Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector
Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector
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DOI：
10.1038/nbt957
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发表时间：
2004-05-01
影响因子：
46.9
通讯作者：
Vignali, DAA
中科院分区：
文献类型：
--
作者：
Szymczak, AL;Workman, CJ;Vignali, DAA
为基因治疗和生物医学研究生成表达多种基因的可靠和通用载体的尝试取得了有限的成功。在这里，我们使用小核糖核酸病毒的“自我切割”2A肽，或来自其他病毒的2A样序列（1-3），产生多顺反子逆转录病毒载体，有效翻译四个顺反子。使用t细胞受体：CD3复合物作为测试系统，我们发现单个2A肽连接的逆转录病毒载体可用于生成所有四种CD3蛋白（CD3epsilon, gamma, delta, zeta），并恢复CD3缺陷小鼠的t细胞发育和功能。我们还展示了使用基于荧光共振能量转移的系统在多种细胞类型（包括血液、胸腺、脾脏、骨髓和早期干细胞祖细胞）中完成2A肽介导的“切割”和两种荧光蛋白的化学计量生产。
Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses(1-3), to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor: CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.