The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes
The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes
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DOI：
10.1038/sj.gt.3300506
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发表时间：
1997-10-01
期刊：
影响因子：
5.1
通讯作者：
Sadelain, M
中科院分区：
文献类型：
--
作者：
Gallardo, HF;Tan, C;Sadelain, M
对于过继细胞疗法的改进至关重要的是高效地产生大量的人类原代II细胞，这些细胞可靠地表达与药物敏感性有关的自杀基因，如单纯疱疹病毒胸苷激酶(HSVtk)。我们在这里展示了一个包含脑心肌炎病毒(EMCV)内部核糖体进入位点(IRES)的优化双顺反子载体在每个细胞平均携带一个整合载体拷贝的人初级II淋巴细胞中具有功能。我们证明了标记NTP和HSVtk的可靠共表达，NTP是人类低亲和力神经生长因子受体的非活性突变体。在双顺反子载体NIT中，NTP表达为帽子依赖的标记，HSVtk表达为非选择的IRES依赖基因。NTP在细胞表面的表达足以使转导细胞高效、快速地浓缩到高纯度。在这些纯化的淋巴细胞中，分别有97+/-4%和92+/-6%的淋巴细胞在1.0和0.1mU M更昔洛韦的存在下被选择性地消除，这表明EMCV IRES确保了两个基因在人原代T细胞中的高效和充分的表达。
It is essential for the improvement of adoptive cell therapies to generate efficiently large populations of human primary II cells that reliably express a suicide gene conferring drug sensitivity, such as herpes simplex virus thymidine kinase (HSVtk). We show here that an optimized dicistronic vector containing the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) is functional in human primary II lymphocytes that bear on average one integrated vector copy per cell. We demonstrate reliable coexpression of the marker NTP, an inactive mutant of the human low-affinity nerve growth factor receptor, and HSVtk. In the dicistronic Vector NIT, NTP is expressed as a cap-dependent marker and HSVtk as a nonselectable IRES-dependent gene. Cell-surface expression of NTP is sufficient to allow for the efficient and rapid enrichment of the transduced cells to high purity. Of these purified lymphocytes, 97 +/- 4% and 92 +/- 6% are selectively eliminated when cultured in the presence of 1.0 or 0.1 mu M ganciclovir, respectively, establishing that the EMCV IRES ensures efficient and sufficient expression of two genes in human primary T cells.