Adeno-associated virus: enabling genomic medicine
Quantification of fully loaded and functional titers is crucial for AAV potency determination. The most common method is qPCR, because it is simple and robust under suitable and ideal conditions. Droplet Digital PCR is a viable alternative for rAAV titering with several inherent benefits. First, the method can deliver absolute quantification of VG titer in the absence of a standard curve. The lack of suitable vector reference standards and potential issues with using plasmid DNA makes this feature of ddPCR technology extremely attractive. When using different primer sets targeting various segments of the genes, ddPCR technology is unaffected by different primer sets of secondary conformation of the VG. In addition, ddPCR technology can determine the integrity of the vector genome using a 2-D approach, where two different probes targeting different positions of the rAAV genome are utilized. Indeed the titer of intact rAAV measured by 2-D Droplet Digital PCR was highly correlated with functional virus (rAAV transduction activity) in an accelerated stability study.