Investigation on gene therapy for congenital bleeding tendency

先天性出血倾向的基因治疗研究

• 批准号: 03454523
• 负责人: SAITO Hidehiko
• 金额: $ 3.58万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454523/
• 关键词: factor IX; expression vector; retroviral vector; pL9RNL; gene therapy; VII因子; レトロウイルスベクター; 第IX因子; レトロウイルスベクタ-

We constructed a new factor IX expression vector containing a full length factor IX cDNA.The Moloney Murine Leukemia Virus (MoMLV)-based retroviral vector pLRNL was cloned with the entire coding region of human factor IX down stream of the promoter of Rous sarcoma virus, givinh rise to the construct pL9RNL.The GP+E 86 packaging cell was transfected with pL9RNL and two mouse fibroblast cell lines (PA317 and NIH3T3) were infected with the virus generated from PA317 cell. During the 24 hr culture period, the maximum 1.2mug of factor IX was secreted into the medium from 10^6 of GP+E 86 cells. Factor IX in the culture media had the relatively normal procoagulant activity and was barium-citrate precipitated. Western blotting analysis of the barium-citrate precipitated revealed that a single chain 50Kd protein indistinguishable from the plasma-derived factor IX was produced in these three cells. The retroviral expression system that we utilized herein may contribute to the study of recombinant wild type or various mutant factor IX,and to the basic study for the future gene therapy.

构建了含有人凝血因子IX全长cDNA的表达载体pLRNL，将人凝血因子IX的全长编码区克隆到Rous肉瘤病毒启动子下游，构建成pL9RNL逆转录病毒载体pLRNL。在24小时培养期间，10^6的GP+E86细胞分泌的因子IX最大值为1.2mug。培养上清液中的凝血因子IX具有相对正常的促凝活性，且为柠檬酸钡沉淀物。免疫印迹分析显示，在这三种细胞中都产生了与血浆衍生因子IX难以区分的单链50Kd蛋白。我们所利用的逆转录病毒表达系统为重组野生型或各种突变型因子IX的研究奠定了基础，也为今后的基因治疗奠定了基础。

项目成果

Matsushita, T.: "Construction and its expression of a new retroviral vector containing a human blood coagulation factor XI cDNA." Thromb Res in press.

Matsushita, T.：“含有人凝血因子 XI cDNA 的新型逆转录病毒载体的构建及其表达。”

Saito, H.et al: "Construction and its expression of a new retroviral vector containing a human blood coagulation factor IX cDNA." Thromb Res. 69. 387-393 (1993)

Saito, H.等人：“含有人凝血因子 IX cDNA 的新型逆转录病毒载体的构建及其表达。”

Yamamoto, K.: "Impaired secretion of the elongated mutant of protein C(protein C-Nagoya): Molecular and cellular basis for hereditary protein C deficiency." J Clin Invest. 90. 2439-2446 (1992)

Yamamoto, K.：“蛋白 C 延长突变体（蛋白 C-名古屋）的分泌受损：遗传性蛋白 C 缺乏症的分子和细胞基础。”

Ichinose, A.: "Two types of abnormal genes for plasminogen in families with a predisposition for thrombosis." Proc Natl Acad Sci USA. 88. 115-119 (1991)

Ichinose, A.：“有血栓形成倾向的家族中有两种类型的纤溶酶原异常基因。”

Yamamoto, K.: "Homozygous protein C deficiency: identification of a novel missense mutation that causes impaired secretion of the mutant protein C." J Lab Clin Med. 119. 682-689 (1992)

Yamamoto, K.：“纯合蛋白 C 缺陷：鉴定出一种新的错义突变，该突变会导致突变蛋白 C 的分泌受损。”