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LIPID MEMBRANE STRUCTURE HAVING INTRANUCLEAR LOCALIZATION PROPERTY

Foreign code F150008584
File No. (S2014-0299-N0)
Posted date Nov 26, 2015
Country WIPO
International application number 2014JP084034
International publication number WO 2015098907
Date of international filing Dec 24, 2014
Date of international publication Jul 2, 2015
Priority data
  • P2013-264927 (Dec 24, 2013) JP
Title LIPID MEMBRANE STRUCTURE HAVING INTRANUCLEAR LOCALIZATION PROPERTY
Abstract A lipid membrane structure for delivering a substance into the nucleus of a cell. The lipid membrane structure can deliver a nucleic acid into the nucleus of an immunocyte such as a dendritic cell with high efficiency. In the lipid membrane structure, a lipid membrane is modified with (a) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 1 and/or (b) a polypeptide which comprises an amino acid sequence produced by the deletion and/or substitution and/or insertion of one or several amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 and which has an activity of promoting the migration of the lipid membrane structure into the nucleus of a cell.
Outline of related art and contending technology BACKGROUND ART
An agent that specifically binds to the affected area as a means of transporting the lipid membrane structure encapsulating the drug in liposomes has been proposed a method. In particular, the treatment of malignant tumors in the field of anti-tumor agent is encapsulated in the liposomes and the effectiveness of many reported. In addition, the lipid membrane structure can be used for gene expression as a multifunctional envelope-type nano-structures (MEND: Multifunctional envelope-type nano device; hereinafter, is abbreviated as' MEND ' herein. Such as for example see Drug Delivery System, 22-2, pp.115-122, 2007) is proposed. This structure may be, selectively in a particular cell such as a gene for the delivery of a drug delivery system can be used as, for example, such as gene therapy of the tumor has been known to be useful.
The structure may be used lipid membrane drug, nucleic acid, peptide, polypeptide, or the target substance such as sugars such as tumor specific target organ site as a means for delivery, the surface of the lipid membrane structure is modified with functional molecules have been proposed many methods. Of an agent such as an anti-tumor agent containing a lipid membrane structure is a target cell by endocytosis and reaches into the cell to be contained within endosomes and the state is, then, enzymatic hydrolysis of lysosomes in response to the action or the like included in the drug has been released into the cytoplasm. Has been absorbed in the endosome in order to improve the drug release from the liposome, the surface of the liposome to the peptide (GALA: modified MEND Biochemistry, 26, pp.2964-2972, 1987) (Biochemistry, 43, pp.5618-5623, 2004) or liposome (Japanese Patent Application JP-2006-28030) is proposed.
In addition, nucleic acid and the like of a target substance containing a lipid film structure is transferred into the nucleus of the target cell as means for, for example, the external surface of the liposome in the liposome octaarginine (International Publication WO2005/32593; Journal of Controlled Release, 98, pp.317-323, 2004), nuclear translocation peptide modified liposomes having a lipid membrane layer membrane 2 (International Publication WO2006/101201), of monosaccharides such as galactose or mannose surface of the liposome (International Publication WO2007/102481) is proposed. Multiple lipid membrane modified saccharide structure (T-MEND) is fuzed to a lipid membrane and nuclear membranes and shows, in vitro gene expression in the test result was improved efficiency thereof.
On the other hand, immune cells, in particular antigen presenting dendritic cells has the effect of an antigenic protein in the nucleus of introducing a nucleic acid encoding it, which is transcribed and translated from the nucleic acids in dendritic cells and the antigen-presenting dendritic cell surface protein can be, immunity against the proteins with the living body can be obtained. From this point of view, an immune cell such as dendritic nucleic acid in the nucleus of a technique for efficient delivery is demanded.
However, such as a lipid membrane structure described above using MEND dendritic cells in the nucleus of acid in the case of introducing a nucleic acid introduction efficiency is, other cells, for example tumor cells and compared with the case of hepatocytes is not sufficient. Finally the introduced nucleic acid expression in the nucleus until, uptake into, endosomal escape, nuclear import, nuclear transcription and a variety of intracellular kinetics is need to undergo processes, non-dividing cells such as dendritic cells in a core film layer membrane 2 is always present and intact, the nuclear envelope lipid membrane structure does not block the ability of the nuclear import is conjectured. Therefore, the structure may be used lipid membrane to the nucleus of dendritic cells in order to deliver nucleic acids, each of the above processes, in particular a core-layer membrane 2 how to break through the barrier film is to be, extremely becomes an important problem.
27 Peptide KALA polypeptide referred to as amino acid residues are known and, using the plasmid DNA itself cationic charge can be forming a complex have been reported (Biochemistry, 36, pp.3008-3017, 1997). This peptide KALA liposomes lipid membrane by modifying the surface of the structure of the lipid membrane structure could promote nuclear import (International Publication WO 2011/132713) have been reported.
Scope of claims (In Japanese)請求の範囲 [請求項1]
細胞の核内に物質を送達するための脂質膜構造体であって、脂質膜が下記のポリペプチド(I):
アミノ酸残基数が8~26個のポリペプチドであって、ペプチド鎖中にテトラペプチド:Lys-Ara-Leu-Araの繰り返し単位を連続して2~4個含むポリペプチド(ただし該繰り返し単位のうちのいずれか1個又は2個以上においてLysがArgとなっていてもよく、及び/又は該繰り返し単位のうちのいずれか1個においてLysとLeuに挟まれるAraがHisとなっていてもよい)
で修飾された脂質膜構造体。

