title: AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications.
abstract: The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293 T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 μg DNA per ml of media (21 μg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) and 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer.
authors.affiliation: CND, Department of Neurology, Emory University, Atlanta, GA 30322, United States. Electronic address: xhuang4@emory.edu.
authors.forename: Xinping
authors.initials: X
authors.lastname: Huang
authors.forename: Antja-Voy
authors.initials: AV
authors.lastname: Hartley
authors.forename: Yishi
authors.initials: Y
authors.lastname: Yin
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Herskowitz
authors.forename: James J
authors.initials: JJ
authors.lastname: Lah
authors.forename: Kerry J
authors.initials: KJ
authors.lastname: Ressler
mesh_terms: D002460:Cell Line; D002499:Centrifugation, Density Gradient; D000229:Dependovirus / Q000254:growth & development* / Q000302:isolation & purification*; D003956:Dialysis; D006801:Humans; D009584:Nitrogen / Q000032:analysis; D010758:Phosphorus / Q000032:analysis; D011094:Polyethyleneimine / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D019562:Viral Load; D014776:Virus Cultivation / Q000379:methods
chemical_list: D010758:Phosphorus; D011094:Polyethyleneimine; D009584:Nitrogen
keywords: AAV; AAV2-GFP; N/P ratio; Optimization; Polyethylenimine (PEI); Recombinant virus