Zhang B, Xia H Q, Cleghorn G, Gobe G, West M, Wei M Q
Department of Medicine, Prince Charles Hospital, Brisbane, Queensland, Australia.
Gene Ther. 2001 Nov;8(22):1745-51. doi: 10.1038/sj.gt.3301587.
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 degrees C. 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 3,000 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.
用泡状口炎病毒糖蛋白（VSV-G）假型化的慢病毒载体正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50,000g下超速离心2小时。在这种超高速下，目前使用的转子通常容量较小。因此，制备大量高滴度载体既耗时又费力。在本研究中，从载体生产细胞（VPCs）收获的病毒载体上清液用不同量的聚-L-赖氨酸（PLL）进行预处理，并通过低速离心进行浓缩。当向载体上清液收获物中加入0.005%（w/v）的PLL，随后孵育30分钟并在4℃下以10,000g离心2小时时，确定了最佳条件。与超速离心的直接比较表明，新方法在一轮浓缩中始终能产生更大体积（6ml）的高滴度病毒载体，滴度为1×10⁸转导单位（TU）/ml（来自约3000ml的上清液）。电子显微镜分析表明，PLL/病毒载体形成了复合物，这可能有助于在低速浓缩（10,000g）时容易沉淀，而这种速度通常不能有效地沉淀病毒颗粒。用慢病毒载体/PLL复合物在体外转染几种细胞系并在体内肝脏中转导，证明了有效的基因转移且没有任何明显的毒性迹象。这些结果表明，新方法为收获大量高滴度慢病毒载体提供了一种方便的手段，有助于在大型动物或人类基因治疗试验中进行基因治疗实验，在这些试验中大量慢病毒载体是一个先决条件。
