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 plas
mid that provides the AAV Pep and cap genes. The transfected cells must als
o be overinfected with a helper virus, e.g., adenovirus (Ad), which deliver
s multiple helper functions necessary for rAAV production. Therefore, rAAV
stocks produced using these protocols are contaminated with helper adenovir
us. The generation of a novel packaging/helper plasmid, pDG, containing all
AAV and Ad functions required for amplification and packaging of AAV vecto
r 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 pac
kaging plasmid were up to Ill-fold higher than those achieved using convent
ional protocols for rAAV production. Replacement of the AAV-2 p5 promoter b
y an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; howeve
r, expression of the VP proteins was significantly increased compared with
VP levels from standard packaging plasmids. Immunofluorescence analyses sho
wed that the strong accumulation of VP proteins in pDG-transfected cells re
sulted in enhanced AAV capsid assembly, which is limiting for efficient rAA
V production. Furthermore, using a monoclonal antibody highly specific for
AAV-2 capsids (A20), an rAAV affinity purification procedure protocol was e
stablished. The application of the tools described here led to a significan
t improvement in recombinant AAV vector production and purification.