Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome
Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome
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DOI：
10.1002/jgm.316
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发表时间：
2003-01-01
影响因子：
3.5
通讯作者：
Kim, S
中科院分区：
文献类型：
--
作者：
Hong, YT;Yu, SS;Kim, S
背景：亨特氏综合征是一种致死性溶酶体贮积性疾病，作为传统治疗方法的替代方法，我们开发了使用一系列逆转录病毒载体的基因传递载体。本研究的目的是开发一种安全有效的逆转录病毒载体，并优化使用我们的载体高效转导人骨髓CD34+干细胞的条件。方法构建3种基于mlv的逆转录病毒载体，表达Hunter's综合征患者缺乏的iduronate-2-sulfatase (IDS): MIN-IDS和MIN-IDS，分别与细菌neo和人MDR基因一起表达IDS， MT-IDS缺乏任何可选择的标记物。从包装线PG13衍生出不同的生产者线，并比较病毒滴度和基因表达水平。经过比较，我们使用逆转录病毒载体MT-IDS在不同MOIs下将人骨髓CD34+干细胞转导到纤维连接蛋白上。结果相比之下，MT-IDS不仅产生最高的病毒滴度（接近10(7)cfu/ml），而且在各种转导实验和RNA分析中也显示出最高的基因表达水平。当1.5 × 10(5)人CD34+骨髓细胞在最优MOIs下用MT-IDS转导时，约80%的总集落形成单位含有IDS cDNA。结论不含选择性标记物和病毒编码序列的小体积逆转录病毒载体可驱动基因高水平表达，从产线高效生产，并能高频率进入造血细胞。我们的数据表明，在利用人类CD34+干细胞作为靶细胞的亨特氏综合征基因治疗试验中，使用基于mt的载体具有巨大的潜力。版权所有:John Wiley Sons, Ltd。
Background As an alternative method to the conventional therapies for Hunter's syndrome, which is a lethal lysosomal storage disorder, we have developed gene delivery vehicles using a series of retroviral vectors. The objective of this study was to develop a safe and efficient retroviral vector and to optimize conditions for efficient transduction of human bone marrow CD34+ stem cells using our vector.Methods We constructed three types of MLV-based retroviral vectors expressing iduronate-2-sulfatase (IDS) which is deficient in patients suffering from Hunter's syndrome: MIN-IDS and MIM-IDS, which express IDS along with bacterial neo and human MDR genes, respectively, and MT-IDS lacking any selectable marker. Respective producer lines were derived from the packaging line, PG13, and compared for viral titer and levels of gene expression. After comparing, the retroviral vector, MT-IDS, was used to transduce human bone marrow CD34+ stem cells on fibronectin under various MOIs.Results In comparison, MT-IDS not only produced the highest viral titer (close to 10(7) cfu/ml), but also showed the highest level of gene expression in various transduction assays and RNA analysis. When 1.5 x 10(5) human CD34+ bone marrow cells were transduced with MT-IDS under the most optimal MOIs, about 80% of total colony forming units were shown to contain the IDS cDNA.Conclusions Minimum-sized retroviral vector that contains no selective marker as well as a viral coding sequences could drive a high level of gene expression, be produced efficiently from the producer line, and enter hematopoietic cells at a high frequency. Our data suggest the great potential for using MT-based vector(s) in a gene therapy trial for Hunter's syndrome utilizing human CD34+ stem cells as target cells. Copyright (C) 2002 John Wiley Sons, Ltd.