Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

Abstract

Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications1. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time consuming. Here we report a method to titer AAV with GelGreen(R) dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (~ 30 minutes) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid Elisa results are comparable to each other. We also showed that GelRed(R) dye, a red fluorescence dye from Biotium, can be used to directly "visualize" AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable and cost-efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.