AAV1-CAG-GLuc

AAV viruses belong to the parvovirus family, specifically the dependoparvovirus subfamily. Members of this subfamily require a helper virus, such as adenovirus (Ad) or herpes simplex virus, for efficient infection and replication. The wild-type genome contains three open reading frames encoding replication protein (Rep), capsid protein (Cap), and assembly activation protein, flanked by two inverted terminal repeats. In 1984, Hermonat and Muzyczka published the first paper on a recombinant AAV vector capable of expressing foreign genes in mammalian cells.

Early methods for producing rAAV vectors relied on transient co-transfection of two plasmids: one plasmid carrying an expression cassette for the gene of interest flanked by inverted terminal repeats and the other plasmid carrying the viral rep and cap sequences. These production protocols utilized a helper virus, such as adenovirus, to provide helper functions. A major advance in AAV manufacturing was the identification of the Ad region that mediates AAV vector replication and encapsidation and its cloning into a helper plasmid, enabling a triple plasmid transfection approach. The system uses the human-derived HEK293 cell line and transiently transfects three plasmids: (i) a rep/cap plasmid, (ii) a recombinant vector genome plasmid, and (iii) a helper plasmid expressing adenoviral genes. In addition to being a helper-free approach, the triple transfection protocol offers the flexibility of switching from one serotype to another simply by changing the rep/cap plasmid.