| Project/Area Number |
08044128
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | TOKYO INSTITUTE OF TECHNOLOGY |
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Principal Investigator |
AKAIKE Toshihiro TOKYO INSTITUTE OF TECHNOLOGY,DEPARTMENT OF BIOMOLECULAR ENGINEERING,PROFESSOR, 生命理工学部, 教授 (30101207)
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| Co-Investigator(Kenkyū-buntansha) |
STEPHENE Benedict UNIVERSITY OF KANSAS,DEPARTMENT OF MICROBIOLOGY,ASSOCIATE PROFESSOR, Department of Micro, Associate
KIM Sung Wan UNIVERSITY OF UTAH,CENTER FOR CONTROLLED CHEMICAL DELIVERY,PROFESSOR AND DIRECTO, Center for Controlled, Director a
MARUYAMA Atsushi TOKYO INSTITUTE OF TECHNOLOGY,DEPARTMENT OF BIOMOLECULAR ENGINEERING,ASSISTANT P, 生命理工学部, 助手 (40190566)
BENEDICT Ste Department of Microbiology, University of, Associate
JOZEFOWICZ M Univ. of ParisーNord, 教授
YOU Han Bae Kwangju Inst. of Tech., 教授
水野 喬介 財団法人, 化学及び血清療法研究所, 部長
山田 修平 信州大学, 医学部, 医局員
KIM JinーSeok ユタ大学, 薬学部, 助手
SUNG Wan Kim ユタ大学, 薬学部, 教授
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1997: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1996: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | DNA / low density lipoprotein / polylysine / smooth muscle cells / gene therapy / Transfection / 平滑筋細胞 / 低密度リポタンパク / 遺伝子キャリヤ- / LDL / 血管平滑筋細胞 / グラフト共重合体 / 3重鎖DNA / 肝類洞内皮細胞 |
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| Research Abstract |
In vitro gene expression was studied using a teplex gene delivery system consisting of plasmid DNA,low density lipoprotein, and hydrophobized poly-L-lysine on A7R5 murine smooth muscle cells (SMC). A positively charged giodegradable polymer with hydrophobic side chain (25 mol % of C18-stearyl group) was synthesized using poly-L-lysine as a backbone. A terplex system was formed from various weight ratios of DNA/H-PLL/LDL and its in vitro trahsfection efciency was tested on SMC.The terplex system showed a 2-to 5-fold increase in transfection efficiency of plasmid DNA on A7R5 cells in the presence of serum, compared to the complex of DNA with H-PLL,or DNA with LipofectinTM.Transfection efficacy of the terplex system in the absence of serum also showed a 1.5-fold increase, comopared to LipofectinTM formulation. Pretreatment of the cells with 100 micro M chloroquine for 30 min prior to the transfection resulted in a 30% increase in transfection efficiency of the terplex system. Flow cytometric analysis indicates that DNA should be bound to H-PLL for efficient cellular binding and uptake, and LDL helps internalization of the terples via receptor-mediated endocytosis. The association and/or internalization of the DNA terplex system into cells was inhibited either by the presence of 100 micro M EDTA or excess amount of free LDL,suggesting that LDL plays an important role in the endocytosis of the terplex system by a receptor-mediated fashion. Considerable cytotoxicity due to the H-PLL was not observed at the concentration range used for this experiment when H-PLL was complexed with LDL.In conclusion, this result indicates that a significant degree of exogenous gene delivery and its intracellular expression was achieved by the H-PLL/DNA/LDL terplex system, providing useful information for the development of efficient and safe gene delivery vectors for in vivo gene therapy.
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