[請求項2]
ポリペプチド(I)がLys-Ara-Leu-Araの繰り返し単位を連続して2個含むか、又はArg-Ara-Leu-Araの繰り返し単位を連続して2個含むポリペプチドである請求項1に記載の脂質膜構造体。

[請求項3]
ポリペプチドが請求項2に記載された連続した2個の繰り返し単位のN末端にTrp-Glu-Ara-Lys-Leu-Ara又はTrp-Glu-Ara-Arg-Leu-Araの配列が結合したポリペプチドである請求項2に記載の脂質膜構造体。

[請求項4]
ポリペプチドがTrp-Glu-Ara-Lys-Leu-Ara-Lys-Ara-Leu-Ara-Lys-Ara-Leu-Ara又はTrp-Glu-Ara-Arg-Leu-Ara-Arg-Ara-Leu-Ara-Arg-Ara-Leu-Araである請求項1に記載の脂質膜構造体。

[請求項5]
脂質膜構造体の脂質成分として第3級アミン及びジスルフィド結合を有する脂質を含む請求項1ないし4のいずれか1項に記載の脂質膜構造体。

[請求項6]
脂質膜構造体がリポソームである請求項1ないし5のいずれか1項に記載の脂質膜構造体。

[請求項7]
細胞が免疫細胞である請求項1ないし6のいずれか1項に記載の脂質膜構造体。

[請求項8]
上記ポリペプチド(I)が疎水性基で修飾されており、前記疎水性基が脂質膜に挿入された請求項1ないし7のいずれか1項に記載の脂質膜構造体。

[請求項9]
連続した複数個のアルギニン残基を含むポリペプチドを表面に有する請求項1ないし8のいずれか1項に記載の脂質膜構造体。

[請求項10]
ポリアルキレングリコールを表面に有する請求項1ないし9のいずれか1項に記載の脂質膜構造体。

[請求項11]
免疫細胞表面に提示すべき抗原ポリペプチドをコードする核酸を該細胞の核内に導入するために用いる請求項1ないし10のいずれか1項に記載の脂質膜構造体。

[請求項12]
送達すべき物質が内部に封入された請求項1ないし10のいずれか1項に記載の脂質膜構造体。

[請求項13]
内部に核酸及びカチオン性ポリマーが封入された請求項12に記載の脂質膜構造体。

[請求項14]
送達すべき物質が免疫細胞表面に提示すべき抗原ポリペプチドをコードする核酸である請求項12又は13に記載の脂質膜構造体。

[請求項15]
上記抗原ポリペプチドに対する免疫療法に用いるための請求項14に記載の脂質膜構造体。

[請求項16]
該核酸がDNAであり、該DNAがCpGを含まないDNAである請求項13又は14に記載の脂質膜構造体。

[請求項17]
請求項12ないし16のいずれか1項に記載の脂質膜構造体を有効成分として含む医薬組成物。

[請求項18]
悪性腫瘍の予防及び/又は治療のために用いる請求項17に記載の医薬組成物。

[請求項19]
細胞の核内への脂質膜構造体の移行を促進するために用いるポリペプチドであって、下記のポリペプチド(I):
アミノ酸残基数が8~26個のポリペプチドであって、ペプチド鎖中にテトラペプチド:Lys-Ara-Leu-Araの繰り返し単位を連続して2~4個含むポリペプチド(ただし該繰り返し単位のうちのいずれか1個又は2個以上においてLysがArgとなっていてもよく、及び/又は該繰り返し単位のうちのいずれか1個においてLysとLeuに挟まれるAraがHisとなっていてもよい)

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY
  • Inventor
  • HARASHIMA Hideyoshi
  • AKITA Hidetaka
  • MIURA Naoya
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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