A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors.
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.
Zhang
B
B
Department of Medicine, Prince Charles Hospital, Brisbane, Queensland, Australia.
Xia
H Q
HQ
Cleghorn
G
G
Gobe
G
G
West
M
M
Wei
M Q
MQ
D000818:Animals; D002412:Cations; D002460:Cell Line; D002498:Centrifugation; D005822:Genetic Vectors / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011107:Polylysine; D014161:Transduction, Genetic / Q000379:methods; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation
D002412:Cations; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011107:Polylysine