title: 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.
authors.affiliation: Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany. markus.schmitt@dkfz.de
authors.forename: Markus
authors.initials: M
authors.lastname: Schmitt
authors.forename: Michael
authors.initials: M
authors.lastname: Pawlita
authors.forename: Jürgen
authors.initials: J
authors.lastname: Kleinschmidt
mesh_terms: D001709:Biotechnology / Q000379:methods*; D000229:Dependovirus / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors / Q000235:genetics*; D005838:Genotype; D057166:High-Throughput Screening Assays / Q000379:methods; D010957:Plasmids / Q000032:analysis / Q000235:genetics; D011786:Quality Control; D012680:Sensitivity and Specificity