项目名称： hFIXm-Fc融合基因修饰间充质干细胞治疗血友病B小鼠的研究

项目编号： No.81500108

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 严红

作者单位： 上海交通大学

项目金额： 18万元

中文摘要： 血友病B是凝血因子IX（FIX）单基因缺失引发的遗传性出血性疾病，临床上主要以FIX替代治疗为主，患者需终身频繁输注FIX制品。基因治疗为血友病B提供了一种治愈可能，但常因FIX表达水平低影响治疗效果。本申请拟利用突变型人凝血因子IX与Fc片段（hFIXm-Fc）融合表达的方法，同时增加FIX活性与半衰期，以改善血友病B基因治疗中FIX表达水平低的现状。我们拟用hFIXm-Fc融合基因修饰的血友病B小鼠骨髓间充质干细胞对血友病B小鼠实施治疗，综合应用多种技术，检测hFIXm-Fc融合蛋白在治疗小鼠体内的表达、分布、生物活性和半衰期等指标，评价hFIXm-Fc融合基因用于基因治疗的可行性；并与野生型hFIX基因相比较对血友病B小鼠表型的改善作用，分析hFIXm-Fc融合基因是否增加FIX的表达水平。本研究将为Fc融合表达应用于基因治疗和血友病B基因治疗新策略的探索提供理论与技术基础。

中文关键词： 血友病B；高凝血活性突变型hFIX；hFIXm-Fc融合基因；间充质干细胞

英文摘要： Hemophilia B is a hemorrhagic hereditary disease characterized by the single gene deletion of clotting factor IX (FIX). The current treatment for hemophilia B is based on replacement therapy using plasma derived or recombinant FIX. Hemophilia B patients require frequent intravenous injections of FIX products through their lifetime. Gene therapy is a promising option for curing hemophilia B, but the low expression level affects its therapeutic outcome. This project aims to use mutant hFIX(hFIXm) fusion express with the FC fragment to simultaneously increase hFIX activity and half-life, thus improve the low expression level of FIX in current gene therapy in treating hemophilia B. We will firstly transfect the MSCs of hemophilia B mice with lentivirus expressing hFIXm-Fc gene. Then, we will inject the modified MSCs back to hemophilia B mice to evaluate the therapeutic effects. We will apply the technologies of cell biology, molecular biology, and hematology to demonstrate the feasibility of in vivo expression of hFIX-FC fusion protein on individual animal level. We will analyze the in vivo expression level, distribution, bioactivity and half-life of hFIX-Fc protein. All these efforts will demonstrate the feasibility and safety of in vivo expression of hFIX-FC fusion protein for gene therapy. We will evaluated the beneficial effect of hFIX-FC fusion protein by setting the negative control group (MSCs of hemophilia B model mouse with lentivirus expressing wild type hFIX gene). This will demonstrate MSCs transfected with lentivirus expressing hFIXm-Fc gene can improve the in vivo expression level of FIX. This project will also provide theoretical and technical foundations for applying FC fusion expression in gene therapy and exploring new therapeutic approaches of hemophilia B.

英文关键词： Hemophilia B;high blood clotting activity hFIX mutant;hFIXm-Fc fusion gene;mesenchymal stem cells