AAV belongs to the Dependovirus genus, meaning that it depends on a co-infecting helper virus (usually adenovirus) for productive infection to occur. Additionally, the recombinant AAV genome has two essential genes removed to prevent integration and replication to make AAV a safe and effective tool for gene delivery. Therefore, in order to generate more AAV particles, essential genes must be provided in trans.
We use the triple transfection strategy for AAV packaging, which involves co-transfecting the packaging cell line (usually HEK293T) with the recombinant AAV plasmid containing the gene of interest (GOI), a plasmid containing the essential rep and cap genes, and a third adenovirus-derived helper plasmid supplying genes needed for replication.
For our large-scale and preclinical AAV packaging services, the AAV particles are purified using iodixanol (IDX) gradient ultracentrifugation to remove impurities and empty capsids. Viral titer is then determined using qPCR with quantification based on the inverted terminal repeats (ITRs) present in the viral genome. The AAV particles are then shipped overnight and can be used immediately for in vitro or in vivo research.