Genotyping of AAV Plasmid Stocks: Quality Control in Adeno-Associated Virus Vector Production

Abstract

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.

A Bead-Based Multiplex Assay for the Detection of DNA Viruses Infecting Laboratory Rodents

“…Over the last years, we have already developed several multiplex PCR followed by bead-based Luminex detection using specific probes to detect multiple agents simultaneously in a high-throughput fashion, e.g. human papillomaviruses [27], [28], bovine papillomaviruses [29], human polyomaviruses [30], adeno-associated viruses [31] and cell culture contaminations [32].…”