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RECOMBINANT EXPRESSION VECTOR AND LIPID MEMBRANE STRUCTURE HAVING SAID VECTOR ENCAPSULATED THEREIN

Foreign code F170009107
File No. (S2016-0168-N0)
Posted date Jun 15, 2017
Country WIPO
International application number 2016JP085098
International publication number WO 2017090763
Date of international filing Nov 28, 2016
Date of international publication Jun 1, 2017
Priority data
  • P2015-230498 (Nov 26, 2015) JP
Title RECOMBINANT EXPRESSION VECTOR AND LIPID MEMBRANE STRUCTURE HAVING SAID VECTOR ENCAPSULATED THEREIN
Abstract [Problem] To provide a novel expression vector capable of effectively expressing a target protein in mitochondria and suppressing undesirable expression of the target protein in cell organelles other than mitochondria.
[Solution] The present invention provides a recombinant expression vector for expressing a target protein in mitochondria of animal cells, and a lipid membrane structure having the vector encapsulated therein, wherein the recombinant expression vector has a promoter sequence exhibiting a transcription activity in the nuclei of animal cells, and has, under the control of the promoter sequence, a coding region which codes a target protein and includes one or more TGAs as codons corresponding to tryptophan. The recombinant expression vector according to the present invention can more efficiently and selectively express a target protein in mitochondria, and can be used as a more safe and effective drug for treating mitochondrial diseases.
Outline of related art and contending technology BACKGROUND ART
1 Is one of the mitochondria of organelles in a cell, cell nucleus has independent genomic DNA. Between a mutation in the genomic DNA of a variety of diseases (brain fibromyalgia, neurodegenerative disease, cancer, such as diabetes) has been reported a large number of related, these are generically referred to as a mitochondrial disease.
As a treatment method of mitochondria, mitochondrial protein in the expected therapeutic effect can be expressed in gene therapy and for the purpose. As a method of realizing the method according to claim, expressed in a cell nucleus to cause the transfer of the protein of interest using a method in the mitochondria, and mitochondrial directly within the target protein can be expressed foreign gene expression system has been proposed a method of using.
The target protein is expressed in the cell nucleus to be transported to the mitochondria in connection with the method, a wide variety of expression vectors have been developed. Many of, the genome of the cell nucleus encoded mitochondrial protein has, mitochondrial migration signal peptide that utilizes (mitochondrial targeting signal peptide, MTS). Specifically, the target protein upstream of the MTS (MTS added protein) an expression vector encoding were delivered into the nucleus, additional MTS protein expressed in the cytoplasm, although it is delivered to the mitochondria.
In this method is a type of target protein may be useful, gene therapy is expected to be used is, mitochondrial protein encoded by the mitochondrial genome DNA (mitochondrial endogenous proteins) has a problem in the applicability to the display. Is in the cytoplasm and mitochondria of an endogenous protein is insoluble in many for, expressed in the cytoplasm and mitochondria of agglomeration of the endogenous protein, into the mitochondria transition becomes insufficient in some cases.
Patent Document 1 is, the water-soluble increased mitochondrial specific MTS MTS or added protein from an endogenous protein and encoding using an expression vector, aggregation in the cytoplasm of the protein of interest is a method has been disclosed. However, the endogenous protein is mitochondria, subcellular organelles other than mitochondria and therefore it is cytotoxic to the, method described in Patent Document 1 can be used limit the range of the protein of interest.
In addition, the method comprises using MTS added protein, intracellular transport of an endogenous protein in the mitochondria of the original interference or by competing with the cell and possibly damage the lethal, concern remains as a tool for gene therapy.
On the other hand, mitochondrial directly within the target protein can be expressed in the employment of a foreign gene expression system, a necessary and sufficient amount in the first mitochondrial transcript expression of a protein of interest is required. So as to be adapted for such purpose, a promoter derived from a gene present in the mitochondrial genome DNA, for example selected HSP (heavy strand promoter), which encodes a target protein under its control is placed some DNA designed expression vector. The expression vector may, in the mitochondrial genome DNA triplet devised such as using a high frequency of use is also added. However, to date, the expression level of the expression vector are necessary and sufficient for the treatment of other region has not yet been attained.
For example, the non-patent document 1, the inside of the artificial viral vector MTS HSP is added under the control of the transcriptional induction in mitochondria DNA are sealed, directly to the viral vector is introduced into the mitochondrial target protein are to be expressed be method have been proposed. This method is, introduced from the viral vector of the expression of the target protein level is relatively high on the other hand has the advantage that, due to a viral vector in addition to entailing safety problems, as expected the protein of interest expressed in the mitochondria of the verification or not completely spoken.
Scope of claims (In Japanese)[請求項1]
動物細胞の細胞核内で転写活性を示すプロモーター配列、及び目的タンパク質をコードするコーディング領域であって、トリプトファンに対応するコドンとして1つ又は複数のTGAを含むコーディング領域を前記プロモーター配列の制御下に有する、動物細胞のミトコンドリア内で目的タンパク質を発現させるための組換え発現ベクター。
[請求項2]
前記目的タンパク質をコードするコーディング領域の5’末端側に、ミトコンドリアゲノムDNAのコーディング領域をさらに有する、請求項1に記載の組換え発現ベクター。
[請求項3]
前記目的タンパク質をコードするコーディング領域の3’末端側にミトコンドリアtRNAに対応する塩基配列を有する、請求項1又は2に記載の組換え発現ベクター。
[請求項4]
プロモーター配列がサイトメガロウイルスプロモーター、シミアンウイルス40プロモーター、ラウスサルコーマウイルスプロモーター、EF1αプロモーター、βアクチンプロモーター及びT7プロモーターよりなる群から選択されるプロモーターの塩基配列である、請求項1~3のいずれかに記載の組換え発現ベクター。
[請求項5]
プロモーター配列がサイトメガロウイルスプロモーター又はラウスサルコーマウイルスプロモーターの塩基配列である、請求項4に記載の組換え発現ベクター。
[請求項6]
前記目的タンパク質をコードするコーディング領域におけるトリプトファンに対応するコドンの全てがTGAである、請求項1~5のいずれかに記載の組換え発現ベクター。
[請求項7]
前記ミトコンドリアゲノムDNAのコーディング領域がNADHデヒドロゲナーゼ、サブユニット4のコーディング領域である、請求項2~6のいずれかに記載の組換え発現ベクター。
[請求項8]
請求項1~7のいずれかに記載の発現ベクターを封入した脂質膜構造体。
[請求項9]
スフィンゴミエリンを脂質膜の構成脂質として含む、請求項8に記載の脂質膜構造体。
[請求項10]
配列番号13で示されるアミノ酸配列からなるペプチドを脂質膜表面に有する、請求項8又は9に記載の脂質膜構造体。
[請求項11]
ジオレイルホスファチジルエタノールアミンとスフィンゴミエリンとを脂質膜の構成脂質として含有し、かつ配列番号13で示されるアミノ酸配列からなるペプチドを脂質膜表面に有する、動物細胞のミトコンドリアに核酸を導入するための脂質膜構造体。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • HOKKAIDO UNIVERSITY
  • Inventor
  • HARASHIMA Hideyoshi
  • YAMADA Yuma
  • ISHIKAWA Takuya
  • AKITA Hidetaka
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 DJ 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 KW 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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