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GENE EXPRESSION CASSETTE AND PRODUCT THEREOF

Foreign code F170009080
File No. (S2016-0007-N0)
Posted date May 29, 2017
Country WIPO
International application number 2016JP079219
International publication number WO 2017061354
Date of international filing Oct 3, 2016
Date of international publication Apr 13, 2017
Priority data
  • P2015-198160 (Oct 6, 2015) JP
  • P2016-059297 (Mar 23, 2016) JP
Title GENE EXPRESSION CASSETTE AND PRODUCT THEREOF
Abstract The purpose of the present invention is to provide a gene expression cassette, for stable and high capacity production of a protein of interest. Provided is a gene expression cassette having a structure in which the gene of interest and a DNA construct (X) including a poly A addition sequence lies between a promoter (P) and an enhancer (P'), wherein the gene expression cassette is characterized by further comprising a transposon sequence (T), upstream of the promoter (P) and downstream of the enhancer (P'). Furthermore, in the foregoing, the protein of interest can more effectively be produced stably and in large quantities by combining and suitably arranging, in addition to the transposon sequence (T), a nuclear matrix binding sequence (M), upstream of a replication initiation sequence (S). For example, HRG, PD-1, EMMPRIN, NPTNβ, EMB, RAGE, MCAM, ALCAM, ErbB2, and antibodies can be produced stably and in large amounts.
Outline of related art and contending technology BACKGROUND ART
In order to increase the efficiency of gene expression, promoters such as CAG promoter or CMV promoter expression of various genes have been developed (Patent Document 1-3). Developed by the inventors of the present invention a promoter, and optimize a combination of enhancers and the like, most of the mammalian cells to increase the expression of the gene in one of them is a system 1 (patent document 4, non-patent document 1, reference 2).
Expression of the gene at higher efficiency of the system which can be attempted a development, the combination of the various genes of a promoter by a comparison of the activities of a promoter or enhancer, by considerations that, downstream of a promoter with a gene to be expressed (hereinafter, 'the gene of interest' also referred to as.) And poly A addition sequences and enhancer or downstream of the first DNA construct comprising a promoter linked to the 2, by using the cassette for expression of a gene, gene expressed at high efficiency can be found by the present inventors, have been reported (patent document 4, non-patent document 1, reference 2). However this vector, transient expression of the vector in a cell effective and, currently, the objectives sought in the field of pharmaceutical production and stabilize the protein high-producing mammalian cells in order to manufacture a, was necessary to improve the further vector.
In general, by the method of genetically stable and high target protein in order to obtain cells that can be produced, a gene of interest within a host cell chromosome integration, using a gene amplification, cells with multiple copies of the gene of interest is constructed. Are the most frequently used as a method of, dihydrofolate reductase (DHFR) deficient strain CHO-DG44 method were used. Specifically, a gene of interest and introduced into a cell with the vector containing DHFR, cells with multiple copies of the gene of interest is a method of acquiring and (non-patent document 3, reference 4). However, according to the method using a DHFR-deficient cells, DHFR inhibitor methotrexate (MTX) while increasing the concentration of the culturing step is necessary, a high concentration of MTX-resistant clones are selected to obtain the cloned by time-consuming, general-purpose applicability to other cells and low problems discussed.
As mentioned above, gene expression techniques for to increase the efficiency, the development of various promoters and the like, has been improved. However the production of proteins in mammalian cell systems, such as E. coli or yeast as compared to other host in which it is difficult to obtain sufficient protein. Is in the field of biotechnology, these conventional techniques may also be used, depending on the type of gene type of cell, hardly occur in the gene expression, or a problem that an extremely small amount of the expressed protein is routinely generated. In addition, this problem is, gene expression in the development of a diagnostic or therapeutic medical used, are a major barrier.
For example, is HRG(Histidine-rich glycoprotein), year 1972 identified by Heimburger et al (1972) plasma protein of molecular weight of about 80 kDa. A total of 507 amino acids, wherein the histidine 66 and histidine-containing proteins present in high, mainly synthesized in the liver, as high as about 100-150μg/ml in human plasma at a concentration believed to be present. However, in order to study the clinical significance of HRG, by gene recombinant technology is a method of producing a sufficient amount of the HRG has been desired.
