Do Your AAVs Contain DNA?

Adeno-associated viruses (AAVs) are frequently used as viral vectors in gene therapies to address human diseases, with over 200 studies around the world conducting active clinical Phase 1-3 trials. As gene delivery systems, AAVs include a gene of interest encoded in plasmid DNA that can be up to 5kb in length. AAVs can exist as a heterogeneous population, giving a final  sample that is, for example, 30% full (including the desired plasmid DNA) and 70% empty or partially empty (devoid of the desired plasmid DNA). These empty or partially empty AAV particles can impact potency and immunogenicity and thus are unwanted byproducts of the AAV manufacturing bioprocess. Traditional analytical tools such as transmission electron microscopy  (TEM), analytical ultracentrifugation (AUC), and ion-exchange chromatography (IEX) can be used to characterize capsid content but are complex, labor-intensive, and pose challenges in data reproducibility, throughput, and scalability. Therefore, there exists a need for better methods and systems for the efficient and sensitive quantification of Empty/Full status of AAV samples to be used for gene delivery.

In this application note, we developed a novel method for quantifying the DNA content of AAV particles with Simple Western™, a next-generation biomolecular analytical tool that seamlessly combines capillary electrophoresis and immunodetection with conventional Western blot antibodies.