Dose finding with retroviral vectors:: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population
Dose finding with retroviral vectors:: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population
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DOI：
10.1182/blood-2003-05-1424
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发表时间：
2003-12-01
期刊：
影响因子：
20.3
通讯作者：
Fehse, B
中科院分区：
文献类型：
--
作者：
Kustikova, OS;Wahlers, A;Fehse, B
逆转录病毒载体常用于临床基因治疗，但最近观察到的插入癌基因激活在临床前和临床设置迫使其安全性的讨论。在这里，我们使用K562白血病和原代CD 34（+）细胞研究了大规模培养中逆转录病毒转导效率与单细胞中整合载体拷贝的实际数量之间的关系。我们发现整合数的指数增加与基因转移率和线性增加的表达水平与插入频率。平均而言，我们在每个转导细胞中检测到一个载体插入，基因转移小于30%，60%为3个，90%为约9个（在K562中）。克隆分析显示，转基因拷贝数（在原代细胞中超过20倍）和表达水平的变化显着增加与更高的转导。因此，可以建议将逆转录病毒基因转移限制在约30%，以避免产生含有多个插入的克隆。(C)2003年，美国血液学会。
Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34(+) cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions. (C) 2003 by The American Society of Hematology.