Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: potential for gene therapy of hemophilia B.
Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: potential for gene therapy of hemophilia B.
复制标题
通过逆转录病毒介导的基因转移在兔肝细胞中表达人因子 IX：血友病 B 基因治疗的潜力。
DOI：
10.1073/pnas.87.16.6141
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发表时间：
1990
影响因子：
11.1
通讯作者：
Woo,SL
中科院分区：
文献类型：
--
作者：
Armentano,D;Thompson,AR;Darlington,G;Woo,SL
血友病B(圣诞节病)是一种染色体X连锁的凝血障碍，当因子IX缺乏或功能缺陷时会导致这种疾病。这种酶是在肝脏中合成的，这种遗传病的动物模型的存在将使旨在将功能基因转移到肝脏的体细胞基因治疗方案的开发成为可能。我们构建了一个基于氮气的重组逆转录病毒载体NCMVFIX，用于在原代兔肝细胞中高效转移和表达人凝血因子IX基因。在这个构建中，人巨细胞病毒即刻早期启动子指导因子IX的表达。分离3周龄新西兰大白兔肝细胞，感染重组病毒，分析活性因子IX的分泌情况。受感染的兔肝细胞产生的人凝血因子IX与正常人血浆中的酶难以区分。重组蛋白经过充分的伽马羧化，并且在凝血试验中具有功能活性。这些结果确立了利用感染的肝细胞表达该蛋白的可行性，并朝着通过肝脏基因转移纠正血友病B的目标迈进了一步。
Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. We report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently gamma-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.