Systematic determination of the packaging limit of lentiviral vectors
Systematic determination of the packaging limit of lentiviral vectors
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DOI：
10.1089/104303401753153947
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发表时间：
2001-10-15
影响因子：
4.2
通讯作者：
Sutton, RE
中科院分区：
文献类型：
--
作者：
Kumar, M;Keller, B;Sutton, RE
基于人类免疫缺陷病毒1型（HIV）的载体具有转导非分裂细胞的能力，因此在将基因治疗性递送到细胞中具有很大的潜力。我们在这里描述了一个系统的研究艾滋病毒为基础的载体包装限制。在慢病毒载体的三个不同位置克隆了限制性内切酶产生的不同长度的细菌染色体DNA片段。制备了水疱性口炎病毒G蛋白（VSV G）假型慢病毒颗粒，并在哺乳动物细胞上进行了不同克隆的滴度测定。我们观察到基因组5‘和3’端的限制性内切酶位点位置对于外源DNA的插入能力是优越的。在所有情况下，病毒滴度随着载体长度的增加呈半对数递减。似乎没有绝对的包装限制，因为即使原病毒长度超过18kb，也可以获得可测量的滴度。滴度的降低似乎发生在病毒囊化水平，尽管我们不能排除原RNA核输出的限制。这些结果表明，基于艾滋病毒的载体可能比肿瘤逆转录病毒载体具有次要优势，因为它们具有更大的包装限制，尽管大型载体的极低滴度的效用有限。
Because of their ability to transduce nondividing cells, human immunodeficiency virus type 1 (HIV)-based vectors have great potential for the therapeutic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated bacterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the different clones were titered on mammalian cells. We observed that the restriction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The reduction in titer appears to occur at the level of viral encapsidation, although we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although the very low titers of the larger vectors will be of limited utility.