In addition, PD-1(Programmed cell death 1), EMMPRIN(extracellular matrix metalloproteinase inducer), NPTN β (neuroplastin β), EMB (embigin), RAGE(receptor for advanced glycation end products), MCAM(melanoma cell adhesion molecule), ALCAM(activated leukocyte cell adhesion molecule), proteins such as ErbB2(Receptor tyrosine-protein kinase erbB-2) is, to suppress the immune cells or expressed in a tumor cell are known, these proteins are the medical field and the research and development, pharmaceutical, pharmaceutical, diagnostic agent or reagent that can be used as a candidate for the considered important protein. For these proteins, with a sufficient amount of each protein by gene recombination technology has been desired a method of producing.
Scope of claims (In Japanese)[請求項1]
目的遺伝子及びポリA付加配列を含むDNA構築物(X)が、プロモーター(P)とエンハンサー(P')で挟まれる構造を有する遺伝子発現用カセットにおいて、さらにプロモーター(P)の上流及びエンハンサー(P')の下流にトランスポゾン配列(T)を含むことを特徴とする、遺伝子発現用カセット。
[請求項2]
さらに、プロモーター(P)の上流及び/又はエンハンサー(P')の下流に、複製開始配列(S)が配置されていることを特徴とする、請求項1に記載の遺伝子発現用カセット。
[請求項3]
プロモーター(P)、目的遺伝子及びポリA付加配列を含むDNA構築物(X)、エンハンサー(P')及びトランスポゾン配列(T)が含まれ、更に選択的に複製開始配列(S)を含む遺伝子発現用カセットが、以下の1)~4)のいずれかに示す順序で含まれる、請求項1又は2に記載の遺伝子発現用カセット:
1)(T)、(P)、(X)、(P')、(T);
2)(T)、(S)、(P)、(X)、(P')、(T);
3)(T)、(P)、(X)、(P')、(S)、(T);
4)(T)、(S)、(P)、(X)、(P')、(S)、(T)。
[請求項4]
複製開始配列(S)の上流に核マトリックス結合配列(M)が配置されていることを特徴とする請求項2又は3に記載の遺伝子発現用カセット。
[請求項5]
トランスポゾン配列(T)、プロモーター(P)、目的遺伝子及びポリA付加配列を含むDNA構築物(X)、エンハンサー(P')、核マトリックス結合配列(M)、複製開始配列(S)及びトランスポゾン配列(T)を含む、遺伝子発現用カセット。
[請求項6]
複製開始配列(S)が、ROIS及び/又はARSである請求項2~5のいずれかに記載の遺伝子発現用カセット。
[請求項7]
プロモーター(P)が、CMVプロモーター、CMV-iプロモーター、SV40プロモーター、hTERTプロモーター、βアクチンプロモーター及びCAGプロモーターからなる群から選択されるプロモーターである、請求項1~6のいずれかに記載の遺伝子発現用カセット。
[請求項8]
目的遺伝子及びポリA付加配列を含むDNA構築物(X)の下流に連結したエンハンサー(P')が、hTERTエンハンサー、CMVエンハンサー及びSV40エンハンサーから選択されるいずれか1種又は複数種を含む、請求項1~7のいずれかに記載の遺伝子発現用カセット。
[請求項9]
目的遺伝子及びポリA付加配列を含むDNA構築物(X)において、目的遺伝子が、HRG、PD-1、EMMPRIN、NPTNβ、EMB、RAGE、MCAM、ALCAM、ErbB2及び抗体のいずれかから選択されるタンパク質の部分又は全体をコードする遺伝子を含む、請求項1~8のいずれかに記載の遺伝子発現用カセット。
[請求項10]
請求項1~9のいずれかに記載の遺伝子発現用カセットを含む、遺伝子発現用プラスミド。
[請求項11]
請求項1~9のいずれかに記載の遺伝子発現用カセットを含む、遺伝子発現用ベクター。
[請求項12]
請求項1~9のいずれかに記載の発現用カセットを用いて目的遺伝子を発現させる方法。
[請求項13]
請求項1~9のいずれかに記載の遺伝子発現用カセットを用いて産生されたタンパク質。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
  • Inventor
  • SAKAGUCHI, Masakiyo
  • NISHIBORI, Masahiro
  • KUMON, Hiromi
  • MURATA, Hitoshi
  • YAMAMOTO, Kenichi
  • KINOSHITA, Rie
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 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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