cell-penetrating sequence and a polycationic sequence comprising:
(a) a continuous series of 5 or more residues of lysine (K), arginine (R) or histidine (H);
(b) a continuous series of 7 or more residues of lysine (K), arginine (R) and/or histidine (H); or
(c) 3 to 20 repeat sequences of KH; and
forms a complex with the nucleic acid, in which the ratio of the number of amine groups from the carrier peptide and the number of phosphate groups from the nucleic acid (N/P ratio) is 0.5 to 2.

[claim18]
18. The kit according to Claim 17,
(i) which further comprises a carrier peptide comprising an organelle transit sequence and a polycationic sequence; and/or
(ii) wherein the cell-penetrating sequence is BP100 or Tat2; and/or
(iii) wherein the polycationic sequence comprises a sequence of 5 to 20 consecutive Rs.
  • Applicant
  • RIKEN
  • Inventor
  • NUMATA, Keiji
  • YOSHIZUMI, Takeshi
  • KODAMA, Yutaka
  • OHTANI, Misato
  • DEMURA, Taku
IPC(International Patent Classification)
Specified countries Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

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