Adeno-Associated Viruses Serotype 2-Mediated RNA Interference Efficiently Inhibits Rabies Virus Replication In Vitro and In Vivo

WU, Hong-Xia; WANG, Hua-Lei; GUO, Xiao-Feng; YANG, Yu-Jiao; MA, Jin-Zhu; WANG, Tie-Cheng; GAO, Yu-Wei; ZHAO, Yong-Kun; YANG, Song-Tao; XIA, Xian-Zhu

AAV vector production: rAAV-N796 or rAAV-Neg vectors were produced by liposome-mediated co-transfection of pAAV-N796 or pAAV-Neg, pAAV-RC and pHelper in human embryonic kidney (HEK) 293 cells according to the protocol. Briefly, 2 hr before transfection, each 10-cm-diameter plate of human 293 cells (80% confluent) was fed 10 ml of fresh DMEM containing 10% FbS without antibiotics. A total of 25 mg of plasmid DNA were dissolved in 1 ml of Liposome 2000 (Invitrogen, Carlsbad, CA, U.S.A.) and then mixed and added to the cells after incubation for 25 min. At 4 to 6 hr after transfection, the medium was replaced with fresh DMEM containing 2% FbS and antibiotics. The cells and suspensions were harvested at 72 hr post infection. After low-speed centrifugation on a tabletop centrifuge, the cell pellets were resuspended in 1 ml of 100 mM NaCl-10 mM Tris-HCl (pH 8.5) and subjected to four cycles of freeze-thaw and removal of cell debris. The rAAV particles were then Transduction with rAAV and infection with RABV in NA cells: NA cells were plated at 1 × 10 6 cells/well in 6-well plates and incubated overnight. The rAAV was inoculated into cells with different viral titers. After transduction for 2 hr, the medium was replaced with fresh DMEM containing 2% FbS and antibiotics. After 24 hr of transduction, the cells were infected with 0.01 MOI of the RAbV CVS-11 virus for 48 hr. Another method was infection of cells with 0.01 MOI of the RAbV CVS-11 virus for 6 hr and then inoculation with rAAV.