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Reagent for introduction of protein or gene

外国特許コード F110003065
整理番号 134-US
掲載日 2011年5月25日
出願国 アメリカ合衆国
出願番号 31264807
公報番号 20100291672
公報番号 8338643
出願日 平成19年11月26日(2007.11.26)
公報発行日 平成22年11月18日(2010.11.18)
公報発行日 平成24年12月25日(2012.12.25)
国際出願番号 JP2007073266
国際公開番号 WO2008062911
国際出願日 平成19年11月26日(2007.11.26)
国際公開日 平成20年5月29日(2008.5.29)
優先権データ
  • 特願2006-317841 (2006.11.24) JP
  • 2007JP073266 (2007.11.26) WO
発明の名称 (英語) Reagent for introduction of protein or gene
発明の概要(英語) The present invention provides a reagent for introducing a protein or gene into a cell.
The reagent of the present invention is, for example, a reagent for introducing a protein or gene into a cell, which comprises a composition comprising a cationic amino acid type lipid represented by the following formula (I)-1: (wherein in formula (I)-1: L is a single bond, —CONH—, or —S—S—; M1 is —(CH2)k— or —(CH2CH2O)k— (wherein k is an integer between 0 and 14); and m1 and m2 are each independently an integer between 11 and 21 (in this regard, when providing a reagent for introducing a gene into a cell, the case where both m1 and m2 are 15 is excluded)).
従来技術、競合技術の概要(英語) BACKGROUND ART
Technologies for encapsulating a useful substance in a vesicle or liposome, which is formed of an artificial bilayer membrane, are actively studied in the fields of pharmaceutical drugs, cosmetics, foods, dyes and the like.
Widely used lipids for forming a membrane of a liposome include, for example, a mixed lipid of a membrane-forming lipid such as diacylphosphatidylcholine, cholesterol or the like and a negatively charged phospholipid such as diacylphosphatidylglycerol, diacylphosphatidylinositol, diacylphosphatidylserine or the like.
Recently, it has been studied to introduce a gene into a cell by forming a complex of the gene and an independent cationic lipid or a liposome containing the cationic lipid (Hong Sung Kim et al., Gene-transferring efficiencies of novel diamino cationic lipids with varied hydrocarbon chains.
Bioconjugate Chem. 2004, 15, 1095).
Most of existing cationic lipids for gene introduction, such as 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP), 3beta -N-(N',N'-dimethyl-aminoethane)-carbamoyl cholesterol (DC-Chol), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecyldimethylammonium chloride (DODAC), N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium (DOTMA), didodecyldimethylammonium bromide (DDAB) and Lipofectamine, have quaternary amine for imparting positive charge to the hydrophilic portion of lipid component.
Unlike gene introduction carriers using a cationic polymer such as polyethyleneimine, gene introduction systems using these cationic lipids do not have the proton sponge effect in which a complex with DNA leaves from an endosome.
Therefore, existing cationic lipids for gene introduction are usually mixed with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), which is a membrane-fusogenic lipid.
A cationic lipid has the action to concentrate and stabilize DNA, and DOPE promotes fusion with an endosome membrane, thereby releasing the cationic lipid-DNA complex to the outside of the endosome membrane.
Thus, introduction of DNA which is concentrated and stabilized by the cationic lipid into the nucleus is accomplished (L. Stamatatos et al., Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes.
Biochemistry 1988, 27, 3917; Japanese National-phase PCT Laid-Open Patent Publication No. 11-504631).
However, it is not easy to synthesize the above-described cationic lipid for gene introduction because of its complicated chemical structure, and under present circumstances, such reagents for gene introduction are expensive.
Moreover, lipids for gene introduction having quaternary amine tend to cause aggregation with serum component because the lipids are strongly cationic, and this may cause significant reduction in the gene introduction efficiency in the presence of serum.
Furthermore, these cationic lipids strongly interact with phospholipid on the surface of cell membrane, and therefore cell cytotoxicity thereof is high.
Meanwhile, a carrier is also required in the case where a protein is introduced from the outside into a cell.
As such carriers, lipid-based reagents (e.g., Profect P-1, BioPORTER, and SAINT-MIX), peptide-based reagents (e.g., Profect P-2, and Chariot), etc. are available.
However, since most of these carriers are simply mixed with a protein to form a complex and it is added to a cell for use, it is often impossible to control the size of the complex.
Further, particularly in the case of lipid-based carriers, the efficiency of introduction of a protein of interest is significantly reduced when serum protein is contained in a cell culture solution, and therefore it is required to make an experiment using a medium without containing serum.
Moreover, when an excess amount of free lipid carrier is present, significant cell cytotoxicity may appear.

