Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

IL-10 delivery by AAV5 vector attenuates inflammation in mice with pseudomonas pneumonia

“…59 To facilitate pseudotyping, pAAV.Cb-mIL10 was co-transfected with pXYZ5, an adenoviral helper plasmid with AAV2 rep/AAV5 cap. 60 Anion-exchange chromatography was used to purify the AAV5 pseudotyped vector, and the dot-blot assay was used to determine the titer of rAAV virions harboring vector genomes. The pseudotyped packaged vector was then designated as AAV5.Cb-mIL10.…”

Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization

“…The recombinant vector AAV2.sFLT01 was produced by triple transfection of 293 cells protocol using helper plasmids p5rep-D-CMVcap and pHelper (Stratagene, La Jolla, CA, USA), and purified according to the protocol using an iodixanol step gradient and HiTrap Heparin column (GE Healthcare Life Sciences, Piscataway, NJ, USA) on an Ä KTA FPLC system (GE Healthcare Life Sciences, Piscataway, NJ). 32,33 The AAV2.sFLT01 viral preparation had a titer of 2.2 Â 10 12 drps (DNase resistant particles) per ml. Viral titers were determined using a real-time TaqMan PCR assay (ABI Prism 7700; Applied Biosystems, Foster City, CA, USA) with primers that were specific for the BGH poly A sequence.…”

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…Iodixanol has a much lower toxicity compared to CsCl; it also prevents aggregation of rAAV particles and does not reduce infectivity. 49,50,52 The recently described multistep AAV purification methods employ a discontinuous iodixanol gradient centrifugation as a prepurification step, followed by an affinity virus purification by a heparinized support matrix chromatography 50 or ion-exchange chromatography. 52 The resulting rAAV stocks have significantly higher purity compared to purification via two rounds of CsCl gradient.…”