Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors

Standard protocols for the generation of adenoassociated virus type 2 (AAV-2)-based vectors for human gene therapy applications require cotransfection of cells with a recombinant AAV (rAAV) vector plasmid and a packaging plasmid that provides the AAV rep and cap genes. The transfected cells must also be overinfected with a helper virus, e.g., adenovirus (Ad), which delivers multiple helper functions necessary for rAAV production. Therefore, rAAV stocks produced using these protocols are contaminated with helper adenovirus. The generation of a novel packaging/helper plasmid, pDG, containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids, is described here. Cotransfection of cells with pDG and an AAV vector plasmid was sufficient for production of infectious rAAV, resulting in helper virus-free rAAV stocks. The rAAV titers obtained using pDG as packaging plasmid were up to 10-fold higher than those achieved using conventional protocols for rAAV production. Replacement of the AAV-2 p5 promoter by an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; however, expression of the VP proteins was significantly increased compared with VP levels from standard packaging plasmids. Immunofluorescence analyses showed that the strong accumulation of VP proteins in pDG-transfected cells resulted in enhanced AAV capsid assembly, which is limiting for efficient rAAV production. Furthermore, using a monoclonal antibody highly specific for AAV-2 capsids (A20), an rAAV affinity purification procedure protocol was established. The application of the tools described here led to a significant improvement in recombinant AAV vector production and purification.