Optimizing ddPCR assay for characterizing AAV vector genome integrity

Recombinant adeno-associated virus (rAAV) is a promising gene therapy vector to deliver DNA as a treatment for numerous human diseases. Accurate quantification of the rAAV vector genome titer and characterization of its integrity are critical for determining the clinical dose and ensuring product safety and efficacy. Genome integrity can be defined as the intactness of the vector genome both in relation to its expected size and sequence. The replication and packaging stages of rAAV production can potentially result in the incorporation of truncated or partial genomes compromising genome integrity. Droplet digital PCR (ddPCR) using two sets of primers-probe pairs that target the 5′ beginning and 3′ end of vector genomes allows for estimation of percentage linkage between the targets when a double positive signal is detected. Here we describe ddPCR method optimization for rAAV vector genome characterization and show that omitting a heating step in the ddPCR workflow improves the estimated percentage linkage.

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