Genotyping of AAV plasmid stocks: quality control in adeno-associated virus vector production

Recombinant adeno-associated virus (rAAV) vectors are a promising tool for gene therapy. When multiple serotypes are handled in the same laboratory during the AAV vector production, it is essential to have means to identify the serotype in a sample and to confirm the absence of cross-contaminating AAV sequences in plasmid stocks as well as end products. Here, we describe the development of a Multiplex AAV Genotyping (MAG) assay to type sensitively and specifically DNA from AAV serotypes 1-12 and to detect AAV2 serotype DNA sequences encoding peptide insertions used to modify tissue tropism. MAG is based on multiplex PCR using type-specific primers and subsequent multiplex hybridization by Luminex. The assay is highly specific, and can easily identify plasmid cross-contaminations. Using 10-fold dilution series, the detection limit was below 10 AAV genomes per PCR. In artificial cross-contamination experiments with a 1,000-fold excess of one AAV serotype versus another one, the contaminating type could be still detected with 10-100 AAV genomes. In a first application, MAG identified successfully cross-contaminated AAV plasmid stocks. In conclusion, MAG is a powerful high-throughput tool in assessing the purity and identity of AAV DNA plasmids and other starting materials used for AAV vector production.