Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer.
AAV AAV production Iodixanol gradient Tangential flow filtration PEGylation Digital droplet PCR
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The methodology outlined herein represents the accumulated knowledge accrued over years of trial and error, and could not be possible without the help of countless people. We would especially like to recognize: Mark Potter, Vince Chiodo, Shannon Boye, and Sanford Boye at the University of Florida. Kevin Nash at the University of South Florida.
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