Adeno-associated virus (AAV) triple transfection for virus production is a widely used method for generating recombinant AAV particles, which are pivotal for gene therapy applications. In Adenovirus helper-free systems, this method involves the co-transfection of three plasmids into producer cells, usually human embryonic kidney 293 (HEK293) cells, which then produce AAV vectors capable of delivering genetic material to target cells. 

The Rep/Cap Plasmid carries the AAV rep capsid (cap) genes. The rep genes are essential for replication of the AAV genome, and the cap gene codes for the viral capsid proteins, determining the AAV serotype and thus its tissue tropism. Adenovirus helper plasmid contains essential helper genes from adenovirus (or herpes simplex virus) that support AAV replication and packaging. These genes complement the functions missing in the rep/cap plasmid and the AAV genome plasmid. 

Encoded within the AAV genome plasmid (or cis-plasmid) is the transgene flanked by AAV inverted terminal repeats (ITRs). The ITRs are necessary for maintenance of episomal stability, replication, and in the presence of Rep protein, packaging into pre-formed AAV capsids and integration of the transgene into the host genome (wilmot). For natural AAV the rep and cap genes are flanked by the ITRs, however, for gene therapy applications, replacing rep and cap to a separate plasmid and inserting the gene of interest (GOI or “gene therapy”) between the ITRs has given rise to recombinant AAVs (rAAV).

In this white paper, our experts dive into the methods used for the detection of recombinant AAVs and the proven and robust detection methods developed at SK pharmteco for several AAV serotypes.