Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors
Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors
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DOI：
10.1038/sj.gt.3301653
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发表时间：
2002-04-01
期刊：
影响因子：
5.1
通讯作者：
Klingmüller, U
中科院分区：
文献类型：
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作者：
Ketteler, R;Glaser, S;Klingmüller, U
肿瘤逆转录病毒载体已成功地应用于基因治疗试验，但低转导率和转基因表达的损失仍然是其应用的主要障碍。为了克服这些问题，我们通过用脾病灶形成病毒LTR替换3' LTR并插入土拨鼠肝炎B病毒翻译后调节元件来修饰广泛使用的莫洛尼鼠白血病病毒衍生的逆转录病毒载体pMX。为了比较高效转基因表达的关键要求，我们产生了杂合逆转录病毒载体pMOWS和pOWS，其具有完整的鼠胚胎干细胞病毒（MESV）前导序列或不包含引物结合位点（PBS）和剪接供体（SD）的缩短的MESV前导序列。应用这些逆转录病毒载体显着增强造血细胞系和祖细胞中的转基因表达。对于鼠胚胎干（ES）细胞的转导，携带MESV-PBS和-SD的逆转录病毒载体pMOWS是上级的，导致65%的表达绿色荧光蛋白（GFP）的ES细胞。令人惊讶的是，在鼠和人原始造血祖细胞（HPC）中，用pOWS实现了高达66%的GFP表达细胞的最高效率，pOWS是一种保留与MoMuLV-PBS一致的负调控元件的逆转录病毒载体。总之，我们的杂交逆转录病毒载体促进多能细胞中显著改善的转基因表达，因此具有在疾病模型的原代细胞中重建基因以及用于基因治疗的巨大潜力。
Oncoretroviral vectors have been successfully used in gene therapy trials, yet low transduction rates and loss of transgene expression are still major obstacles for their application. To overcome these problems we modified the widely used Moloney murine leukemia virus-derived retroviral vector pMX by replacing the 3' LTR with the spleen focus-forming virus LTR and inserting the woodchuck hepatitis B virus post-translational regulatory element. To compare requirements crucial for efficient transgene expression, we generated the hybrid retroviral vectors pMOWS and pOWS that harbor the complete murine embryonic stem cell virus (MESV)-leader sequence or a shortened MESV-leader not comprising primer binding site (PBS) and splice donor (SD). Applying these retroviral vectors significantly augmented transgene expression in hematopoietic cell lines and pro- genitor cells. For transduction of murine embryonic stem (ES) cells the retroviral vector pMOWS that harbors the MESV-PBS and -SD was superior resulting in 65% green fluorescent protein (GFP) expressing ES cells. Surprisingly, in murine and human primitive hematopoietic progenitor cells (HPC), the highest efficiency of up to 66% GFP expressing cells was achieved with pOWS, a retroviral vector that retains the negative regulatory element coinciding with the MoMuLV-PBS. In summary our hybrid retroviral vectors facilitate significantly improved transgene expression in multipotent cells and thus possess great potential for reconstituting genes in primary cells of disease models, as well as for gene therapy.