Novel Tools for Production and Purification of Recombinant Adeno-Associated Viral Vectors

Original standard protocols for the generation of recombinant adeno-associated viruses (rAAVs) have generally involved the cotransfection of 293 cells with a rAAV plasmid vector (pAAV) and a helper/packaging plasmid, followed by over-infection with a helper virus, normally an adenovirus (Ad) at low multiplicity of infection (MOI) (1 ). The pAAV vector contains the transgene of interest flanked by the AAV inverted terminal repeats (ITRs) and the helper/packaging plasmid contains the rep and cap genes of wild-type AAV. The drawback with this procedure is the presence of contaminating Ad helper, together with wild-type-like AAV particles in the derived virus vector stocks. This is because of recombination between the rAAV plasmid and rep and cap genes on the packaging plasmid resulting in the restoration of a wild-type virus. In addition, Ad infection can result in lysis of more than 50% of cells prematurely, releasing rAAV particles into the supernatant and therefore reducing virus particle yields from cellular extracts (2 ).