Huang Xin, Wilber Andrew, McIvor R Scott, Zhou Xianzheng
Department of Pediatrics, The Cancer Center, University of Minnesota, Minneapolis, MN, USA.
Methods Mol Biol. 2009;506:115-26. doi: 10.1007/978-1-59745-409-4_9.
Genetic modification of peripheral blood T lymphocytes (PBL) or hematopoietic stem cells (HSC) has been shown to be promising in the treatment of cancer (Nat Rev Cancer 3:35-45, 2003), transplant complications (Curr Opin Hematol 5:478-482, 1998), viral infections (Science 285:546-551, 1999), and immunodeficiencies (Nat Rev Immunol 2:615-621, 2002). There are also significant implications for the study of T cell biology (J Exp Med 191:2031-2037, 2000). Currently, there are three types of vectors that are commonly used for introducing genes into human primary T cells: oncoretroviral vectors, lentiviral vectors, and naked DNA. Oncoretroviral vectors transduce and integrate only in dividing cells. However, it has been shown that extended ex vivo culture, required by oncoretroviral-mediated gene transfer, may alter the biologic properties of T cells (Nat Med 4:775-780, 1998; Int Immunol 9:1073- 1083, 1997; Hum Gene Ther 11:1151-1164, 2001; Blood 15:1165-1173, 2002; Proc Natl Acad Sci U S A, 1994). HIV-1-derived lentiviral vectors have been shown to transduce a variety of slowly dividing or nondividing cells, including unstimulated T lymphocytes (Blood 96:1309-1316, 2000; Gene Ther 7:596-604, 2000; Blood 101:2167-2174, 2002; Hum Gene Ther 14:1089-1105, 2003). However, achieving effective gene transfer and expression using lentivirus vectors can be complex, and there is at least a perceived risk associated with clinical application of a vector based on a human pathogen (i.e., HIV-1). Recently it has been found that oncoretroviral and lentiviral vectors show a preference for integration into regulatory sequences and active genes, respectively (Cell 110:521-529, 2002; Science 300:1749-1751, 2003). Additionally, insertional mutagenesis has become a serious concern, after several patients treated with an oncoretroviral vector for X-linked SCID developed a leukemia-like syndrome associated with activation of the LMO2 oncogene (Science 302:415-419, 2003). Naked DNA-based genetic engineering of human T lymphocytes also requires T cells to be activated prior to gene transfer (Mol Ther 1:49-55, 2000; Blood 101:1637-1644, 2003; Blood 107:2643-2652, 2006). In addition, random integration by electroporation is of low efficiency. We have recently reported that the Sleeping Beauty transposon system can efficiently mediate stable transgene expression in human primary T cells without prior T cell activation (Blood 107:483-491, 2006). This chapter describes methodology for the introduction of SB transposons into human T cell cultures with subsequent integration and stable long-term expression at noticeably high efficiency for a nonviral gene transfer system.
对外周血T淋巴细胞（PBL）或造血干细胞（HSC）进行基因改造已被证明在癌症治疗（《自然综述：癌症》3:35 - 45，2003年）、移植并发症（《血液学当前观点》5:478 - 482，1998年）、病毒感染（《科学》285:546 - 551，1999年）以及免疫缺陷治疗（《自然综述：免疫学》2:615 - 621，2002年）方面颇具前景。这对于T细胞生物学研究也具有重要意义（《实验医学杂志》191:2031 - 2037，2000年）。目前，有三种常用于将基因导入人原代T细胞的载体：致癌逆转录病毒载体、慢病毒载体和裸DNA。致癌逆转录病毒载体仅在分裂细胞中进行转导和整合。然而，研究表明，致癌逆转录病毒介导的基因转移所需的延长体外培养可能会改变T细胞的生物学特性（《自然医学》4:775 - 780，1998年；《国际免疫学》9:1073 - 1083，1997年；《人类基因治疗》11:1151 - 1164，2001年；《血液》15:1165 - 1173，2002年；《美国国家科学院院刊》，1994年）。源自HIV - 1的慢病毒载体已被证明可转导多种缓慢分裂或不分裂的细胞，包括未受刺激的T淋巴细胞（《血液》96:1309 - 1316，2000年；《基因治疗》7:596 - 604，2000年；《血液》101:2167 - 2174，2002年；《人类基因治疗》14:1089 - 1105，2003年）。然而，使用慢病毒载体实现有效的基因转移和表达可能较为复杂，并且基于人类病原体（即HIV - 1）的载体临床应用至少存在一种可感知的风险。最近发现，致癌逆转录病毒载体和慢病毒载体分别倾向于整合到调控序列和活性基因中（《细胞》110:521 - 529，2002年；《科学》300:1749 - 1751，2003年）。此外，在几名接受致癌逆转录病毒载体治疗X连锁重症联合免疫缺陷的患者出现与LMO2癌基因激活相关的白血病样综合征后，插入诱变已成为一个严重问题（《科学》302:415 - 419，2003年）。基于裸DNA的人T淋巴细胞基因工程也要求在基因转移前激活T细胞（《分子治疗》1:49 - 55，2000年；《血液》101:1637 - 1644，2003年；《血液》107:2643 - 2652，2006年）。此外，通过电穿孔进行的随机整合效率较低。我们最近报道，睡美人转座子系统可在无需预先激活T细胞的情况下，高效介导人原代T细胞中的稳定转基因表达（《血液》107:483 - 491，
