Two-Step Small Scale Purification of Recombinant Adeno-Associated Viruses

Recombinant adeno-associated viruses (AAVs) are robust and versatile tools for in vivo gene delivery. Natural and designer capsid variations in AAVs allow for targeted gene delivery to specific cell types. Low immunogenicity and lack of pathogenesis also add to the popularity of this virus as an innocuous gene delivery vector for gene therapy. AAVs are routinely used to express recombinases, sensors, detectors, CRISPR-Cas9 components, or to simply overexpress a gene of interest for functional studies. High production demand has given rise to multiple platforms for production and purification of AAVs. However, most platforms rely heavily on large amounts of starting material and multiple purification steps to produce highly purified viral particles. Often, researchers require several small-scale purified AAVs. Here, we describe a simple and efficient technique for purification of recombinant AAVs from small amounts of starting material in a two-step purification method. In this method, AAVs are released into the packaging cell medium using high salt concentration and pelleted by ultracentrifugation to remove soluble impurities. Then, the resuspended pellet is purified using a protein spin-concentrator. The two-step purification consisting of ultracentrifugation and spin-concentration eliminates the need for fraction collection and the time-consuming evaluation of individual fractionated aliquots for titer and purity. In this method, the resulting AAV preparations are comparable in titer and purity to commercially available samples. This simplified process can be used to rapidly generate highly purified AAV particles in small scale, thereby saving resources.