A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors
A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors
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DOI：
10.1038/sj.gt.3301587
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发表时间：
2001-11-01
期刊：
影响因子：
5.1
通讯作者：
Wei, MQ
中科院分区：
文献类型：
--
作者：
Zhang, B;Xia, HQ;Wei, MQ
用水泡性口炎病毒糖蛋白（VSV-G）假型化的慢病毒载体正在成为体外和体内基因治疗研究的首选载体。然而，目前收获慢病毒载体的方法依赖于在50 000 g下超离心2 h。在这种超高速下，目前使用的转子通常具有小的体积容量。因此，制备大量的高滴度载体是费时费力的。在本研究中，从载体产生细胞（VPC）收获的病毒载体上清液用不同量的聚-L-赖氨酸（PLL）预处理并通过低速离心浓缩。当将0.005%的PLL（w/v）加入到载体上清液收获物中，随后孵育30 min并在4 ℃下以10 000 g离心2 h时，建立了最佳条件。与超离心法的直接比较表明，新方法在一轮浓缩中以1 × 10（8）转导单位（TU）/ml（来自约3000 ml上清液）持续产生更大体积（6 ml）的高滴度病毒载体。电子显微镜分析表明，PLL/病毒载体形成复合物，这可能有助于在低速浓度（10 000 g），通常不会有效沉淀病毒颗粒的速度容易沉淀。用慢病毒载体/PLL复合物在体外转染几种细胞系并在肝脏中体内转导，证明了有效的基因转移，而没有任何明显的毒性迹象。这些结果表明，新方法提供了一种方便的手段，收获大量的高滴度慢病毒载体，促进基因治疗实验中的大型动物或人类基因治疗试验，其中大量的慢病毒载体是先决条件。
Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.