title: A helper virus-free packaging system for recombinant adeno-associated virus vectors.
abstract: Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection 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 onto human 293 cells with a vector plasmid in the absence of Ad infection, the 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 plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43614-5804, USA.
authors.forename: R F
authors.initials: RF
authors.lastname: Collaco
authors.forename: X
authors.initials: X
authors.lastname: Cao
authors.forename: J P
authors.initials: JP
authors.lastname: Trempe
mesh_terms: D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D019065:Virus Assembly