A helper virus-free packaging system for recombinant adeno-associated virus vectors

Adeno-associated virus (AAV) is a human parvovirus that is currently receiv ing widespread attention for its potential use as a gene therapy vector. Co nstruction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recom binant genome into an infectious virion. Most current protocols for generat ing rAAV entail the co-transfection of a vector plasmid and a packaging pla smid that expresses the viral replication and structural genes onto adenovi rus (Ad) infected cells growing in culture. Limitations of this procedure i nclude (1) contamination of rAAV with the Ad helper virus, (2) low yields o f rAAV and (3) production of replication-competent AAV. In this report we d escribe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infect ion requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected o nto human 293 cells with a vector plasmid in the absence of Ad infection, t he rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasm ids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more wi despread use of the AAV vector system because of its simplicity and high yi elds. This system will be especially useful for preclinical analyses of mul tiple rAAV vectors. (C) 1999 Elsevier Science B.V. All rights reserved.