Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells
Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells
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DOI：
10.1016/s1525-0016(03)00104-7
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发表时间：
2003-06-01
影响因子：
12.4
通讯作者：
Cheng, LZ
中科院分区：
文献类型：
--
作者：
Yu, XB;Zhan, XC;Cheng, LZ
慢病毒载体与传统的肿瘤逆转录病毒载体相比有几个优点。LVS有效地转导缓慢分裂的细胞，包括造血干细胞(HSCs)，从而实现稳定的基因转移和表达。此外，最近发展起来的自失活(SIN)LV允许启动子特异性的转基因表达。对于许多基因转移应用，需要转导多个基因。我们在尝试在单个双顺反子LV转录本中共表达由内部核糖体进入位点(IRES)元件连接的两个转基因时，得到了不一致的结果。在我们构建的包含感兴趣基因、IRES和GFP报告基因的六个以上的双顺反子LV中，在转导的细胞中没有检测到GFP荧光。因此，我们研究了如何通过LV实现两个转基因的一致和高效的共表达。在含有延伸因子1α启动子的SIN LV中，我们包括来自巨细胞病毒的第二个启动子，即磷酸甘油酸激酶基因，或人类白细胞抗原-DRα基因。使用含有两个构成启动子的单个LV，我们实现了两种转基因在转导的CD34(+)HSCs及其后代中以及在其他类型的人类细胞中的高效和持续表达。因此，这种双启动子LV可以在单个靶细胞中高效地共表达多个转基因，并将使多种基因转移应用成为可能。
Lentiviral vectors (LVs) offer several advantages over traditional oncoretroviral vectors. LVs efficiently transduce slowly dividing cells, including hematopoietic stem-progenitor cells (HSCs), resulting in stable gene transfer and expression. Additionally, recently developed self-inactivating (SIN) LVs allow promoter-specific transgene expression. For many gene transfer applications, transduction of more than one gene is needed. We obtained inconsistent results in our attempts to coexpress two transgenes linked by an internal ribosomal entry site (IRES) element in a single bicistronic LV transcript. In more than six bicistronic LVs we constructed containing a gene of interest followed by an IRES and the GFP reporter gene, GFP fluorescence was undetectable in transduced cells. We therefore investigated how to achieve consistent and efficient coexpression of two transgenes by LVs. In a SIN LV containing the elongation factor 1alpha promoter, we included a second promoter from cytomegalovirus, the phosphoglycerate kinase gene, or the HLA-DRalpha gene. Using a single LV containing two constitutive promoters, we achieved strong and sustained expression of both transgenes in transduced engrafting CD34(+) HSCs and their progeny, as well as in other human cell types. Thus, such dual-promoter LVs can coexpress multiple transgenes efficiently in a single target cell and will enable many gene transfer applications.