特許請求の範囲(英語) [claim1]
1. A reagent for introducing a protein into a cell, which comprises a composition comprising a cationic amino acid type lipid represented by the following formula (I)-1:

wherein: L is a single bond, -- CONH -- , or -- S -- S -- ; M1 is -- (CH2)k -- or -- (CH2CH2O)k -- ; k is an integer between 1 and 11; and m1 and m2 are each independently an integer between 11 and 21.
[claim2]
2. A reagent for introducing a gene into a cell, which comprises a composition comprising a cationic amino acid type lipid represented by the following formula (I)-1:

wherein: L is a single bond, -- CONH -- , or -- S -- S -- ; M1 is -- (CH2)k -- or -- (CH2CH2O)k -- ; k is an integer between 1 and 11; and m1 and m2 are each independently an integer between 11 and 21; with the proviso that both m1 and m2 are not an integer of 15.
[claim3]
3. The reagent according to claim 1 or 2, wherein the composition further comprises at least one substance selected from the group consisting of diacylphosphatidylcholine, cholesterol, and an amphipathic molecule to which a polyethylene glycol chain is bound.
[claim4]
4. The reagent according to claim 3, wherein one or two acyl chains of diacylphosphatidylcholine is an oleoyl group.
[claim5]
5. The reagent according to claim 1 or 2, wherein the composition is a molecular assembly dispersed in an aqueous medium.
[claim6]
6. The reagent according to claim 5, wherein the molecular assembly forms a bilayer membrane vesicle structure, and wherein an inner aqueous phase thereof includes a protein or gene.
[claim7]
7. The reagent according to claim 1 or 2, wherein the composition is a dry powder product.
[claim8]
8. The reagent according to claim 7, wherein the dry powder product can form a bilayer membrane vesicle structure including a protein or gene when dispersed in an aqueous medium.
[claim9]
9. The reagent according to claim 1 or 2, wherein the cell is: a cell constituting a part of body tissue; plasma, serum or blood; or a cell cultured in a medium containing a part of components thereof.
[claim10]
10. A kit for introducing a protein or gene into a cell, which comprises the reagent according to claim 1 or 2.
[claim11]
11. A cationic amino acid type lipid represented by the following formula (I)-3:

wherein: M2 is -- (CH2)k' -- or -- (CH2CH2O)k' -- ; k' is an integer between 1 and 14; and p1 and p2 are each independently an integer between 11 and 21.
[claim12]
12. A reagent for introducing a protein into a cell, which comprises a composition comprising a cationic amino acid type lipid represented by the following formula (I)-1:

wherein: L is -- CONH -- , or -- S -- S -- ; M1 is -- (CH2)k -- or -- (CH2CH2O)k -- ; k is an integer between 2 and 14; and m1 and m2 are each independently an integer between 11 and 21.
[claim13]
13. A reagent for introducing a gene into a cell, which comprises a composition comprising a cationic amino acid type lipid represented by the following formula (I)-1:

wherein: L is -- CONH -- , or -- S -- S -- ; M1 is -- (CH2)k -- or -- (CH2CH2O)k -- ; k is an integer between 2 and 14; and m1 and m2 are each independently an integer between 11 and 21; with the proviso that both m1 and m2 are not an integer of 15.
[claim14]
14. The reagent according to claim 12 or 13, wherein the composition further comprises at least one substance selected from the group consisting of diacylphosphatidylcholine, cholesterol, and an amphipathic molecule to which a polyethylene glycol chain is bound.
[claim15]
15. The reagent according to claim 14, wherein one or two acyl chains of diacylphosphatidylcholine is an oleoyl group.
[claim16]
16. The reagent according to claim 12 or 13, wherein the composition is a molecular assembly dispersed in an aqueous medium.
[claim17]
17. The reagent according to claim 16, wherein the molecular assembly forms a bilayer membrane vesicle structure, and wherein an inner aqueous phase thereof includes a protein or gene.
[claim18]
18. The reagent according to claim 12 or 13, wherein the composition is a dry powder product.
[claim19]
19. The reagent according to claim 18, wherein the dry powder product can form a bilayer membrane vesicle structure including a protein or gene when dispersed in an aqueous medium.
[claim20]
20. The reagent according to claim 12 or 13, wherein the cell is: a cell constituting a part of body tissue; plasma, serum or blood; or a cell cultured in a medium containing a part of components thereof.
[claim21]
21. A kit for introducing a protein or gene into a cell, which comprises the reagent according to claim 12 or 13.
[claim22]
22. The reagent according to claim 1, wherein k is an integer between 3 and 5.
[claim23]
23. The reagent according to claim 2, wherein k is an integer between 3 and 5.
  • 発明者/出願人(英語)
  • TAKEOKA SHINJI
  • TAKEDA NAOYA
  • KURUMIZAKA HITOSHI
  • SAKANE ISAO
  • IKEGAYA NAMIKO
  • OBATA YOSUKE
  • SAITO SYUNSUKE
  • WASEDA UNIVERSITY
国際特許分類(IPC)
米国特許分類/主・副
  • 562/624
  • 564/123
  • 568/22
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