High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells.
High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells.
复制标题
人脐带血干细胞慢病毒转导后高水平的β-珠蛋白表达和优选的基因内整合。
DOI：
10.1172/jci21838
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Humphries,RKeith
中科院分区：
文献类型：
--
作者：
Imren,Suzan;Fabry,MaryE;Westerman,KarenA;Pawliuk,Robert;Tang,Patrick;Rosten,PatriciaM;Nagel,RonaldL;Leboulch,Philippe;Eaves,ConnieJ;Humphries,RKeith
遗传校正的自体造血干细胞移植是治疗镰状细胞病和β-地中海贫血的一种有吸引力的方法。在这里，我们用编码抗镰状化β A-T87 Q-珠蛋白转基因的自失活慢病毒载体感染人脐带血细胞，并分析了将感染细胞直接移植到亚致死剂量照射的NOD/LtSz-scid/scid小鼠后6个月内产生的转导后代。大约一半的人红系和髓系祖细胞在含有转基因的小鼠中再生，并且从这些体内再生细胞体外衍生的红系细胞产生高水平的β A-T87 Q-珠蛋白。接头介导的PCR分析在所有分析的小鼠中鉴定了多个转基因阳性克隆，每个细胞具有2.1 ± 0.1个整合的前病毒拷贝。对含有载体片段的基因组测序显示，86%的前病毒插入物发生在基因内，包括与人类白血病有关的几个基因。这些发现表明用候选治疗性慢病毒载体有效转导非常原始的人脐带血细胞，导致治疗相关水平的β-珠蛋白蛋白的长期和稳健的红细胞特异性产生。然而，前病毒整合在调节造血的基因内的频率表明需要额外的安全性修饰。
Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and β-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling βA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scidmice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo–regenerated cells produced high levels of βA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 ± 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of β-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.