Clinical-scale lentiviral vector transduction of PBL for TCR gene therapy and potential for expression in less-differentiated cells.
Clinical-scale lentiviral vector transduction of PBL for TCR gene therapy and potential for expression in less-differentiated cells.
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DOI：
10.1097/cji.0b013e31818817c5
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发表时间：
2008-11
期刊：
影响因子：
--
通讯作者：
Morgan RA
中科院分区：
文献类型：
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作者：
Yang S;Rosenberg SA;Morgan RA
在人类基因治疗应用中，慢病毒载体可能比伽马逆转录病毒载体更具优势，因为它们具有转导未分裂细胞的能力，对基因沉默的抗性，以及缺乏整合位点的偏好。在本研究中，我们利用携带特异性抗肿瘤T细胞受体(TCR)的VSV-G伪型第三代慢病毒载体，建立了临床规模的慢病毒转导PBL的方法。与基于RetroNectin的方法相比，在鱼精蛋白硫酸盐存在下接种(1000×g，32℃，2小时)是最有效和最经济的转导大量PBL的方法。在一个6孔板中，每孔高达2000万个细胞被有效地转导，并在两周内经历了平均50倍的扩张。TcR转导的PBL介导的特异性抗肿瘤活性包括干扰素-γ的释放和细胞裂解。与伽马逆转录病毒载体相比，TCR转基因可以在分化程度较低的细胞群体中优先表达。
In human gene therapy applications, lentiviral vectors may have advantages over gamma-retroviral vectors because of their ability to transduce non-dividing cells, their resistance to gene silencing, and a lack of integration site preference. In this study, we utilized VSV-G pseudotype third generation lentiviral vectors harboring specific anti-tumor T-cell receptor (TCR) to establish clinical-scale lentiviral transduction of PBL. Spinoculation (1000 × g, 32°C for 2 h) in the presence of protamine sulfate represents the most efficient and economical approach to transduce a large number of PBLs compared to RetroNectin-based methods. Up to 20 million cells per well of a 6-well plate were efficiently transduced and underwent an average 50-fold expansion in two weeks. TCR transduced PBL mediated specific anti-tumor activities including IFN-γ release and cell lysis. Compared to gamma-retroviral vectors, the TCR transgene could be preferentially expressed on a less-differentiated cell population.