Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids.

Abstract

Adeno-associated virus (AAV) has shown great potential in gene therapy due to its low immunogenicity, lack of pathogenicity to humans, and ability to provide long-term gene expression in vivo. However, there is currently a need for fast, high-throughput characterization systems that require low volumes for the determination of its sample composition in terms of full and empty capsids since empty capsids are a natural byproduct of AAV synthesis. To address this need, the following study proposes a high-throughput electrophoresis-mediated microfluidics approach that is independent of sample input concentration to estimate the composition of a given sample by combining its protein and ssDNA information relative to a standard. Using this novel approach, we were able to estimate the percentage of full capsids of six AAV8 samples with an average deviation from the actual percentage of 4%. The experiments used for these estimations were conducted with samples of varying percentages of full capsids (21–75%) and varying concentrations (5 × 1011–1 × 1012 VP/mL) with a total volume requirement of 3–10 μL for triplicate analysis of the sample. This method offers a rapid way to evaluate the quality and purity of AAV products. We believe that our method addresses the critical need as recognized by the gene and molecular therapy community.