Transposase-Mediated Construction of an Integrated Adeno-Associated Virus Type 5 Helper Plasmid

Adeno-associated viruses (AAVs) are replication-defective parvoviruses that require helper virus functions for efficient productive replication. The AAVs are currently premier candidates as vectors for human gene therapy applications. In particular, much recent interest has been expressed concerning recombinant AAV serotype 5 (rAAV-5) vectors, as they appear to utilize cellular receptors distinct from those of the prototypical AAV serotype (AAV-2) and have been reported to have transduction properties in vivo that differ significantly from those of the prototype. One of the most popular current methods for the production of rAAVs involves co-transfection of human 293 cells with three plasmids: (i) an adenovirus (Ad)-derived helper plasmid containing Ad genes required for AAV replication, (ii) an AAV-derived plasmid encoding complementing AAV genes (i.e., the viral rep and capgenes), and (iii) a target plasmid containing a transgene of interest flanked by AAV inverted terminal repeats (ITRs) that confer packaging and replication capabilities upon the ITR-flanked heterologous DNA. Here we describe novel plasmid reagents designed for convenient and efficient production of rAAV-5. An integrated helper plasmid containing all Ad genes required for the efficient production of recombinant AAV, as well as the complementing AAV genes on the same plasmid backbone, was constructed via transposase-mediated insertion into an Ad helper plasmid of a transposable element containing the AAV-5 rep and cap genes linked to a selectable marker. This simple strategy can be used in the rapid and efficient construction of integrated helper plasmids derived from any reported AAV serotype for which a molecular clone exists.